INFORMATION TO USERS

This was produced from a copy of a document sent to us for microfilming. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the material submitted.

The following explanation of techniques is provided to help you understand markings or notations which may appear on this reproduction.

1.The sign or “target" for pages apparently lacking from the document photographed is “Missing Page(s)". If it was possible to obtain the missing pagefs) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting through an image and duplicating adjacent pages to assure you of complete continuity.

2. When an image on the film is obliterated with a round black mark it is an indication that the film inspector noticed either blurred copy because of movement during exposure, or duplicate copy. Unless we meant to delete copyrighted materials that should not have been filmed, you will find a good image of the page in the adjacent frame.

3. When a map, drawing or chart, etc., is part of the material being photo­ graphed the photographer has followed a definite method in “sectioning" the material. It is customary to begin filming at the upper left hand comer of a large sheet and to continue from left to right in equal sections with small overlaps. If necessary, sectioning is continued again—beginning below the first row and continuing on until complete.

4. For any illustrations that cannot be reproduced satisfactorily by xerography, photographic prints can be purchased at additional cost and tipped into your xerographic copy. Requests can be made to our Dissertations Customer Services Department.

5. Some pages in any document may have indistinct print. In all cases we have filmed the best available copy.

University Microfilms international 300 N ZEEB ROAD. ANN ARBOR. Mf 48106 18 BEDFORD ROW. LONDON WC1 R 4EJ, ENGLAND 8100160

G o w e r , W i l l i a m R o b e r t , Jr .

DEOXYGUANOSINE KINASE: THE IDENTIFICATION OF MULTIPLE FORMS IN MAMMALIAN TISSUES. PURIFICATION AND CHARACTERIZATION OF THE MITOCHONDRIAL ISOZYME FROM BOVINE LIVER

The Ohio State University PH.D. 1980

University Microfilms International300 N. Zeeto Road, Aim Arbor, MI 48106 PLEASE NOTE: In all cases this material has been filmed 1n the best possible way from the available copy. Problems encountered with this document have been Identified here with a check mark .

1. Glossy photographs 2. Colored Illu stratio n s 3. Photographs with dark background 4. Illustrations are poor copy _ _ _ _ _ 5. °r1nt shows through as there 1s text on both sides of page ______6. Indistinct, broken or small p rint on several pages

7. Tightly bound copy with print lost 1n spine _ _ _ _ 8. Computer printout pages with Indistinct print _ _ _ _ 9. Page(s) lacking when material received, and not available from school or author 10. Page(s) ______seem to be missing 1n numbering only as text follows 11. Poor carbon copy ______12. Not original copy, several pages with blurred type _ _ _ _ 13. Appendix pages are poor copy 14. Original copy with light type 15. Curling and wrinkled pages ______16. O ther ____

University Microfilms International 300 N £555 *D. ANN ARBOR Ml 48106 '3131 761-4700 DEOXYGUANOSINE KINASE:

THE IDENTIFICATION OF MULTIPLE FORMS IN MAMMALIAN TISSUES,

PURIFICATION AND CHARACTERIZATION OF THE MITOCHONDRIAL

ISOZYME FROM BOVINE LIVER

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the

Degree of Doctor of Philosophy in the Graduate School of

The Ohio State University

By

William Robert Gower Jr., B.A., M.S.

• * • «

The Ohio State University

1980

Reading Committee: approved B

Professor David H. Ives Professor George S. Serif Professor Lee F. Johnson Department of Biochemistry To Dawn and Bill

ii ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to Dr. David Ives for his patient guidance, support and friendship during my stay at Ohio

State. I would also like to extend my appreciation to the faculty members in the Department of Biochemistry for their friendship and guidance. I would like to thank a very special friend who also toiled in Room 711 and whose teachings were invaluable, my brother-in-law,

Dr. Marty Deibel. I would like to acknowledge others who were both good friends and consultants in and out of the laboratory, Drs. Ed

Machuga, Tam Davis, Doug Prasher, Sue-May Wang, Mike Carr and Doris

Buchanan and Dale Hershberger. I would like to express my sincerest appreciation to Paula Campbell for her diligent work in typing the final draft of the dissertation. Lastly, a very special acknowledgement to my new family, Denise and Elicia who have added a new dimension to my life that has made the difficult times bearable and the good times more than wonderful. VITA

July 5, 1949...... B o m - Hazleton, Pennsylvania

1970...... A.A. , cum laude, Phi Theta Kappa, Who's Who Among Students in American Junior Colleges, Valley Forge Military Junior College, Wayne, Pennsylvania

1972...... B.A. , Biology, Dean's List, Distinguished Military Graduate, University of Delaware, Newark, Delaware, Student Research Fellowship, Temple University, Philadelphia, Pennsylvania

1972-1974...... Graduate Teaching Associates, Department of Biology, vilianova University, Vilianova, Pennsylvania

1976...... M.S., Biology, Vilianova University, Vilianova, Pennsylvania

1974-1980...... Graduate Teaching Associate, Department of Biochemistry, University Senator, Graduate Student Alumni Research Award, The Ohio State University, Columbus, Ohio

PUBLICATIONS

Gower, W.R. Jr., and Ives, D.H. (1978), "Distinct Mitochondrial and Cytoplasmic Forms of Purine and Pyrimidine Deoxynucleoside Kinases," 176th American Chemical Society National Meeting, Abstract No. 80

Gower, W.R. Jr., Carr, W.C., and Ives, D.H. (1979), "Deoxyguanosine Kinase: Distinct Molecular Forms in Mitochondria and Cytosol," J. Biol Chem. 245, 2180-2183 VITA continued

Gower, W.R. Jr.f Carr, M.C., and Ives, D.H. (1979), "Properties of Mitochondrial Dcoxyguanosine Kinase," Xlth International Congress of Biochemistry, Abstract No. 04-7-S84

Gower, W.R. Jr., and Baker, W.W. (1979), "The Partial Purification and Characterization of the NADP-Dependent Isocitrate Dehydrogenase Allozymes of the Mouse," Biochem. Genet. (Submitted for publication)

FIELDS OF STUDY

Major Field: Biochemical Fnzymology

Studies in Biochemical Genetics. Professors Wilbur Baker, George Marzluf and Lee Johnson

Studies in Developmental Biology. Professor Wilbur Baker

Studies in Cell Physiology and Ptetabolism. Professors John Edwards and John Friede

Studies in Nucleic Acid Biochemistry. Professor David Ives

v TABLE OF CONTENTS

Page ACKNOWLEDGEMENTS ...... '...... iii

VITA ...... iv

LIST OF TABLES ...... viii

LIST OF FIGURES ...... x

LIST OF PLATES ...... xv

ABBREVIATIONS ...... xvi

ABSTRACT ...... xx

INTRODUCTION...... 1

1. Occurrence and Nature of Multiple Forms of Enzymes I 2. Multiple Forms of Mammalian Pyrimidine 10 Deoxynucleoside Kinases

a. Thymidine Kinase 10 b. Deoxycytidine Kinase 15

3. Mammalian Purine Deoxynucleoside Kinases 17 4. Enzyme Regulation and Hysteretic Enzymes 28

EXPERIMENTAL PROCEDURE ...... 35

1. Materials 35 2. Methods 37

a. Analysis of Bases, Nucleosides and Nucleotides 37 b. Enzyme Assays 38 c. Analytical Methods 43 d. Tissue Preparation and Subcellular Fractionation 49

RESULTS ...... 52

1, Electrophoretic Survey of Mammalian Tissues For 52 Deoxynucleoside Kinases 2. Calf Thymus Deoxyguanosine Kinase Activities 61

a. Preliminary Separation of Cytosol and 66 Mitochondrial Deoxyguanosine Kinase Activities

vi Table of Contents continued

Page

b. Partial Purification of Cytosol and 69 Mitochondrial Deoxyguanosine Kinase Activites c. Properties of Cytosol and Mitochondrial 76 Deoxyguanosine Kinase Activities

i. Isoelectric Focusing 80 ii. Molecular Weight Determination 80 iii. Nucleoside Specificity 80 iv. Identification of Enzyme Product 86 v. Apparent For dGuo 91 vi. Effect of Nucleotides on Enzyme Activity 91

3. Purification of Deoxyguanosine Kinase From Bovine 96 Liver

4. Properties of Deoxyguanosine Kinase From Bovine 121 Liver

a. Enzyme Assay 121 b. Analysis of the Hysteretic Effect 137 c. ATP*Mg Optima 157 d. Phosphate Donor Specificity 160 e. Divalent Cation Specificity 160 f. Phosphate Acceptor Specificity 165 g. pH Optimum 167 h. Effect of Triton X-100 167 i. Molecular Weight Determinations 174 j. Heat Lability 177 k . Isoelectric Focus ing 186 1. Effects of Nucleotides on Enzyme Activity 186 m. Preliminary Kinetic Analysis 194

DISCUSSION...... 208

APPENDIX ...... 218

A. Electrophoretic Survey of Various Mammalian Tissues 218 For Pyrimidine Deoxynucleoside Kinases B. Purification of the Cytosol Deoxycytidine 235 Phosphorylating Activity From Calf Thymus

BIBLIOGRAPHY ...... :237

vii LIST OF TABLES

Table Page

1. A Representative Sample of Enzymes Exhibiting 3 Polymorphism in Mammalian Tissues Demonstrating Various Mechanisms of Variant Etiology

2. Properties of Human dCyd and dThd Kinases 11 Isolated From Blast Cells of Acute Myelocytic Leukemia

3. Sources and Some Physico-Chemical Properties 25 of Mammalian Enzyme Preparations Exhibiting dGuo Kinase Activity

4. Paper Chromatographic Systems Utilized For 39 Separation of Various Bases, Nucleosides and Nucleotides

5. Purification of Calf Thymus Mitochondrial dGuo 79 Kinase

6 . Nucleoside Specificity of dGuo Kinase Activity 85 From Calf Thymus

7. Effects of Various Pyrimidine Nucleotides on 94 dGuo Kinase Activities From Calf Thymus

8. Effects of Various Purine Nucleotides on dGuo 95 Kinase Activities From Calf Thymus

9. Distribution of Mitochondrial Marker Enzymes and 103 Deoxyguanosine Kinase in the Sequential Differential Centrifugation Fractions From Bovine Liver

10. Sedimentation of Mitochondrial dGuo Kinase From 107 Bovine Liver After Osmotic, Triton X-100 and Sonic Lysis of Mitochondria

11. Purification of Bovine Liver Mitochondrial dGuo 118 Kinase

12. Properties of the Bovine Liver Mitochondrial dGuo 142 Kinase Reaction

viii List of Tables continued

Table Page

13. Sunmary of Results From Bovine Liver Mitochondrial 152 dGuo Kinase Reaction Time Courses: Effect of Preincubation of the Enzyme With Various Agents

14. Phosphate Donor Specificity of the Bovine Liver 163 Mitochondrial dGuo Kinase

15. Divalent Cation Specificity of the Bovine Liver 164 Mitochondrial dGuo Kinase

16. Nucleoside Specificity of the Bovine Liver 166 Mitochondrial dGuo Kinase

17. Effects of Various Nucleoside Monophosphates on 191 Bovine Liver Mitochondrial dGuo Kinase

13. Effects of Various Nucleoside Diphosphates on 192 Bovine Liver Mitochondrial dGuo Kinase

19. Effects of Various Nucleoside Triphosphates on 193 Bovine Liver Mitochondrial dGuo Kinase

ix LIST OF FIGURES

General Pathways For Nucleotide Salvage and 18 Interconversion in Mammalian Tissues

Electrophoretic Profile of Deoxynucleoside S3 Kinases in Adult Rat Liver and Thymus

Electrophoretic Profile of Deoxynucleoside 55 Kinases in Adult Rat Liver Mitochondria

Electrophoretic Profile of Deoxynucleoside 57 Kinases in Adult Rat Spleen Mitochondria

Electrophoretic Profile of Deoxynucleoside 59 Kinases in Adult Bovine Liver and Calf Thymus Cytosol and Mitochondrial Fractions

Electrophoretic Profile of Deoxynucleoside 62 Kinases in Adult Hamster Liver Mitochondria

Electrophoretic Profile of Deoxynucleoside 64 Kinases in Mouse Liver Mitochondria and HeLa Cells

Analytical Disc PAGE of the Calf Thymus dGuo 67 Kinase From Cytosol and Mitochondrial Fractions Generated by Sequential Differential Centrifugation

Chromatography of Calf Thymus Ammonium Sulfate 70 Fractions on DEAE-Cellulose

Elution Profile of Calf Thymus Mitochondrial 74 dGuo Kinase From Sephacryl S-200

Blue Sepharose CL-6B Affinity Chromatography 77 of Calf Thymus Mitochondrial dGuo Kinase (Fraction III)

Isoelectric Focusing (pH 5-7) of the Calf 81 Thymus dGuo Kinase Activities

x List of Figures continued

Figure Page

13. Molecular Weight Determination of the Calf Thymus 83 Mitochondrial dGuo Kinase By Gel Filtration on Sephacryl S-200

14. Identification of the Tritium-labeled Produce of 87 the Calf Thymus Mitochondrial dGuo Kinase Reaction by Ion Exchange Paper Chromatography

15. Identification of the Phosphorylated Deoxyguanosine 89 Product of the Calf Thymus Mitochondrial dGuo Kinase Reaction

16. Effect of Varying dGuo Concentration on the Reaction 92 Rate of Calf Thymus dGuo Kinase Activities

17. Time Dependent Release of dGuo Kinase, dThd Kinase 105 and Glutamate Dehydrogenase From Mitochondrial By Sonication

18. Elution Profile of Bovine Liver Mitochondrial dGuo 109 Kinase From DEAE-Sephacel

19. Elution Profile of Bovine Liver Mitochondrial dGuo 112 Kinase from Sephacryl S-200

20. Elution Profile of Bovine Liver Mitochondrial dGuo 114 Kinase From Blue Sepharose CL-6B

21. Analytical Disc PAGE Gel of Bovine Liver Mitochondrial 119 dGuo Kinase fFraction VII)

22. Scan of an SDS PAGE Gel of Bovine Liver dGuo Kinase 122 (Fraction VII)

23. Molecular Weight Determination of the Two Mnjor 124 Protein Components in Fraction VII By SDS PAGF

24. Reaction Time Course of Bovine Liver Mitochondrial 126 dGuo Kinase: Effect of Varying Enzyme Concentration on Length of Lag Period

25. Effect of Varying Enzyme Concentration on the Length 128 of the Lag Period

xi List of Figures continued

Figure Page

26. Effect of Preincubating the Bovine Liver . 131 Mitochondrial dGuo Kinase With Various Components of the Assay Mixture on the Reaction Time Course

27. Reaction Time Course of Bovine Liver dGuo Kinase: 133 Comparison of DE-81 Disk Assay Method With Chromatographic Quantitation of Product

28. Dependence of Bovine Liver Mitochondrial dGuo 135 Kinase Activity on Enzyme Concentration

29. Identification of the Tritium-labeled Product 138 of the Bovine Liver dGuo Kinase Reaction By Ion Exchange Paper Chromatography

30. Identification of the Phosphorylated Deoxyguanosine 140 Product of the Bovine Liver dGuo Kinase Reaction

31. Reaction Time Course of Bovine Liver Mitochondrial 143 dGuo Kinase: Temperature Dependence of the Lag Phenomenon

32. Reaction Time Course of Bovine Liver Mitochondrial 145 dGuo Kinase: Effect of Varying Preincubation Time With ATP on the Length of the Lag Period

33. Reaction Time Course of Bovine Liver Mitochondrial 148 dGuo Kinase: Effect of Varying ATP Concentration in the Preincubation Mixture on the Lag Period

34. Analysis of ATP By Ion Exchange Paper Chromato­ 150 graphy After Incubation With Bovine Liver dGuo Kinase

35. Analysis of ATP By Paper Electrophoresis After 150 Incubation With Bovine Liver dGuo Kinase

36. Reaction Time Course of Bovine Liver Mitochondrial 153 dGuo Kinase: Effect of Preincubation of Enzyme With Various Ligands

xii List of Figures continued

Figure Page

37. Reaction Time Course of Bovine Liver Mitochondrial 155 dGuo Kinase: Effect of Preincubation of Enzyme With AMPPCP.

38. Glycerol Gradient Centrifugation of Bovine Liver 158 Mitochondrial dGuo Kinase

39. Effect of MgCl2 Concentration on the Bovine Liver 161 dGuo Kinase Activity at Feveral Fixed ATP Concentrations

40. Effect of pH on the Activity of the dGuo Kinase 168 From Bovine Liver Mitochondria

41. Effect of Varying Concentrations of Triton X-100 170 on the Bovine Liver Mitochondrial dGuo Kinase

42. Reaction Time Course of Bovine Liver Mitochondrial 172 dGuo Kinase: Effect of Enzyme Preincubation at 4°C and 37 °C, With or Without Triton X-100

43. '*olecular Weight Determination of the Bovine 175 Liver Mitochondrial dGuo Kinase By Gel Filtration on Sephacryl S-200

44. Molecular Weight Determination of the Bovine 178 Liver Mitochondrial dGuo Kinase Using Disc PAGE

45. Effect of Temperature on the Stability of Bovine 180 Liver Mitochondrial dGuo Kinase

46. Heat Inactivation of Bovine Liver dGuo Kinase at 182 50°C and 65°C in the Presence of ATP

47. Stability of Bovine Liver Mitochondrial dGuo 184 Kinase at 65°C In the Presence of Various Ligands

48. Isoelectric Focusing (pH 3.5-10) of Bovine 187 Liver Mitochondrial dGuo Kinase

49. Isoelectric Focusing (pH 5-7) of Bovine Liver 189 Mitochondrial dGuo Kinase

xiii List of Figures continued

Figure Page

50. Effect of Varying dGuo Concentration on the 195 Reaction Rate of Bovine Liver dGuo Kinase

51. Effect of Varying dGuo Concentration on the 197 Reaction Rate of the Bovine Liver dGuo Kinase; Inhibition by dlno

52. Replot of Data From Figure 51, Slopes Versus 200 dlno Concentration

53. Effect of Varying dGuo Concentration on the 202 Reaction Rate of the Bovine Liver dGuo Kinase; Inhibition by dGTP

54. Effect of Varying ATP Concentration on the 204 Reaction Rate of the Bovine Liver dGuo Kinase; Inhibition by dGTP

55. Replot of Data From Figure S4. Slopes Versus 206 dGTP Concentration

56. Electrophoretic Profile of dThd and dCyd Kinases 219 in Whole Fetal Rat Cytosol and Mitochondrial Cell Fractions

57. Electrophoretic Profile of dThd and dCyd Kinases 221 in Mitochondria Isolated From Adult Pat Liver, Kidney and Spleen

58. Electrophoretic Profile of dThd and dCyd Kinases 223 in Mitochondria Isolated From Adult Rat Brain and Testis

59. Electrophoretic Profile of dThd and dCyd Kinases 225 in Cytosol of Whole Fetal PD-4 HamsteT Tissue

60. Electrophoretic Profile of dThd and dCyd Kinases 227 in Cytosol of C3H Mouse Spleen

61. Electrophoretic Profile of dThd and dCyd Kinases 229 In Cytosol of C3H Mouse Liver

62. Electrophoretic Profile of dThd and dCyd Kinases 231 In a Whole Extract of HTsv_4o Cells

63. Electrophoretic Profile of dThd and dCyd Kinases 233 In a Whole Cell Extract of WI-38 Cells

xiv LIST OF PLATES

Plate Page

I. Electron Micrograph of the Crude Bovine 99 Liver Mitochondrial Fraction (P-16.7): Magnification X7,250.

II. Electron Micrograph of the Crude Bovine 101 Liver Mitochondrial Fraction (P-16.7): Magnification X24.000.

xv ABBREVIATIONS

Ado Adenosine

ADP Adenosine 5'-diphosphate

AMP Adenos ine 5'-monopho sphate

AMPPCP Adenosine 5'(6,y -methylene)-triphosphate

ANS Aminonaptholsulphonic acid ara-A 1-8 -D-arabinofuranosyladenine ara-C 1- 6 -D-arabinofuranosylcytosine ara-G 1- 8 -D-arabinofuranosylguanine

ATP Adenosine 5'-triphosphate bis N,N'-methylene-bis bis-MSB p-bis-(o-methylstyryl)-benzene

Brdlfrd 5-bromo-2 'deoxyuridine

BSA Bovine serum albumin

CDP Cytidine S'-diphosphate

CMP Cytidine 5’-monophosphate

CTP Cytidine 5'-triphosphate

Cyd Cytidine dAdo 2’-deoxyadenosine dADP 2’-deoxyadenos ine 5f-diphosphate dAMP 2*-deoxyadenosine 5'-monophosphate dATP 2’-deoxyadenosine 51 -triphosphate dCDP 2’-deoxycytidine 5’-diphosphate

xvi Abbreviations continued

dOtP 2 r-deoxycytidine 5r-monophosphate

dCTP 2 f-deoxycytidine S r-triphosphate

dCyd 2'-deoxycytidine

DHAE Diethylaminoethyl

dGDP 2'-deoxyguanosine S 1-diphosphate

dGMP 2*-deoxyguanosine 5'-monophosphate

dGTP 2 ’-deoxyguanosine 5'-triphosphate

dGuo 2'-deoxyguanosine

dlno 2'-deoxyinosine dIDP 2*-deoxyinosine 5'-diphosphate dIMP 2'-deoxyinosine 5'-monophosphate disc D i scont inuous dr TP 2'-deoxyinosine 5'-triphosphate

DNA Deoxyribonucleic acid dTDP 2'-deoxythymidine 5 ’-diphosphate dThd Z 1-deoxythymidine dTMP 2’-deoxythymidine 5'-monophosphate

DTE Dithioerythritol

DTT Dithiothreitol dTTP 2'-deoxythymidine 5’-triphosphate dUMP 2’-deoxyuridine 5'-monophosphate dUDP 2’-deoxyuridine S *-diphosphate Abbreviations continued dUTP 2 *-deoxyuridine 5'-triphosphate dUrd 2 *-deoxyuridine

EHNA erythro-9-(2-hydroxy-3-nonyl) adenine

BETA Ethylenediaminetetraacetate

FdUrd 5-fluoro-2'deoxyuridine

GDP Guanosine 5'-diphosphate

CMP Guanosine 5'-monophosphate

GTP Guanosine S'-triphosphate

Guo Guanosine

Hx Hypoxanthine

HM Homogenizing medium

IDP Inosine 5'-diphosphate

IMP Inosine 5f-monophosphate

ITP Inosine 5f-triphosphate

NAD 6-nicotinamide adenine dinucleotide, oxidized form

NADH 6-nicotinamide adenine dinucleotide, reduced form

NADP 6-nucotinamide adenine dinucleotide phosphate, oxidized form

CM Osmolysis medium

PAGE Polyacrylamide gel electrophoresis

IWSF Phenylmethysulfonyl fluoride

PPO 2,5-diphenyloxazole

j xviii Abbreviations continued

PRPP 5-phosphoribosyl-1-pyrophosphate

(d)R-l-P fdeoxy-) ribose-X-phosphate

RNA Ribonuc 1 e ic ac id

SDS Sodium dodecyl sulfate

TCA Trichloracetic acid

THtED N,N,N;N'-tetramethylethylenediamine

a-TGJR a-2'-deoxy-6-thioguanosine

S-TGdR g-2'-deoxy-6-thioguanosine

Tris Tris-(hydroxymethyl)-aminomethane

UDP Uridine 51-diphosphate

UMP Uridine 5'-monophosphate

Urd Uridine

UTP Uridine 5'-triphosphate

YADH Yeast alcohol dehydrogenase

xix ABSTRACT

DEOXYGUANOSINE KINASE:

THE IDENTIFICATION OF MULTIPLE FORMS IN MAM1ALIAN TISSUES,

PURIFICATION AND CHARACTERIZATION OF THE MITOCHONDRIAL

ISOZYME FROM BOVINE LIVER

By

William Robert Gower Jr., Ph.D.

The Ohio State University, 1980

Professor David H. Ives, Advisor

Analytical discontinuous polyacrylamide gel electrophoresis has been used to determine the occurrence of multiple forms of both purine and pyrimidine deoxynucleoside kinases in manmalian tissues. Three forms of deoxyguanosine (dGuo) kinase activity have been identified.

A unique dGuo kinase activity was found in mitochondria, and most species examined had an additional constitutive dGuo kinase in the cytosol. However, in bovine tissues, the cytosol dGuo kinase was associated with a rapidly migrating deoxycytidine (dCyd) and deoxyaden­ osine (dAdo) kinase which was found only in proliferating cells. The calf thymus mitochondrial and cytosol forms were partially purified by

DEAE-cellulose, gel filtration and affinity chromatography on Blue

Sepharose CL-6B. In addition to differences in sub-cellular location and electrophoretic mobility, the two forms could be differentiated by chromatographic behavior on DEAE-cellulose and Blue Sepharose CL-6B, nucleoside specificity, apparent for dGuo, isoelectric pH, and

xx end-product inhibition. The mitochondrial dGuo kinase was primarily

specific for dGuo and deoxyinosine (dlno). Unlike the cytosol enzyme, which proved to be the broadly specific dCyd kinase studied by others,

the mitochondrial enzyme did not phosphorylate dCyd. Its apparent

Kflj for dGuo, 6.0 x 10"6 M, was two orders of magnitude lower than that of the cytosol enzyme. The mitochondrial enzyme was strongly inhibited by dGTP and dITP and activated up to 6-fold by dTDP and UDP when assayed at pH 8.0. In contrast to the cytosol enzyme, neither dCTP nor dATP had much effect on the mitochondrial enzyme. The mitochondrial enzyme was purified 1,857-fold from bovine liver using DEAE-Sephacel, Sepha­ cryl S-200, thermodenaturation, Blue Sepharose affinity chromatography and preparative gel electrophoresis. The enzyme reaction exhibits an

initial lag period with a half-life of 2 to 5 minutes. The length of the lag was inversely proportional to enzyme concentration and could be completely eliminated by prior incubation with ATP, CTP or dGTP for 30 minutes at 37*C, The ATP activation effect was found to be dependent upon temperature, time and ATP concentration used in preincubation. The mitochondrial activity required the presence of a divalent cation and a nucleoside triphosphate. Maximal rate was achieved with ATP »

1.5 x 10"3 M) as the donor with CTP and dTTP being the only other effec­ tive donors. The enzyme had a molecular weight of 5S,000 as determined by gel filtration and glycerol gradient centrifugation and 65,000 as determined by polyacrylamide gel electrophoresis. Isoelectric pH of the enzyme was determined to be 5.75. Optimal enzyme activity was ob­ served at pH 5.0 in the presence of Triton X-100 and a sulfhydryl protecting agent. The rate of the first order heat inactivation of the

xxi enzyme at 65 “C was significantly decreased by ATP, dTDP and dGTP. The enzyme is subject to end-product inhibition by dGTP and the structural analogue dITP. Inhibition by dGTP was non-competitive with respect to dQao and competitive with respect to ATP with an apparent of 2.5 x

10“7 M. At pH 5.0, dTDP and UDP were also inhibitory, whereas at pH

8.0 1.0 mM dTDP stimulated activity almost 3-fold. The enzyme prepara­ tion phosphorylated dGuo and dlno exclusively. The nonpreincubated enzyme exhibited bimodal kinetics with respect to dGuo, whereas the activated enzyme exhibited linear kinetics with an apparent Kj,, of 4.5 x 10~5 M. dlno was a competitive inhibitor with respect to dGuo with an apparent Kj of 2.5 x 10“4 M. INTRODUCTION

1. Occurrence and Nature of Multiple Forms of Enzymes

The concept of multiple forms of enzymes or isozymes was first intro­ duced by Markert and Miller in 1959 Cl)* Prior to this time most inves­

tigators attributed heterogeneity in enzyme preparations to artifacts.

Today while artifacts remain and erroneous interpretations exist, it is abundantly clear that multiple foims of enzymes and proteins exist within a single organism which represent genetically distinct species with

identical or similar catalytic character.

Since their discovery, variant forms of enzymes have proved to be ubiquitous phenomena that occur as the result of a variety of different mechanisms. These mechanisms can be differentiated into genetic and non- genetic, and for any single enzyme system, more than one mechanism may be operating.

Genetic mechanisms include multiple gene loci and allelic variation.

With the occurrence of multiple gene loci there exist two or more separate genes coding for distinct polypeptides. These variants represent isozymes in their truest sense (2). The occurrence of alleles at any single gene locus will, in the heterozygous condition, provide two different polypep­ tide chains from a single locus. The products produced as a result of either or both mechanisms may form separate monomeric enzymes, or in the case of oligomeric enzymes, associate in a variety of ways to form several combinations. 2

Nongenetic or chemical mechanisms include those processes which in­ volve intracellular action on the polypeptide product, either from a single locus or multiple loci having the same or different alleles.

This would include alterations of the primary structure after its trans­ lation, i.e. proteolytic cleavage and chemical modifications of amino acid residue, including the addition or removal of amide, acyl, carbohy­ drate, phosphate and sulfate groups, to name a few. This category also encompasses secondary rearrangements of the protein’s tertiary structure leading to conformational changes, as well as alterations in quaternary structure of oligomeric species where individual subunits remain cataly- tically active. Enzyme variants that are generated in this fashion are best labeled "pseudo-isozymes," since they represent different states of the same primary structure (3).

Table 1 lists nine representative enzyme systems which exhibit multi­ ple forms in mammalian tissues. These enzymes demonstrate the variability in the type and number of variant forming mechanisms operating in mam­ malian tissues.

L-lactate dehydrogenase (EC 1.1.1.27) catalyzes the interconversion of lactate and pyruvate in the presence of NAD. In mammals as in other vertebrates there are three separate genetic loci (H,M,X) that code for one of the subunits of the tetrameric enzyme (4-7). There are two major types of lactate dehydrogenase which are electrophoretically distinguish­ able. One type is found predominantly in the heart (H) and the other in skeletal muscle (M). The H and M genes are expressed in different degrees in different cell types at different stages of cell differentia­ tion (4). The polypeptide products of the two loci in most tissues give rise, through random association, to a set of five isozymes (H^H^M^Hj

M 2,fW3,M4). A third gene, designated X, codes for a polypeptide subunit Table 1. A Representative Sample of B u y * * Exhibiting Polymorphism in Mawuliam Tissues D e m i s tret ing Various Mechanisms of Variant Etiology9

No. Of MechatiismU) No. Of Allelic B u yn e Variants Detected Otwiating Loci Polymorphism Specificity Ref.

Lactate Ifchydrogenase (EC 1.1,1.27) 6 Genetic-ML 3 yes tissue (4-12) differentiation

Pyruvate Kinase (EC 2.7.1.40) 5 Genetic-ML ND yes tissue (13-16) Qtumical-C.AG cell cycle

Aldolase (1£ 4.1.2.7) Geuetic-M. 3 yes tissue 07-24) Qumical-HGQtl^ differentiation

Creatine Kinase (EC 2.7.1.2) >6 Genctic-ML 2* ND tissue (25*32) Chemical-RSI subcellular differentiation

J-Galuctosidasc (EC 3.2.1.23) >4 □temical -SA,RC0Hl2 1 yes tissue (33-36) suhcellular

Isocitratu Dehydrogenase 3 Genetic-ML 3 yes tissue (37-4S) (KID-linked, IX 1.1.1.41) subcellular (WUIP-linked, EC 1.1.1.42)

Glutamine Hiosplioribosylpyrophospliate 2 (lumical-AG 1 ND ND (46*50) AmiJotransferasc (EC 2.4.2.14) thymidine Kinase (IX 2.7.1.21) 2 Genetic-M, 2 ND subcellular (51*54) cell cycle

IK-oxycytidinu Kinase (EC 2.7.1.24) 2 Genet1C-ML 2 M) subcellular (04-97) cell cycle

“Abbreviations used ere: M. • Multiple loci; NGONIj- aside modification; RSI - sulfbydryl modification; SA • sialic acid modification; Nil - not determined; AG * aggregation; C ■ conformational 4

that, in vivo, combines solely with itself to form a tetramer (X^) located

exclusively in primary spermatocytes (8), In addition to differences in

tissue distribution and electrophoretic mobility, the two major isozymes

differ in amino acid composition, imnunological and catalytic properties

CO-11). Primarily, the catalytic differences are that the enzyme has

both a lower K^, K± and turnover number, The tissue specificity, differ­

ences in catalytic properties and constancy of the lactate dehydrogenase

isozymes suggest that they have physiological significance, but the true meaning for why three genes for the enzyme are maintained is unclear (12),

Pyruvate kinase (EC 2.7.1.40) catalyzes the transfer of phosphate

from phosphoenolpyruvate to ADP. In rat there appears to be three basic

kinase isozymes, designated K, M and L which are electrophoretically dis­

tinguishable. The K form is found predominantly in fetal tissues, kidney

and other adult organs, the M foim in adult muscle and brain, and the L

isozyme in adult liver and erythrocytes (13-15). These major forms can be differentiated by pi, molecular weight, catalytic properties and sen­

sitivity to inhibition by various triphosphonucleotides (15). Isoelec­ tric focusing has shown that the K isozyme exhibits pi values of 6.4 or

6.8, the L isozyme 5.4 or 5.7 and the M type 7.S (13). Focusing results have demonstrated that in liver 90% of the total pyruvate kinase activity is in the L form with the remaining 101 in the K form, while in kidney cortex 80% is in the K form and 201 in the L form (13). However, in muscle the M isozyme was shown to represent total cellular activity (13).

Evidence has demonstrated that the pi 5.4 and 5.7 forms of the liver

(L) isozyme are probably R and T confoimers of a single protein (IS).

Hie pi 5.7 form exhibits sigmoidal kinetics with respect to phosphoenol- pyruvate and is activated by fructose 1,6-diphosphate, whereas the pi 5,4 5

enzyme is not (15). In addition, when the pi 5.7 form was incubated with fructose 1,6-diphosphate it was converted to the pi 5.4 form.

Ibsen, et aL (15) also demonstrated that the pi 6.4 variant of the

K isozyme exists in an equilibrium between dimer and tetrameric forms

in which magnesium produces a shift towards the tetrameric form. The magnesium effect was also produced by fructose 1,6-diphosphate with the

isozyme isolated from human erythrocytes (16).

Aldolase (EC 4.1.2.7) catalyzes the reversible aldol condensation between fructose 1,6-diphosphate and glyceraldehyde 3-phosphate. In marmalian tissues there are three electrophoretically distinct types of the tetrameric enzyme, designated A, B and C (17). Aldolase hetero­ geneity, like lactate dehydrogenase, is regulated by tissue specific and developmental mechanisms. Aldolase A is found predominantly in adult skeletal muscle, heart, spleen and brain, B in liver and C in brain

(17). Kidney contains both A and B forms with three heteropolymers, the product of random association of the A and B subunits. Brain contains both A and C forms with three heteropolymers completing the character­

istic five isozyme set. However in embryonic skeletal muscle both A and

C forms are found. During myogenesis there is a transition from an

"embryonic" isozyme pattern produced by the two forms to an "adult" pat­ tern where the A form is found exclusively (18).

In addition to the differences in electrophoretic mobility, the three major forms differ in molecular weight, amino acid composition, immunological specificity and catalytic properties (19-22). Aldolase A appears to be the form primarily involved in glycolysis. This conclusion is based on the findings that the enzyme is inhibited by ATP and 6 diethylstilbestrol and cleaves fructose 1,6-diphosphate fifty-times fas­ ter than fructose 1-phosphate (17). Aldolase B splits both substrates equally, is not inhibited by ATP o t diethylstilbestrol and has a lower

for triose phosphates consistent with its proposed role in gluconeo- genesis in liver (17). Hie properties of aldolase C are intermediate with the A and B forms, thus making its physiological role unclear.

Besides the genetic distinction of the three major forms it has been demonstrated that aldolase A exhibits heterogeneity in its subunits upon isoelectric focusing (23). In rabbit muscle the aldolase A hetero­ geneity was found to be the direct result of deamidation of a single asparagine residue resulting in a random distribution of tetramers containing from zero to four deamidated subunits. The extent of deami­ dation was found to be directly proportional to the age of the organism

(24). Whether this process is a result of the tertiary structure or an endogenous enzymological activity remains undefined.

Creatine kinase (EC 207.3.2) catalyzes the reversible transfer of phosphate between creatine and ATP. In most vertebrates there are four principal electrophoretic forms of creatine kinase. Three cytosol isozymes occur in characteristic tissue-specific distributions while the fourth occurs in mitochondria (25,26). The majoT cytosol isozymes, desig­ nated NM, MB and BB, are dimers having a molecular weight of approximately

80,000, formed by the association of two types of subunits (27,28). The fW type appears predominantly in skeletal muscle, the BB type predominant­ ly in brain and the MB type in heart. The three major cytosol forms plus the mitochondrial isozyme exhibit significant differences in kinetic properties, amino acid composition and inraunological specificity (26,28-30). 7

Similar to the aldolase isozyme transition in skeletal muscle, the isozyme pattern of creatine kinase also changes with muscle differenti­ ation both in vivo and in vitro (25,31). The first catalytically active form in embryonic muscle is the BB form. After eleven days gestation in chick all three forms (BB,MB,NM) are present, and by fifteen days there has been an almost complete transition to the NM form (25).

The purified mitochondrial isozyme, while appearing as a single band upon SDS electrophoresis, has recently been found to exhibit hetero­ geneity upon electrophoresis of the native enzyme (32). It was demon­ strated that one form was converted to the other by incubating the enzyme with 2-mercaptoethanol, indicating that the mechanism of interconversion involved reversible oxidation of sulfhydryl groups.

6-galactosidase (EC 3.2.1.23) catalyzes the hydrolytic cleavage of

0-galactosides to yield free galactose. In rat, mouse and human tissues the activity is found to exist in at least two molecular forms, one having an acid pH optimum and the other a neutral pH optimum (33-36).

Recent studies in mouse have revealed that the enzyme activity is coded for by one genetic locus and the multiple forms found are the result of various secondary or chemical modifications to the dimeric protein mole­ cule (36). Upon gel filtration the liver enzyme exhibited two molecular weight foTms. At pH 8.1 the molecular weight appeared to be 110,000, whereas at pH 5.2 the molecular weight shifted to approximately 230,000 dal tons. At intermediate pH values both species were observed, indicating a pH dependent aggregation of the 110,000 subunit. In liver, lung and spleen the majority of activity was localized in lysosomes, while in kidney and brain approximately 30 to 40% of the enzyme activity was associated with the microsomal fraction with the remainder in lysosomes (36). 8

These tissues, in addition, contained multiple forms as determined by electrophoresis or isoelectric focusing. The forms differed in net charge but not molecular weight. The source of charge differentiation was not determined, but it was suggested that the loss of amide groups in the older enzyme molecules was a possibility. It was demonstrated that the tissue differences in enzyme heterogeneity was due to differ­ ential sialylation of the enzyme. After neuraminidase treatment the electrophoretic mobility of the enzyme from all tissues was reduced to a conmon pattern identical with that exhibited by the kidney. The sialylation was found to be the most extensive in liver and decreased in other tissues to apparently none in kidney. All of the variant forms appeared to have similar molecular weights, thermolabilities and kinetic properties. In addition, a mutation in the structural gene of B-galacto- sidase, located on chromosome 9, affected the electrophoretic mobility of all the variant forms (36).

IsocitTate dehydrogenase catalyzes the reversible transformation of isocitrate to 2-oxoglutarate. In mammalian tissues it is found to exist as two distinct types. One form of the enzyme has a specific coenzyme requirement for MAD (EC 1.1.1.41) and the other for NADP (EC 1,1.1.42),

The NAD-specific enzyme is located solely in mitochondria, whereas two forms of the NADP-specific enzyme are found, one in mitochondria and the other in the extramitochondrial cytosol (37,38). Besides its exclusive intramitochondrial location and cofactor requirement, the NAD-specific enzyme has been found to differ from the NADP-specific forms in K for tn isocitrate, optimal pH, molecular weight and stability (38). In addition, the NAD-specific isocitrate dehydrogenase is stimulated by AMP and ADP 9

but is inhibited by ATP and NADH which are both competitive with NAD

(38,39), Atkinson demonstrated that the NAD-specific enzyme responded directly to the adenylate energy charge similar to the response of other

ATP producing enzymes in the cell (40). In light of its intracellular energy control mechanism it is believed that this form of the enzyme is responsible for the oxidation of isocitrate in the tricarboxylic acid cycle in organisms which maintain the three forms. In addition to dif­ ferent intracellular locations, the NADP-specific isozymes have been found to differ in their electrophoretic mobility, genetic control, tis­ sue distribution and heat lability (41-43), The physiological role of the two forms is still under speculation. One theory suggests that the cytoplasmic isozyme may supply reduced triphosphopyridine nucleotide for fatty acid synthesis (44,45),

Glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) catalyzes the 8 substitution of the amido group of glutamine on C-l of

PRPP. This reaction is the initial rate-limiting step in de novo purine biosynthesis (46). The enzyme has been isolated and characterized from several sources including pigeon (47,48) and human (46,49,50). Kineti- cally the enzyme exhibits cooperative binding of PRPP, with classical sig­ moid kinetics accentuated by the negative effector AMP, The inhibition produced by AMP, as well as other purine ribonucleotides, can be reversed by increasing the relative concentration of PRPP (46). The enzyme from pigeon liver has been shown to demonstrate hysteretic behavior, i.e. there was a variable lag phase in the reaction time course before maximal velo­ city was achieved (48). This lag period could be reduced significantly by preincubating the enzyme with PRPP, In searching for a molecular 10 mechanism for this kinetic behavior it was determined that the human enzyme could be separated into two molecular weight species by either gel filtration or sucrose gradient sedimentation (46). It was demon­ strated that the low molecular weight form (133,000) could be converted to the high molecular weight form (270,000) by incubating the smaller form with purine ribonucleotides (AMP,GMP), When the high molecular weight species was incubated with PRPP and sedimented in the presence of PRPP, only the small form was found in the gradient. It was also shown that as AMP concentrations increased and enzyme activity decreased there was a concomitant decrease in the relative concentration of the low molecular weight enzyme. From these results it was concluded that the low molecular weight form is the catalytically active species and the high molecular weight form inactive.

2. Multiple Forms of Mamnalian Pyrimidine Deoxynucleoside Kinases

a. Thymidine Kinase

Thymidine kinase (EC 2.7.1.21) catalyzes the transfer of phosphate from ATP to dThd, dUrd and various halogenated analogues of both to form the monophosphonucleotide. Two forms of the enzyme have been demon­ strated in mammalian tissues (51-54). One isozyme is located in the cytosol and the other in the mitochondria. In human the two forms differ in electrophoretic mobility, elution from dThd-Sepharose, pi, molecular weight, apparent activation energy, Km foT dThd and ATP, nucleoside specificity, phosphate donor specificity, sensitivity to inhibition by distal end-products and genetic control (54-56). Table 2 lists some of these specific differences found between the two isozymes isolated from human blast cells of acute myelocytic leukemia. Table 2. Properties of Huaan dCyd and dT)id Kinases Isolated Fraa Blast Cells of Acute Myelocytic leukosis (54,55,90)

dThd J&L Property (Vtosol Mitochondria Cytosol Mitochondria licleosidc Specificity dllid, dllrd dThd. dllrd dCyd, Cyd, ara-C dCyd, dThd

Km (nucleoside) 2.6 /IM 5.2 JJH 3.0 fJM 2S pH

Km (ATP-lfc) 0.22 M 3 0.10 n H 0.016 a M o.o? m

\u 90,000 70,000 70,000 70,000

Uisc 1‘AiUi Kf 0.1 0.6 1.0 O.Stf

Activation tinergy (app) 15.17 kcsl/nol 10.93 kcal/mol 15 kcal/mol 15 kcal/eol

Divalent Cation IV > Ca > H i > hi > Cu > ttt > Ncf* W Qi > Fe > Zn Cs > Pe • 2n pll Option 7.6-7.B 7.4-7.8 ND ND n»sphate Donor ATP - dATP > ara-ATP > AT? > CTP > CTP * CTP > ATP • dGTP> ATI* > CTP > dATP

CTP > Cl? > dCTP ■ dCT?> dATP > CTP > ara-ATP> dTTP > dATP > CTP> WIT • CTP > dCTP v v dWTP dCTP - dCIP > dCTP 1-dCTP > ara-CTP> dCTP > ora-cn* > dCTP > dATP > dCIP dcip> JTn>> I-dCTP

Mtcleotide Inhibitors dTTP, I-dim* dTTP. I-dCTP, dCTP, dCTP, CTP dCTP, dTTP ara-CTP I-dlfTP

Mechanism sequential ping-pong sequential seq u en tial

*(fcmonstrated sigmoidal kinetics for ATP-MB bND - not determined It has been well established that dThd kinase activity is directly related to the growth rate of the tissue from which it is derived.

Bollum and Potter (57) demonstrated that just prior to the initiation of DNA synthesis in regenerating rat liver there is a marked increase in the level of dThd kinase. Increased levels of dThd kinase has been demonstrated in adrenocorticotropic hormone - stimulated hamster adrenal gland, parotid gland of mice injected with isoproterenol and in phyto­ ha emagglut ini n- stimulated human lymphocytes (58-60). This increase in activity was directly correlated to the onset of S phase of the cell cycle. Thymidine kinase activity is enhanced prior to DNA synthesis in cells infected with poxvirus, adenovirus, herpesvirus, pseudorabies virus, polyoma and SV-40 (61-66), where in some cases the induced enzyme is virus specified (67). Elevated levels of dThd kinase have been found in various tumors and fetal tissues (68-71). The activity in these tissues was distinguishable from that found in normal adult tissues. The proper­ ties of the tumor cell enzyme were determined to be similar to those of the "fetal" enzyme, whereas the activity from normal tissue exhibited properties identical to the "adult" form of the enzyme (66,69-71). Sub­ sequently, it was shown that the "fetal" enzyme, that form stimulated during cell proliferation and onset of S phase, was one in the same with the cytosol isozyme, whereas the "adult" form, found in resting cells, was the mitochondrial isozyme (56,69). These data confirmed earlier findings that demonstrated while levels of the cytosol dThd kinase varied with the rate of synthesis, levels of the mitochondrial isozyme were constant and independent of the rate of DNA synthesis (72).

Genetic evidence which demonstrates that the two dThd isozymes are coded for by distinct genetic loci comes from the utilization of somatic 13 cell hybrids and mutant cell lines, Using somatic cell hybrids of human- mouse and monkey-mouse both Kit and Leung (73) and Willecke, et al. (74) have demonstrated that the two primate isozymes are coded for by loci on different nuclear chromosomes. Willeck et al. (74) suggest that the mitochondrial enzyme is coded for by a gene on human chromosomes 2, 13 or

16. Elsevier, et al. (75) have demonstrated that the human cytosol enzyme is coded for by a gene locus on chromosome 17, bands q21-22.

Further evidence for the genetic distinction between the two isozymes, as well as a clue to their physiological role, has come from the use of mutant animal cell lines. Two mammalian cell lines, He La (BU25) and

LMTKj resistant to BrdUrd, have been developed from human HeLa S3 and LM mouse fibroblasts respectively (76,77). Hie HeLa (BU25) and LMHC" cells lack the cytosol dThd kinase but maintain the mitochondrial isozyme (53,78), indicating the cytosol enzyme is not obligatory in cells which have a functioning de novo pathway for the synthesis of dTMP. In the wild type cells the mitochondrial isozyme represents only 1% of the total kinase activity, whereas in the mutants it represents all of the cellular activi­ ty (53). It has been demonstrated by several studies that both mutant cell lines incorporate BrdUrd and t -dThd exclusively into mitochondrial

UNA (52,53,79). In contrast to these findings, Bogenhagen and Clayton (80) provide evidence which suggests that IKTK~ cells may incorporate [3H]-dThd into nuclear DNA at low levels. When LA 9 wild type mouse cells were grown in media containing t3H]-dThd both nuclear and mitochondrial DNA were labeled (53). Berk and Clayton also found that when LA 9 cells were exposed to labeled dThd, as expected, the predominant intracellular labeled dThd nucleotide was dTTP. However, the LMTK- cells under identical 14

conditions failed to concentrate dThd and the intracellular labeled appeared

only as dThd. These data suggest that cytosol dThd kinase is somehow

involved in the mechanism of transport of dThd into the cell, as well as

providing thymidine nucleotides for DNA synthesis.

To elucidate the path(s) of dThd incorporation into mitochondrial DNA,

Berk and Clayton (53) utilized the LWTK' cells, which cannot convert dThd

to dTMP in the extramitochondrial cytosol and LA 9 cells grown in metho­ trexate (inhibits dihydrofolate reductase), which blocks de novo synthe­

sis of dTMP via thymidylate synthetase. When [3H]-dThd was added to cells under these conditions they discovered that the specific activity of

intramitochondrial dTTP was greater than in LNfTK* cells not treated with methotrexate. This is the result expected if one, the dTTP is trans­ ported into mitochondria and two, there is a reduction in the dilution of intramitochondrial labeled dTTP by dTTP synthesized de novo. In addi­ tion, after a pulse of [3H]-dThd, methotrexate-treated cells incorporated more label into mitochondrial ENA than the untreated control cells. In similar experiments they demonstrated that while in LA 9 cells inhibitors of de novo dTMP synthesis (FdUrd and methotrexate) did stimulate utiliza­ tion of exogenous dThd for mitochondrial DNA synthesis, the levels were well below that for the IMTT cells (53,80). From their results they propose a model which provides for the entry of dThd into the intramito- chondrial dTTP pool via the transport of dThd nucleotide, but in addition, a second pathway operates whereby a small fraction enters via dThd with the employment of the mitochondrial dThd kinase and subsequent mono- and diphosphonucleotide kinases. These data suggest that the mitochondrial DNA synthetic system is

independent of the nuclear DNA synthetic system. The existance of this

autonomy has been substantiated by others. In mamnalian cells, mito­

chondrial DNA synthesis appears to occur late in the S phase or in G-2

(81,82). Furthermore, Bogenhagen and Clayton (80) were able to,show

that when LMTK* cells were treated with 10 uM methotrexate and 20 uM

thymidine, mitochondrial DNA synthesis continued at 50 to 60% of the

control rate for an additional ten hours, while nuclear DNA synthesis

was inhibited 96%. Additional evidence in support of the uncoupled nature

of the two DNA synthetic events comes from experiments with drugs (cyclo-

heximide and puromycin) which directly inhibit protein synthesis and

subsequently nuclear DNA synthesis. Kit and Minekawa (S2) demonstrated

that cycloheximide treatment reduced the incorporation of t3H]-dThd into nuclear DNA of normal and virus-transformed monkey, hunan and mouse cells.

In contrast, they found that labeling of mitochondrial DNA was much less affected. In mouse and hunan cells puromycin had the same effect. These experiments indicate that while nuclear DNA synthesis is tightly coupled to drug-sensitive protein synthesis, mitochondrial DNA synthesis is not.

It would appear therefore, that the primary physiological role of the mitochondrial isozyme is to provide thymidine nucleotides to the mito­ chondrial dTTP pool. Its role in providing thymidine nucleotides to the extramitochondrial pool would appear to be insignificant. Thus, the enzyme adds to the autonomy of the mitochondria, providing precursors for its own independent DNA synthesis.

b. Deoxycytidine Kinase

Deoxycytidine kinase (EC 2.7.1.24) catalyzes the phosphorylation of of dCyd to dCMP using a variety of nucleoside triphosphates as phosphate 16

donors. It has been isolated and characterized front a lumber of sources

including calf thymus (83-87), L1210 cells (88), hunan (89,90) and P81S

murine neoplasms (91-93). Like dThd kinase there are two forms of the

activity in mammalian cells, one located in the cytosol and a second

isozyme in mitochondria (90,94-97). In human the two forms differ in

electrophoretic mobility, nucleoside specificity, % for dCyd and ATP,

phosphate donor specificity, sensitivity to inhibition by nucleotides and

genetic control (90,95). Table 2 lists some of these differences between

the two isozymes isolated from hunan blast cells of acute myelocytic

leukemia. Analogous to dThd kinase, the cytosol dCyd kinase is found primarily in proliferating cells such as those of lymphoid origin (thymus

and spleen), bone marrow, tumor cells, fetal tissues and its activity has

been found to be highest during the S phase of the cell cycle (95-98). In

resting cells and adult tissues the mitochondrial dCyd isozyme represents

the major activity (96). The cytosol enzyme is responsible for the phos­

phorylation and subsequent biological activity of the antileukemic drug,

ara-C (94). In cells resistant to ara-C the cytosol dCyd kinase is lacking,

while the mitochondrial isozyme, inactive towards ara-C, is maintained

(4). De Saint-Vincent and Butt in (94) also demonstrated that when ara-C

resistant hamster fibroblasts (CMA 32) were exposed to [ -dCyd it was

incorporated exclusively into mitochondrial ENA.

Recently evidence has been presented to support the hypothesis that

both dCyd and dThd kinase activities from mitochondria reside in the same

protein molecule. Both activities co-purify by dThd elution from dThd-

Sepharose, co-sediment in glycerol gradients and have identical electro­

phoretic mobilities (90,95). Both activities are inhibited by dTTP and

dCTP, and dCyd acts as an inhibitor with respect to dThd for the 17

mitochondrial dThd kinase (90,87). In contrast to these findings, using

product inhibition and initial velocity studies the dCyd kinase appeared

to follow a sequential mechanism while the phosphorylation of dThd by the

same enzyme preparation followed a ping-pong mechanism (Table 2). How­

ever, this might be an indication of a complex enzyme structure, i.e.,

one possessing multiple binding sites for either dCyd or dThd but only one

type of catalytic site for either substrate.

* 3. Mammalian Purine Deoxynucleoside Kinases

The deoxynucleoside kinases are members of a group of enzymes that

collectively function to salvage the deoxynucleoside precursors of ENA

synthesis from pre-existing DNA, RNA and nucleotides. Figure 1 illustrates

those pathways utilized by mammalian cells to salvage and interconvert

nucleotides for DNA synthesis. In the majority of cells this amphibolic

scheme operates in concert with de novo synthetic pathways for purine and

pyrimidine nucleotides.

Another inportant role of the salvage pathway is to provide the bio­

logical activation of a variety of chemotherapeutic agents utilized to

combat diseases whose pathogenesis requires the synthesis of DNA. Of par­

ticular interest is a group of drugs classified as nucleoside antibiotics.

These nucleoside analogues, which include ara-C, ara-A, a-TGdR, B-TGdR,

ribavirin and various 5-substituted pyrimidine deoxynucleosides have * become increasingly important as antitumor and antiviral agents (99-102).

The active metabolites of these nucleoside analogues are their corre­

sponding S'-triphosphates, which are potent inhibitors of DNA synthesis.

With the realization that the deoxynucleoside kinases catalyze the initial

step in the conversion of the nucleoside analogues to their active form has Figure 1. General Pathways for Nucleotide Salvage and Interconversion in Mammalian Tissues. Enzymes indicated: (1) DNAase; (2) RNAase; (3) phosphatases or nucleotidases; (4) nucleoside phosphorylases; (5) phos- phoribosyl transferases (ribose only); (6) nucleoside kinases; (7) nucleoside phosphotransferases; (8) nucleoside monophosphate kinases; (9) nucleoside diphosphate kinases.

18 DNA RNA 1(1.2)

(d)NUCLEOSIDE 3h or5-MONOPHOSPHATE

(3)

(d)NUCLEOStDE Pi (4) (d)R-l-P

BASE Xw^^PRPP / PPJ {d)NUCLEOSIDE 5-MONOPHOSPHATE Nipt (8)1 N O P ^

(d)NUCLEOSIDE 5-DIPHOSPHATE

(d)NUCLEOSIDE 5-TRIPHOSPHATE 20 come an increased interest in the nature of these enzymes in normal and transformed cells.

Recently the purine deoxynucleoside kinases have been implicated in the pathogenesis of at least two inherited ijnntmodeficiency diseases in » man. These diseases are associated with the lack of either adenosine deaminase (EC 3.5.4.4) or purine nucleoside phosphorylase (EC 2.4.2.1).

Adenosine deaminase deficiency results in a severe combined immunodeficiency disease with the loss of both T-cell and B-cell function, and lack of purine nucleoside phosphorylase has been correlated with impairment of

T-cells and associated cellular imnune functions (103,104). While it is apparent that both enzymes are lacking in all tissues examined only the lymphoid cells are affected. Investigations into the nature of the mechanism whereby these enzyme deficiencies affect lymphocyte development have revealed markedly elevated dATP levels in adenosine deaminase- deficient erythrocytes and dGTP levels in purine nucleoside phosphorylase- deficient erythrocytes (105-107). In conjunction with these findings has been the observation of elevated levels of the corresponding deoxyribinu- cleoside in blood and urine (108,109). These results accompanied by the known lynphocytotoxic. effects of both deoxynucleosides in vitro (110-114) have led investigators to suggest inhibition of the enzyme ribonucleotide reductase by dATP or dGTP as the mechanism of cellular toxicity (105,110,

111,114). A recent study by Wilson, et al. (115) with human T-lymphoblasts confirms that dGuo toxicity is mediated via inhibition of ribonucleotide reductase, but suggests that dAdo inhibits DNA synthesis by mechanisms other than, or, in addition to inhibition of ribonucleotide reductase. Hershfield

(116) has provided evidence recently for an additional mode of toxicity by 21

dAdo. He reports that increased dAdo levels in hunan lymphoblasts leads to an accumulation of S-adenosylhomocysteine by suicide inactivation of

S-adenosylhcmocysteine hydrolase, thereby depleting the cell of S-adenosyl- methionine and preventing essential methylation reactions. Although the precise mechanism(s) whereby these enzyme deficiencies affect lymphocyte development and/or function still have not been fully elucidated, evidence for the toxicity of the triphosphonucleotides is cogent and underscores the need for a better understanding of the nature and nunber of deoxynucleoside kinases in lymphoid cells.

In contrast to the pyrimidine system, relatively little is known about mammalian purine deoxynucleoside kinases. In extracts of calf thymus both dAdo and dGuo kinase activities are co-purified with dCyd kinase, even after the latter was purified up to SOO-fold (83-87,117-119). While the prepar­ ation by Durham and Ives (83) continued to show specificity for the three deoxynucleosides even after purification to apparent homogeneity by adding preparative gel electrophoresis to the isolation procedure (120), absolute proof for the existence of a single protein phosphorylating dCyd, dAdo and dGuo has not been presented. However, kinetic analysis demonstrating that dAdo and dGuo compete with dCyd for the same catalytic site (84,85,97) and genetic evidence demonstrating that an ara-C resistant subline of mouse leukemia L1210 cells which lost 981 of dCyd kinase activity relative to the wild type also had lowered dGuo (871) and dAdo (30%) kinase specific activities (121) provides strong support for a single protein model.

The various preparations of the cytosol dCyd kinase all exhibit the ability to phosphorylate ara-C and in some instances Cyd (85,87,118).

While all of the alternate phosphate acceptors produce higher maximum 22 velocities (85), the consistent lower value for dCyd indicates it is the preferred substrate under intracellular conditions. Although the phosphorylation of dCyd has been shown to exhibit bimodal double-reciprocal plots suggesting negative cooperativity, phosphorylation of the alternate substrates demonstrates linear kinetics (84,87).

Deoxycytidine kinase has a very broad specificity with respect to phosphate donor. It appears that all nucleoside triphosphates (ribose or deoxyribose) can function as donors with the exception of dCTP (83).

The mono- and diphosphonucleotides are incapable of acting as phosphate donors.

The phosphorylation of dCyd, as well as other nucleosides, is subject to potent feedback inhibition by its distal end-product dCTP. The enzyme is inhibited to a lesser extent by the corresponding mono- and diphos - phodeoxynucleotides.

In general the enzyme from calf thymus appears to have a broad pH optimum between 6 and 10 (83,118). Molecular weight estimations range from 56,000 to 63,000 (83,118). The enzyme requires a divalent cation with Mg+^ providing maximal activity. Optimal activity and stability require a sulfhydryl reducing agent such as 2-mercaptoethanol or DTT.

There is considerable evidence that additional manvnalian dAdo and dGuo kinase activities exist. Using conventional purification techniques dCyd kinase has been prepared free from purine deoxynucleoside kinase activities in human leukemic granulocytes (89), human blast cells (90) and P815 murine tumors (91). In all cases these activities retained the ability to phosphorylate ara-C, ruling out the possibility of the mito­ chondrial isozyme being the species isolated. Thus it would appear that 23 the deoxynucleoside kinases isolated from rat, mouse and hunan tissues lack the multiple specificity exhibited by the calf thymus cytosol enzyme, and unlike the thymus activities, these enzymes may be separated by standard methods.

Streeter, et al. (122) partially purified an enzyme activity from rat liver that does not phosphorylate dCyd but does phosphorylate Ado, dAdo and ribavirin. Sensitivity to inhibition by p-choloromercuribenzoic acid, optimal pH and MgCl2 concentration, heat lability and kinetic studies indicate that Ado and dAdo kinase activities are located at separate sites, or more likely, separate enzymes. The ^ values for dAdo and Ado, S00 yM and 0.8 uM respectively, suggest that Ado is the preferred substrate.

Whether or not dAdo is phosphory 1 ated by adenosine kinase (EC 2.7.1.20) has been unclear. Adenosine, dAdo and ara-A phosphorylating activities have been associated with the same protein in an earlier study using preparations isolated from rabbit liver and Ehrlich ascites tumor cells

(123). In human tissues Ado and dAdo kinase activities were separated on DE52-cellulose and were determined to vary in specific activity in different tissues (111). In a recent report working with hunan placental

Ado kinase Andres and Fox (124) were able to achieve substantial separation of the two activities by DEAE-cellulose and 5'-AMP-Sepharose 4B. However, despite extensive purification utilizing these media, the Ado kinase preparation was still able to phosphorylate dAdo. The most recent report of an apparent homogeneous Ado kinase from rabbit liver confirms that while they are very poor substrates relative to Ado, both dAdo and ara-A are phosphorylated (125,126). In contrast, Ado kinase recently purified 14,000 fold from L1210 murine leukemia cells did not phosphorylate dAdo or ara-A 24

(127). Nevertheless, in normal cells it would appear likely that Ado kinase and dAdo kinase activities are distinct enzymes, with the former having low affinity for dAdo.

Krygier and Momparler (117-119) have isolated a dAdo kinase from calf thymus which phosphorylates dGuo and Cyd but has very little capacity to phosphorylate dCyd. Their isolation protocol is very similar to that reported by Durham and Ives (83) for purification of dCyd kinase from calf thymus. In light of this and the fact that nucleoside specificity was determined at a single concentration nearly 100 times higher than the for dCyd (83-85), it seems reasonable to suspect the reported nucleoside specificity. Since the level of nucleoside was near the of dAdo it would be anticipated that higher maximum velocity would be achieved for dAdo relative to dCyd. Furthermore, dCTP was found to be approximately 100 times more effective than dATP as an inhibitor of the enzyme, similar to that reported for dCyd kinase from calf thymus (84,87). Thus, it seems reasonable to assume that their preparation represents an activity similar to that prepared by others foT dCyd kinase.

Table 3 lists the sources and some properties of enzyme preparations from marmalian tissues which have been reported to contain dGuo kinase activity. As described previously, dGuo kinase activity is found associated with dCyd and dAdo kinase activities isolated from calf thymus, even after the activity has been purified to apparent homogeneity (120). However, both dGuo kinase and dAdo kinase from tissues other than thymus, are separable from dCyd kinase by conventional techniques. Meyers and Kreis

(91) have purified dCyd kinase to homogeneity from P815 murine tumors and in the process separated a dGuo/dAdo phosphorylating activity on DEAE-

Sephadex A50. As described earlier most of the dGuo kinase activity was Table I. Sources and Some Hiysico-Chemical Properties of Maanalian buyne Preparations lahibitinf d & o Kinase Activity.

Nucleoside Specificity, Primary & Ibcltotide Source Apparent Kn fcjH) Phosphate Donors* m Optinua Inhibition Ref.

Calf Ihywis dCyd (14) Cl? > ATP • UFP > Hf ND dCIP > dCDP> (») ara-C (40) dGTP dOR> dAdo (ND) dftjo (Ml)

Calf Ihyais dCyd (5.3, 16) dUTP > ATP > GTP > 56,000 6-10 dCTP > dCDP > 1DP> (03, I ara-C (41) dTTP TOP > dCHP dAdo (73) dGuo (310)

Calf Thymis Cyd (600) ATP > CTP > CTP > 63,000 6.5-1.5 dCTP > dATP > CTP OH, dAdo (700) dTTP (dCXP > dCMP) dfiuo (1,100) dCyd (NO)

Calf Ihyms dCyd (10, 44) d CTP > CTP > ATP ID 7-10 dCTP > CTP > dCIP> (17) cyd (1,400) dATP dGuo (3,000) dAdo (ND)

Calf I b y u dcyd (2) ID ND ND ID («S) ara-C (25) dGuo (110) Cyd (240) dAdo (330)

H A m a i a l dfeo (7.7) ND ID 5.2 dCTP, dGDP, dOP, 0 3 1 ) Marne Skin UDP

fonrioe Skin dGuo (0.32) ATP > CTP - CTP 51,500 0.5 dCTP > dCTP > d ATP (13Q)

*Hhmm Ilnurs $ V M (110) A T P - CTP ID 7.4-1.2 ID 0 » ) Calf I h y w s *dGuo (Ki, 2.4) dCyd (K[. 0.036) ara-C (Ki. 1.7) h b H w donors exblbilint 2711 activity relative to ATP Tteaction product was d l W jND a not dote mined rhirlfication was as described by ft whan and Ives (13) for dCyd Kinase Mcleosida acceptor for K| datnminntiona was f*1QII 26

lost jrrom extracts of an L1210 murine leukemia cell line selected for

resistance to ara-C. Nevertheless, Nakai and LePage (128) found that

such extracts were able to phosphorylate the 6-thio analogues of dGuo,

t»-TGdR and 8-TGdR, at fairly high rates. Purification of the 8-TGdR phosphorylating activity from calf thymus and hmtan leukocytes from chronic

lymphoblastic leukemia utilizing a modified procedure described by

Durham and Ives (83) to isolate dCyd kinase provided a 220-fold increase

in specific activity (128). While ara-C, dGuo and dGTP were competitive

inhibitors for 8-TGdR phosphorylation, surprisingly, dCyd and dCTP were noncompetitive. In addition, the purified preparations lost the capacity to phosphorylate a-TGdR. Therefore, it appears that a dGuo kinase asso­ ciated with dCyd kinase phosphoTylates 6-TGdR but a second enzyme exists that in some tissues can phosphorylate both a- and 6-TGdR.

Additional evidence for the existence of a distinct dGuo/dAdo kinase in calf thymus comes from an analytical disc PAGE study conducted by

Baxter, et al. (129). Three electrophoretic variants of dGuo kinase were detected in a partially purified preparation of calf thymus dCyd kinase isolated by the method of Durham and Ives (83). One activity (Rm 1.0) was associated with both dCyd and dAdo kinase activities and two other activi­ ties (Rju 0.6 and 0.74) associated with dAdo kinase.

Recently, Green and Lewis (130) have prepared a dGuo kinase from por­ cine skin free of dCyd and dAdo phosphorylating activities. Using DEAE-

Sephadex, this activity was separated from two other kinase activities, one which phosphorylated dCyd, dAdo and dGuo and a second dAdo kinase. The

1,000-fold purified dGuo kinase has an obligatory requirement for ATP as the phosphate donor similar to that reported for the calf thymus dCyd kinase. 27

Preference for donor was determined to be ATP > CTP a GTP > UTP with a relatively high ^(app) for ATP-Mg of 3.3 mM. Analogous to the calf thymus dCyd kinase the activity has a molecular weight of 58,S00 and a broad pH optimum between 6 and 7.5. However, significant differences were demonstrated in terms of R^Capp) for dGuo and sensitivity to inhibition by deoxynucleoside triphosphates. The porcine skin dGuo kinase exhibits an apparent ^ for dGuo of 0.32 uM which is well below that reported for dGuo phosphorylation by calf thymus dCyd kinase. While the enzyme demon­ strates sensitivity to dCTP and dATP feedback inhibition, dGTP provides the strongest inhibition. At concentrations of 0.1 nM inhibition was 1001 for dGTP, 62% for dCTP and 48% for dATP. The inhibition by dGTP was competitive with respect to dGuo with an apparent of 21 yM. The enzyme’s exclusive specificity for dGuo, low for dGuo and marked sensitivity to dGTP feed­ back inhibition, suggests that the enzyme, unlike that associated with dCyd kinase, is the preferred dGuo phosphorylating activity under intracellular conditions in this tissue.

In a more recent report Barker and Lewis (131) have partially purified a dGuo phosphorylating activity from neonatal mouse skin. Unlike other reported dGuo kinase activities, this enzyme exhibits a surprisingly low pH optinun of 5.2. While substrate specificity was not reported, the enzyme exhibited a K^app) of 7.7 yM for dGuo. With respect to ATP>tyg kinetics, a bimodal double-reciprocal plot was observed with K^(app) values of 270 yM at low concentrations and 2.0 irM at high concentrations of ATP*Mg.

The reaction product dGMP was a competitive inhibitor of phosphorylation with respect to dGuo (Ki(app) 180 uM). Deoxyguanosine triphosphate was a competitive inhibitor for both dGuo and ATP'Mg with apparent values of 1.9 uM and 0.2 uM respectively. Deoxyguanosine diphosphate displayed a 28 competitive inhibition pattern with respect to ATP-Mg with a K^(app) of

1.3 yM, but demonstrated mixed-type inhibition with respect to dGuo. At high concentrations UDP also inhibited the enzyme competitive with dGuo but exhibited mixed-type inhibition at lower concentrations.

Although the enzyme exhibits a low for dGuo resembling the dGuo kinase isolated from porcine skin, the significantly lower pH optimum and lack of reported nucleoside specificity make the relationship between the two unclear. However, this report accompanied by the previous findings indicate that there is a dGuo phosphorylating activity in manmalian tissues distinct from the tripartite dCyd kinase.

4. Enzyme Regulation and Hysteretic Enzymes

Intrinsic to the maintenance of homeostasis at all levels of life is the regulation of the flow of metabolites through anabolic and catabolic pathways. These pathways must be coordinated and, in addition, must respond both to short-term changes in the external environment such as fluctuation in nutrient levels, as well as to periodic intracellular events such as DNA synthesis. Specific control mechanisms by which cells regulate and coordinate metabolism can be categorized into two major types: control of enzyme activity (catalytic efficiency); and control of enzyme synthesis.

Additionally, evidence has accumulated which suggests that specific enzyme degradation plays an essential role in metabolic regulation.

In terms of enzyme structure and function perhaps the most interesting and intensively studied area has been that of regulatory enzymes. These enzymes generally occupy strategic branch points in the complex network of intermediary metabolic sequences, so that an alteration in their cata­

lytic rate imnediately affects the rate of metabolite flow through an entire pathway, the initial step of which is usually catalyzed by the 29 affected enzyme. The catalytic efficiency of these enzymes is regulated by various effectors or modulators which include substrates, coenzymes, activators or inhibitors.

In general, these enzymes are capable of binding effectors not only at or close to the active site, but at a distal, distinct allosteric site. In most cases kinetic plots of initial velocity versus substrate concentration are sigmoid instead of hyperbolic as expected for an enzyme exhibiting classical Michael is-Menten kinetics. This sigmoid effect is indicative of multiple interacting binding sites. This effect may be defined as homotropic if multiple identical ligands are bound, or heterotropic when different ligands bind to the enzyme. The effect of the regulation is not always to alter the enzyme’s Vi m y * but is very often to increase or diminish its affinity for substrate.

Most allosteric enzymes found to date have a relatively high molecular weight and are composed of multiple subunits. These subunits or protomers can be identical, each maintaining both catalytic and allosteric sites or may be different, i.e. one type contains the catalytic site and the other the allosteric site. In both cases interaction between the protomers is affected by the presence of allosteric ligands.

Two classical models have been presented to explain the mechanism whereby allosteric regulation occurs. Monod, et al. (132] describe a model of an allosteric enzyme which is multimeric, with a symmetrical arrangement of identical protomers. Protomers may be monomers or some arrangement of identical or non-identical monomers. The enzyme exists in dynamic equilibrium between two conformational states, one with lower affinity for ligands (T) and one with a higher affinity (R), but in each 30 state all binding sites are equivalent. The symmetry of the enzyme mole­ cule, including the symnetry of interactions between promoters, is conserved in transitions between states. In its simplest form control is explained by the effector altering the ratio of the two states by stabilizing one of the forms, i.e. an activator binds to the R state and an inhibitor to the

T state and in both cases prevents the transition to the alternate form.

Koshland, et al. (133) describe a model which replaces alternative confor­ mational states of the enzyme in a pre-existing thermodynamic equilibrium by an "induced-fit" sequential model. In the absence of ligands all enzyme molecules are in the same conformational state. Upon binding a ligand, a subunit undergoes a conformational change and this change may be transmitted to an adjacent subunit through subunit interface inter­ actions. In contrast to the Monod model, the Koshland type allows for sequential changes in subunit conformation, i.e. the binding of a ligand to a subunit does not require conformational change in adjacent subunits.

Activation occurs when the ligand binds and alters the conformation so as to facilitate the proper molecular alignment for catalysis, while inhibition is produced when the ligand holds the polypeptide unit in a catalytically unfavorable mode.

While these two models have been found applicable to the majority of regulatory enzyme systems, there have been others whose kinetic behavior depicts a more complex mechanism. Whereas the previously described models describe struc­ ture-function relations simply in terms of conformational alterations, they have been expanded to include systems in which variant conformational forms of an enzyme do not have the same intrinsic specific activity and enzymes which undergo polymeri- zation-depolymerization tranitions in which different molecular weight forms of the enzyme have different kinetic characteristics (134,135). In addition, 31 the assumption that the rates of interconversion of the different enzyme complexes are rapid prior to the release of product from the enzyme may not be valid in all cases, since evidence has accumulated which indicates that some of these prior steps may be rate determining. Therefore, subunit 4 conformational or association-dissociatian transitions after addition of ' r substrate, but prior to the release of product, may be rate-limiting.

Frieden (136) has explained that if these transitions were slow processes relative to the measurement of the reaction rate, and if the change led to an enzyme state with different kinetic properties, then a lag or burst in the reaction time course would be observed. He labeled this phenomenon hysteresis and distinguished it from those processes which are related to substrate depletion or product accumulation.

Several enzymes have been documented as exhibiting a slow transition in their kinetic behavior after the addition of ligand. Mechanistically, these enzymes can be categorized into three groups: conformational iso­ merization; displacement of ligand; and polymerization-depolymerizatian transitions. Two examples of enzymes in which a slow conformational change between two kinetically distinct species has been invoked to explain lag periods are D-lactate dehydrogenase from Escherichia coli (137) and glycer- aldehyde 3-phosphate dehydrogenase from Saccharorayces cerevisiae (138). A homogeneous preparation of D-lactate dehydrogenase from E. coli demonstrated a lag with a half-life (t^) of several seconds (137). Incubation of the enzyme with pyruvate prior to the start of the reaction eliminated the lag.

Hie length of the lag period was independent of enzyme concentration and the enzyme's molecular weight did not change in either the presence or absence of substrate, indicating that the lag was not the result of an alteration in aggregational state. This and other evidence indicated that the enzyme 32

had two binding sites for pyruvate, one associated with activation and

another with catalysis. Employing the methods of stopped flow and temper­

ature junp with S. cerevisiae glyceraldehyde 3-phosphate dehydrogenase,

Kirschner (138) demonstrated a lag (t^* tenths of a second) in product versus time plots. The lag was evident only when the reaction was, initiated by enzyme or glyceraldehyde 3-phosphate and was shown to be the result of the transition of enzyme from its inactive (T) conformer into its active

(R) conformer induced by NAD.

A few enzymes have been found to exhibit hysteretic behavior. due to vthe slow displacement of a tightly bound ligand from the enzyme. This trans­ ition results in a change in rate of product formation due to the displace­ ment of one ligand (inhibitor) by another, either directly or indirectly as a result of an alteration in conformation. Using stopped flow methods

Frieden demonstrated that a lag (tjj 2.3 seconds) occurs in the reaction time course of glutamate dehydrogenase when enzymatic activity is measured in the presence of the inhibitor GTP and the activator ADP (136). Be concluded that the slow dissociation of the tightly bound GTP (Kj, 0.4 |iM) « by ADP produced an increase in the velocity of the reaction after an initial lag. Rabbit muscle ADP deaminase shows similar behavior with respect to GTP (139). Kinetic studies suggest the enzyme has three inter­ acting purine nucleotide binding sites: an AMP binding active site, an inhibitor site that binds nucleoside triphosphates, and a non-specific acti­ vator site. When the enzyme is incubated in the absence of GTP or in the presence of both GTP and AMP prior to reaction initiation there is no observable lag. However, when the enzyme is preincubated with GTP the reaction time course demonstrates a time-dependent release of inhibition. 33

It is suggested that this phenomenon is the result of either substrate

(AMP) or product (IMP) binding to the activator site and weakening the binding of GTP.

Numerous enzymes have been reported which exhibit hysteretic behavior due to reversible ligand-mediated subunit polymerization or depolymerization.

Enzymes demonstrating ligand-induced aggregation resulting in a slow trans­ ition from an inactive low molecular weight form to an active higher molecular weight species include NADP-linked isocitrate dehydrogenase (43), phosphoribosylpyrophosphate synthetase (140), El of the phosphoenolpyruvate- dependent phosphotransferase system of E. coli (141), phosphoenolpyruvate carboxylase (142), acetyl CoA carboxylase (143)» deoxycytidylate deaminase

(144), a-N-acetylgalactosaminidase (145), anthranilate synthase (146) , phenylalanine 4-hydroxylase (147) and L-threonine dehydratase (148). Those demonstrating ligand-induced depolymerization include glutamine phos­ phoribosylpyrophosphate amidotransferase (46) and glycogen phosphorylase a (149). Characteristically, this type of transition is slower (tjj t minute) than either the conformational isomerization or ligand displacement and the length of the lag period is inversely proportional to enzyme concentration.

This latter characteristic is used in conjunction with molecular size analysis to distinguish between this mechanism and conformational isomeri­ zation or ligand displacement.

It is interesting to note that many of the enzymes exhibiting hysteretic behavior are allosteric or regulatory enzymes. Frieden (136) has suggested that the hysteretic response of these enzymes might confer a selective advantage, in that a slow response of the enzyme to changes in ligand level would lead to a time-dependent buffering of some metabolites. This might provide better regulation of metabolite concentrations or flux in the case 34 of pathways which utilize common intermediates or in which there are multiple branch points. However, whether o t not this hypothesis describes true physiological behavior remains unclear since to date evidence has not been presented which u ^nonstrates that an enzyme undergoes hysteretic response under in vivo, as well as in vitro conditions. 35

EXPERIMENTAL PROCEDURE

I. Materials

All standard bases, nucleosides and nucleotides were obtained from

Sigma, P-L Biochemicals, Calbiochem or Plenum. Coformycin, EHNA, a - and

8-2-deoxythioguanosine and ara-G were generous gifts from Dr. H.H. Machamer

(Warner-Lambert/Park-Davis), Dr. H.J. Schaeffer (Burroughs Wellcome), Mr.

P. Vills (National Cancer Institute) and Mr. Riker (National Cancer Insti­

tute) respectively. UDP-hexano1imine Sepharose was a generous gift from

Dr. Robert Hill (Duke University).

[8-3H]-dAdo (12.96 C i / m m o l e ) [2,8-%]-Ado (34 Ci/mmole), [5-%]-Cyd

(27 Ci/mmole), [8-3H]-dGuo (14.5-26.4 Ci/mmole). [8-3H]-Guo (7.8 Ci/mmole),

[methyl-3H]-dThd (28 Ci/mmole), [5,6-3H]-Urd (41.5 Ci/mmole) and [2,8-%]-

ATP (41.6 Ci/nmole) were purchased from ICN Pharmaceuticals. [8-3H]-ara-A

(20 Ci/mmole) and [5-3H(N)]-dCyd (38.1 Ci/mmole) were purchased from Moravek

Biochemicals and New England Nuclear Corporation respectively. Upon

receipt all radiochemicals were analyzed for purity by paper chromatography utilizing chromatographic systems described in Table 4.

DE-81 ion-exchange paper, No. 1 filter paper, No. 3KM chromatographic paper and DEAE-cellulose (DE-23) were purchased from Whatman, Inc.. Organge

Ribbon chromatography paper and SB-2 ion-exchange paper were obtained from

Shchleicher and Schuell and Reeve-Angel respectively.

Sephacryl S-200 Superfine, Sephadex G-25 Mediun, DEAE-Sephacel, Sepha­

rose CL-6B, Blue Dextran 2000, Phenyl Sepharose CL-4B and Octyl Sepharose 36

CL-4B were purchased from Pharmacia Fine Chemicals. Protein Dye Reagent, acrylamide, TEMED, bisacrylamide, arananiian persulfate, bromphenol blue,

Biogel NT and Bio-Lyte (3-10) were purchased from Bio-Rad Laboratories.

Amnonium chloride, amnonium molybdate, ferrous chloride, ferrous sulfate, sodium borate, sodium fluoride, amnonium hydroxide, hydrochloric acid, ethylene glycol, methyl ethyl ketone, sodium phosphate (dibasic), potas­ sium sodium tartrate, manganous chloride, manganous sulfate, ferric chlor­ ide and cobalt sulfate were obtained from the J.T. Baker Chemical Company.

Sodium bisulfite, sodium acetate, glacial acetic acid, magnesium chloride, sodium hydroxide, methanol, potassium chloride, phosphoric acid, formic acid, glycerol, sodium chloride and potassium hydroxide were obtained from

Mallinckrodt, Inc.. Airmonium acetate, amnonium formate, sodium azide, aminonaptholsulphonic acid and trichloracetic acid were obtained from Fisher

Scientific Company. Cupric chloride, calcium chloride, zinc sulfate and zinc chloride were obtained from Baker and Adamson Chemicals. Cupric sulfate, potassium phosphate (monobasic) and sodiun carbonate were obtained from Matheson, Coleman and Bell. Tris, 2-aminoethanol, W 6 F, imidazol, DTT,

DTE, p-nitrophenyl phosphate, 2-mercaptoethanol, glycine, Coomassie

Brilliant Blue G-250 and R-250, Tween 80, NAEH, 2-oxoglutarate, egg albumin, adenosine deaminase (calf intestinal mucosa), 5-nucleotidase (Crotalus ademanteus), 3’-nucleotidase (rye grass), cytochrome c (horse heart), myo­ globin (whale skeletal muscle), trypsin inhibitor (soybean) hexokinase

(yeast), alcohol dehydrogenase (yeast), apoferritin (horse spleen), glycer­ aldehyde phosphate dehydrogenase (muscle) and a-chymotrypsinogen a (bovine pancreas) were purchased from Sigma Chemical Company. Other reagents were 37 obtained as follows: perchloric acid, EDTA (disodiun) (G. Fredrick Smith

Chemical Company); BSA (crystalline and Fraction V) (Pentex); SOS (BEH

Chemicals, Ltd,); hyaluronidase (bovine testis) (Calbiochem-Behringer

Corporation); Agarose-hexaneadenosine-5'-triphosphate, Agarose-hexane-2 * - oeoxyadenosine-5’-triphosphate, AMPPCP (P-L Biochemicals, Inc.); Lowry

Phenol Reagent (O.S.U. Lab Stores); Triton X-100, Preblend dry fluor 2a60

(91% PPO, 91 bis-MSB), toluene (RPI Corporation); t-butanol (Chemical

Samples); isobutyric acid (Aldrich Chemical Company); amnonium sulfate, sucrose (Schwarz/Mam); hydrogen peroxide (Park-Davis and Company); pH

isolytes (5-7) (Brinkman Instruments, Inc.); Diaflo ultrafiltration mem­ branes (1*1-10 and YM-10) (Amicon); Cibacron Blue 3G-A (Ciba-Geigy Corpora­ tion) .

Liquid scintillation counting was conducted in either a Packard Model

3320 tricarb scintillation spectrometer or a Beckmann Model LS-230 scintil­ lation spectrometer. Slab gel electrophoresis was performed with equip­ ment supplied by Ortec. Power supplies used for electrophoresis and iso­ electric focusing were a Vokam Model 2541 (Shandon), Ortec Model 3-1014A.

All spectrophotometric measurements were performed with a Gilford Model

222A spectrophotometer with attached Rapid Sample (Gilford, Model 2443) and recorder (Sargent-Welch, Model SRLG).

2. Methods

a. Analysis of Bases, Nucleosides and Nucleotides

Bases, nucleosides and nucleotides were chromatographed for evaluation of purity and identification by paper chromatography using systems described by Fink and Adams (150) and Durham and Ives (151). Primarily five systems were utilized. These are listed in Table 4 along with average Rf values 38

Table 4. Paper Chromatographic Systems Utilized For Separation of Various Bases, Nucleosides and Nucleotides. All systems were developed (descending) on 1 x 2 inch paper strips of either Orange Ribbon for systems 1-3 (partition) or Whatman DE-81 for systems 4, S (ion exchange). Development times varied from 1-5 hours for systems 3 and four to 12-15 hours for all others.

Solvent4

Compound 1 2 3 4 5

Rf Rf Rf Rf Rf

Guanine .47 .19 dGuo .54 .36 .70 dCMP .28 .18 CMP .04 Adenine .40 Ado .71 .56 AMP .69 ADP .49 ATP .17 dAdo .77 .61 dAMP .83 Hypoxanthine .66 .35 Ino .44 .28 dlno .52 .34 dIMP .46

(1) 95% ethanol: 1.0 M amnonium acetate (7:3) (2) isobutyric acid:0.5 M amnonium hydroxide (5:3) (3) t-butanol:methyl ethyl ketone:water:amnonium hydroxide (4:3:2:1) (4) 0.1 M amnonium formate, pH 3.1 (5) 95% ethanol: 1.0 M amnonium acetate, 0.01 M EDTA, saturating Na2B407, pH 9.0 (7:3) 39 for compounds routinely analyzed. Radiolabeled compounds were located on the chromatogram by cutting the paper strip into several equal sections, followed by liquid scintillation spectrophotometry to measure radioactivity in each section. Unlabeled compounds were located by visual inspection, beneath short wavelength ultraviolet light.

All solutions of bases, nucleosides and nucleotides were prepared by spectrophotometrie measurements using maximal wavelengths and extinction coefficients reported in the literature (152).

i. Preparation of [3H]-dIno and f3H]-Ino

(3Hl-dIno and [3H]-Ino were prepared by deamination of [8-3H] -dAdo and

[2,8-3H]-Ado with adenosine deaminase. The reaction mixture contained 80 nW KH2P04/Na2HP04 buffer, pH 7.237, 250 uCi of [8-3H]-dAdo (12.96 Ci/mmole) or 250 yCi of [2,8-3H]-Ado (32 Ci/nmole) and 0.4 units of adenosine deaminase

(calf intestinal mucosa) in a total volume of 1.0 ml. The reaction was incubated at 37 "C for 6 hours. The reaction was deproteinized by heating in a boiling water batch for 2 minutes, followed by centrifugation at 1,500 x g for 15 minutes. The supernatant was applied to the top of a 23 x 38 cm paper chromatogram (Whatman 3M1) and developed (descending) overnight in i solvent system 2 or 3 (Table 4). The radioactive products were located by using unlabeled markers chromatographed concomitantly along the extreme edges of the paper. The products were eluted from the paper with 95% ethanol, then dried to a minimal volume under nitrogen,

b. Enzyme Assays

i. Nucleoside Kinases - General Procedure

Nucleoside kinases were assayed by the radiometric method of Ives, et al.

(153). Routinely assays were performed in a total volume of 0.08 ml and 40

contained in addition to enzyme the following: Tris/HCl, 8 .037, 0.05

M; ATP, 10 mM; MgCl2, 10 mM; DTTT or DTE, 10 mM; nucleoside, [3H]-dAdo,

[3H]-Ado, [3H]-dCyd, [3H]-Cyd, [3Hl-dGuo, [3H]-Guo, [^J-dlno, [^l-Ino,

t3H]-dThd, 0.02 mM and 0.10 mM (0.1-0.5 uCi/assay). The reaction was

initiated by adding enzyme and incubated in a closed 1.5 ml plastic vial at 37 “C for 30 minutes. A control vial contained all components with water substituted for enzyme. The reaction was teminated either by

imnersion in boiling water for 2 minutes, followed by dilution with 0.48 ml water or by addition of 0.48 ml of 0.1 N formic acid. The reaction vial was centrifuged at 1,500 x g for 10 minutes to sediment denatured protein. A 0.05 ml aliquot of the supernatant was applied to a 1.5 cm

square of DE-81 or SB-2 ion exchange paper, then the paper disk was washed

in 8 1 of 1.0 mM amnonium formate or sodium chloride that was continually

recycled through a charcoal filter for 30 minutes. This washing removed unreacted nucleoside but allowed nucleotide product to remain bound to the

paper. The disk was rinsed briefly in 95% ethanol then air dried. A unit

of enzyme activity is equivalent to that amount which catalyzes the forma­

tion of 1.0 nmole of nucleoside monophosphate per minute. Where alterations

in the assay conditions occurred, a complete description of the modifica­

tions is stated.

ii. Deoxyguanosine Kinase

A modified version of the general procedure for nucleoside kinase assay

was employed for the bovine liver mitochondrial dGuo kinase. The final

reaction mixture contained 71 mM sodiun acetate buffer, pH 5.037, 10 mM

DTE, 0.5% (v/v) Triton X-100, 10 mM ATP, 30 mM MgCl2 and 0.1 mM [^l-dGuo

(0.1-0.5 uCi/assay) in a total volune of 0.08 ml. Unless otherwise stated 41 the enzyme was preincubated 30 minutes at 37 °C in the 4x concentration

ATP and assay buffer components, i.e. 143 mM sodium acetate buffer, pH

5.037, 20 mM DTE, 1.01 (v/v) Triton X-100 and 20 mM ATP in a final volume of 0.04 ml. At the end of the preincubation period the Mg&2 WAS added and the reaction initiated immediately thereafter by adding the dGuo.

The assay was then processed as described for the general procedure assay method.

iii. Adenosine Deaminase

Assay for adenosine deaminase (EC 3.5.4.4) was conducted using a modi* fied version of the radiometric method described by Coleman and Hutton

(154). The reaction mixture contained 50 mM KH2PO4 buffer, pH 7.037, and

0.1 mM [3H]-Ado (0.1-0.5 uCi/ml) in a final volume of 0.08 ml. After incubation at 37°C for 10-30 minutes 0.2 ml of 0.1 N formic acid was added to stop the reaction. The assay mixture was centrifuged at 1,500 x g for

10 minutes to sediment denatured protein. A 0.02 ml aliquot of the super­ natant was placed on a strip (1 x 23 in) of Whatman DE-81 chromatography paper over unlabeled Ino that had been previously applied at the origin, the strip was developed (descending) in 1.0 mM amnonium formate for 60 min­ utes. The strip was dried, and the Ino spot was cut out and counted by the same procedure used for kinase assays as described in Methods. Using an unwashed control disk (1.5 cm square of Whatman DE-81) and a washed con­ trol disk (Ino spot from reaction mixture with no enzyme) a percent con­ version was calculated. A unit of enzyme is equivalent to the deamination of 1.0 nmole of Ado per minute at 37*C.

iv. Glutamate Dehydrogenase

Assay for glutamate dehydrogenase (EC 1.4.1.2) activity was performed 42 at 25*C in SO nM imidazole buffer, pH 7.325, containing 0.6 M ammoniian acetate, 0.32 nM EDTA (disodiun), 5 nM 2-oxoglutarate and 0.14 nM NADH brought to a final volume of 2.8 ml. The reaction was initiated by addi­ tion of enzyme and followed by monitoring the decrease in absorbance at

340 nm. The concentration of enzyme used in the assay was adjusted so that during the initial 10 minutes the change in absorbance versus time was linear. The change in absorbance during the initial 10 minutes of the reaction was used to calculate the reaction rate. Using a molar extinction coefficient of 6,22 x 103 lcm'^mol"1, a unit of enzyme activity is defined as the oxidation of 1.0 umole of NADH per minute at 25*C.

v. Acid Phosphatase

Assay for acid phosphatase (EC 3.1.3.2) activity was conducted essen­ tially as described by DiPietro and Zengerle (IS5) where the hydrolysis of p-nitrophenyl phosphate is measured spectrophotometrically. The reac­ tion mixture contained 96 mM sodium acetate buffer, pH 5.037 and 1.0 mM p-nitrophenyl phosphate brought to a final volume of 5 ml. The reaction was initiated by addition of enzyme and incubated at 37°C. At S, 10, 15 and 20 minute intervals a 1.0 ml aliquot of the assay mixture was removed and imnediately placed in 0.2 ml of 5 N NaOH to stop the reaction. The aliquots were centrifuged at 1,500 x g for 10 minutes to sediment dena­ tured protein. The absorbance of the supernatant was determined at 405 nm. Activity was calculated from the linear average absorbance change per minute during the initial 10 minutes of the reaction. Using a molar extinc­ tion coefficient of 1.88 x 10* lcnT^mol"1 for p-nitrophenol, a unit of enzyme activity is defined as the hydrolysis of 1.0 umole of p-nitrophenyl phos­ phate per minute at 37*C. 43

vi. Adenosine Triphosphatase (ATPase)

Adenosine triphosphatase (EC 3.6.1.3) was assayed by a modified pro­ cedure of Pougeois, et al. (156). The assay mixture contained 40 mM

Tris/HCl, pH 8 .O30, 8 mM ATP, 4 mM MgCl2 in a final volune of 0.5 ml.

The reaction was initiated by addition of enzyme and incubated at 30°C.

At 2, 5, 10 and 15 minute intervals a 0.1 ml aliquot of the assay mixture was removed and added to 0.05 ml of 50% TCA to terminate the reaction.

After mixing, the aliquot was centrifuged at 1,S00 x g for 10 minutes to sediment denatured protein. Inorganic phosphate released was determined by the method of King (157). To 0.1 ml of the deproteinized supernatant, containing from 0.01 to 0.10 ymoles phosphate, was added 0.2 ml of 60% perchloric acid, 0.167 ml of 5% amnonium molybdate, 0.083 ml of a 0.2%

ANS solution (0.2% ANS, 12% sodium bisulphite and 2.4% sodium sulphite) and 0.45 ml water. After mixing thoroughly, the solution was incubated at roam temperature for 5 minutes, then the abosrbance was read at 660 nm.

Phosphate was quantitated using a standard curve generated with a 0.1 mg/ml

KH2FO4 solution. A unit of enzyme activity is equivalent to 1.0 umole inorganic phosphate released per minute at 30°C.

c. Analytical Methods

i. Analytical Discontinuous Polyacrylamide Gel

Analytical disc PAGE was performed routinely in a 9 x 10 cm slab (0.3 cm thick) basically by the procedure of Laemnli and Favre (158) except that

SDS was omitted. The stacking gel, 0.5 cm in length, contained acrylamide

(4%), bis-acrylamide (0.11%), ammoniun persulfate (0.08%), TBfED (0.05%), glycerol (10%) and 0.125 M Tris/HCl, pH 6 .8^. The separating gel, 7.0 cm in length, contained acrylamide (7%), bis-acrylamide (0.06%), glyceTol (10%) and 0.37S M Tris/HCl, pH 8.84. The electrode buffer contained 0.05 M Tris 44 and 0.384 M glycine, pH 8 .S4 . For enzyme stabilization purposes 2 mM ATP was included in the upper tank buffer. The protein sample was mixed with the sample buffer (0*125 M Tris/HCl, pH 6.8 , 301 glycerol, 2 uM ATP and 0.011 bromphenol blue) in a 5:2 ratio. Ten to 100 yl samples were layered on the top of the channels. Electrophoresis was conducted at 4°C using a constant current of 2 mA per channel for the first 60 minutes, then increased to 4 mA per channel until the bromphenol blue band migrated approximately 1.0 cm from the bottom of the gel. The gel was then removed from the slab cell and the channels were cut from the slab. The gel was stained for protein by the method of Holbrook and Leaver (159). The gel was immersed in 3.5% perchloric acid containing 0.04% Coomassie Brilliant

Blue G-250, stained at room temperature for 90 minutes, then transferred to a 5% acetic acid solution and soaked an additional 30 minutes. For assay purposes the separating gel portion of each channel was sliced into

2 nm sections. The gel sections were assayed in situ in 0.16 ml of enzyme reaction mixture. Incubation was at 37°C for 1 to 18 hours depending upon the experiment. The reaction was terminated by adding 0.48 ml of water, mixing, then applying 0.05 ml on a 1.5 cm square of Whatman DE-81 ion exchange paper. Processing of the paper disks and description of enzyme reaction mixtures were as described elsewhere in this section.

ii. Analytical Continuous Polyacrylamide Gel Electrophoresis

Analytical continuous PAGE was conducted by the same procedure described for disc PAGE with the following changes. Hie gel consisted of acrylamide

(6 or 7%), bis-acrylamide (0.19%), amnaniua persulfate (0.04%), T O O

(0.06%), 10% glycerol, 0.142 M Tris and 0.035 M glycine, pH 9.54 . The electrode buffer contained 0.142 M Tris and 0.035 M glycine, pH 9.54 with 45

the upper tank containing in addition 2 mM ATP, 10 nM 2-mercaptoethanol and 10% glycerol for enzyme stabilization. Excess amnonium persulfate in the polymerized gel was neutralized by pre-electrophoresis with 35 mM thioglycolate in 20% glycerol-at a constant current of 20 mA. Protein samples were deluted with glycerol and bromphenol blue so that the final concentrations were 20% and 0.01% respectively,

iii. Isoelectric Focusing

The procedure for electrofocus ing on a polyacrylamide slab gel was as described by Deibel and Ives (160) with some modifications. The gel was polymerized in a cell identical to that used for analytical disc PAGE.

The gel contained acrylamide (7%), bis-acrylamide (0.19%), amnonium per­ sulfate (0.06%), TEMED (0.06%), glycerol (25%) and 2% total ampholines.

For broad range and narrow range ampholyte was utilized. The upper elec­ trode tank (cathode) contained 0.05 M ethanolamine, and the lower tank (anode) contained 0.05 M phosphoric acid. Protein samples (0.02-0.07S ml) in 50% glycerol were applied to the bottom of the channel wells beneath poly­ merizing gel. Focusing was initiated upon completion of polymerization.

The gel was focused at 4°C at 300 V for 5-8 hours. Current equilibriun at 300 V was achieved by focusing initially at 50 V then increasing the potential by 50 V every 15 minutes for a total of 75 minutes. Following the removal of the gel from the cell the pH gradient was measured by cut­ ting one channel into 2 mm sections, incubating the sections in 0.5 ml of water for at least 1 hour, then measuring the pH of the solutions. For protein staining the sample channel was cut from the slab, rinsed thoroughly

in water for 30 minutes, then stained by the procedure described for ana­ lytical disc PAGE. Assay of enzymes was performed by the in situ technique 46

utilized for analytical disc PAGE with the exception that the assay mix­ ture contained 0.5 M Tris/HCl of 0.5 M sodium acetate,

iv. SDS Polyacrylamide Gel Electrophoresis

SDS PAGE was performed according to a modified procedure of Laemnli and Favre (158) in a 9 x 10 cm slab (0.3 cm thick). The stacking gel,

0.5 cm in length, contained acrylamide (71), bis-acrylamide (0.19%),

0.125 M Tris/HCl, pH 6.832 and 0-1* SDS. The separating gel, 7.0 cm in length, contained acrylamide (12%), bis-acrylamide (0.32%), 0.375 M

Tris/HCl, pH 8.832 0.1% SDS. The electrode buffeT contained 0.05 M

Tris, 0.38 M glycine and 0.1% SDS, pH 8.532. Both gels were polymerized with 0.04% amnonium persulfate and 0.06% TEMED. Protein samples (0.01-0.05 ml) were applied to 0.125 M Tris/Hcl, pH 6 .822, 2% SDS, 20% glycerol, 50 mM

2-mercaptoethanol and 0.01% bromphenol blue. Before application the pro­ tein -solution was heated for 5 minutes in a boiling water bath to completely dissociate the proteins into their subunits. Electrophoresis was conducted at room temperature at 16 mA for the initial 60 minutes and then at 70 mA until the tracking dye reached 1.0 cm from the bottom of the gel. The gel was stained for protein essentially by the method of Weber and Osborn (161).

The gel was irranersed in a solution of 0.25% Coomassie Brilliant Blue R-250,

50% methanol and 9% acetic acid and stained a mininun of 5 hours. The gel was then destained in a solution of 50% methanol and 10% acetic acid and was subsequently stored in a solution of 5% methanol and 7.5% acetic acid.

v. Sephacryl 5-200 Gel Filtration Molecular Weight Determination

Sephacryl S-200 Superfine (Pharmacia) was diluted in distilled water, deaerated by applying a vacuum for IS minutes, then packed into a column 47

(1.5 x 75 an) by gravity at 4°C. The colunn bed (1.5 x 69 an) was equi­ librated with 3 1 of 100 nW Tris/HCl, pH 8 .O4 buffer containing 100 mM

KC1, 10 mM 2-mercaptoethanol, 101 glycerol, 1.0 uM EDTA and 0.05 mM PMSF.

Calibration of the column was performed using Blue next ran 2000 (Pharmacia), cytochrome c (equine heart), myoglobin (whale skeletal muscle), trypsin inhibitor (soybean), ovalbunin (egg), hyaluronidase (bovine testis), BSA, hexokinase (yeast) and alcohol dehydrogenase (yeast). The protein standards were prepared in equilibration buffer at a concentration of 1.0 mg/ml and applied in a volume of 0.5 ml. Elution of the proteins was monitored by absorbance at 280 nm.

vi. Glycerol Gradient Centrifugation

Linear gradients of glycerol (10-30%, v/v) in 100 mM Tris/Hcl buffer,

7. and 10 mM 2-mercaptoethanol were prepared essentially by the pro­ cedure of Martin and Ames (162). Prior to filling the gradient former sufficient light solution was used to flush out and remove trapped air in the connecting tube valve. Two ml of the light solution and 1.85 ml of the heavy solution were placed in the mixing and reservoir chambers respec­ tively. Mixing was achieved by use of a motorized teflon coated wire with a loop at one end. A second similar wire was placed in the reservoir to compensate for the change in volume. The stainless steel outlet capillary was placed at the bottom of a 7/6 x 2 3/8 inch polyallomer tube (Beckman) and the gradient poured by opening the outlet stopcock, insuring flow, then opening the connecting tube valve. Stirring was regulated so as to achieve adequate mixing without forming bubbles or splashing solution on to the chamber wall. Gradients were placed in their respective buckets and allowed to chill to 4*C. The sample (0.1 ml) was layered on top of the 48

gradient (3.85 ml) by hand and the tubes were centrifuged in a SWS6 swinging bucket rotor at 4°C for 20 hours at 56,000 rpm. Two-drop fractions were collected from the top of the tube by pumping 601 (v/v) glycerol and 0.011 bromphenol blue into the bottom of the tube, using a 5 ml glass syringe itted with an 18 gauge deflected-point needle,

vii. Protein Determination

Protein was determined by two methods. Method I is the Lowry pro­ cedure, modified to lessen sulfhydryl interference as described by Geiger and Bessman (163) and Method II is the procedure of Bradford (164) as described by Bio-Rad Laboratories (165).

Method I. The following stock reagents were prepared: (A) 10%

Na2C03 in 0.5 M NaOH; (B) 5.4% K N a C ^ O e ^ ^ O ; (C) 1% CuS04*5H20. An alkaline copper working solution was prepared on the day of assay by mixing equal volumes of stock solutions (B) and (C) and then mixing 1 volume of this solution with 10 volumes of stock solution (A). Diluted Folin-

Ciocalteau reagent was prepared by adding 1 volume of the 2 N reagent to

10 volunes of distilled water. To 1.0 ml of protein solution containing

25 to 150 ug protein was added. 1.0 ml of the alkaline copper working solu­ tion. After mixing, 0.02 ml of 3% H202 was added with vigorous shaking.

This mixture was allowed to react at room temperature for 30 minutes. Sub­ sequently, 2.0 ml of the diluted Folin-Ciocalteau reagent was added and after mixing was allowed to stand 30 minutes at room temperature. The absorbance was then read at 750 nm. Standard curves were generated using a 1.0 mg/ml BSA (Fraction V) solution prepared in the buffer used in the protein solutions to be assayed. A new standard curve was made for every protein determination. This assay was used primarily for determining 49 pTOtein in crude preparations that were utilized for analytical disc PAGE.

Method II. Just prior to assay 1 volume of the Bio-Rad Dye Reagent

Concentrate was diluted with 4 volumes of distilled water and filtered

through Whatman No. 1 paper. .To 0.05 ml of protein solution containing

10 to 70 ug protein was added 2.5 ml of the diluted Dye Reagent. This mixture was stirred gently with a vortex mixer and allowed to stand at

room temperature for 15 minutes. The absorbance then was read at 59S nm.

For solutions containing from 2 to 10 ug protein a modified procedure was utilized. To 0.8 ml of protein solution was added 0.2 ml of the concentrated

Dye Reagent. The solution was stirred gently with a vortex mixer, allowed

to stand at room temperature for 15 minutes, and then the absorbance was

read at 595 nm. Standard curves were generated for both procedures using

a 2.0 mg/ml ovalbumin (crystalline) solution prepared in 50 mM Tris/HCl buffer, pH 7.54> lot glycerol, 10 mM 2-mercaptoethanol, 1 mM EDTA and

50 uM PMSF. A new standard curve was prepared each time the assay was performed. The assay was used routinely for determining protein during

purification.

d. Tissue Preparation and Subcellular Fractionation

i. Liver, Kidney, Spleen, Brain, Testis, Thyroid Gland and Thymus

All steps were performed at 4°C. Cytosol and mitochondrial fractions

were prepared from thymus as described by Tyler and Gonze (166), and from

liver, kidney, spleen, brain, testis and thyroid gland as described by

Beattie (167).

Healthy male Sprague Dawley rats (340-400 g), C3H or Swiss mice (41-48 g)

and PD-4 hamsters (117-152 g) were killed by decapitation and the appropriate

•organs surgically removed. Bovine liver, porcine thyroid gland and calf 50 thymus were obtained fresh from a local abattoir. The tissues were rinsed in cold homogenizing buffer (50 mM Tris/HCl, pH 7.3A, 0.25 M sucrose, 50 mM KC1, 5 mM MgCl2 and 10 mM 2 -mercaptoethanol] and then minced with scis­ sors. Rat, mouse and hamster-tissues were disrupted in a chilled Teflon- glass Potter-Elvehjem homogenizer in 6 volumes (w/v) of the homogenizing buffer. After mincing bovine liver, porcine thyroid gland and calf thymus were homogenized in a Waring Blendor (Model FC-2) in 6 volumes (w/v] of homogenizing buffer for 5 seconds at low speed and 15 seconds at high speed.

Using a Sorvall RC2-B refrigerated centrifuge maintained at 4°C, each crude homogenate was centrifuged at 1,200 x g for 10 minutes. The supernatant was saved and the pellet was resuspended in 3 volumes (w/v] of the homo­ genizing buffer, than centrifuged at 1,200 x g for 10 minutes. The two low speed supematent fractions were pooled and centrifuged at 12,000 x g

(thymus] or 8,700 x g (liver, kidney, spleen, brain, testis, thyroid gland) for 10 minutes. The cytosol fraction was prepared from the supernatant by serial centrifugations at 20,000 x g for 10 minutes and 105,000 x g for 90 minutes. All centrifugations at or greater than 105,000 x g were performed in a Beckman L2-65B preparative ultracentrifuge. The pellet from the

8,700 x g centrifugation was washed 3 times with centrifugation at 5,200 x g for 10 minutes in homogenizing buffer. The pellet from the 12,000 x g centrifugation was washed 3 times with centrifugation at 12,000 x g for 10 minutes in homogenizing buffer. The pellets were resuspended separately in a minimal volune of osmolysis buffer (50 mM Tris/HCl, pH 7.34 and 10 mM

2-mercaptoethanol), then incubated at 4°C overnight. The lysed mitochondria were disrupted further by rapidly freezing and thawing 4 times in acetone- dry ice or sonicating on ice using a Branson Sonifier Model 350 for a total 51 of 3-5 minutes. Irradiation was in S second bursts interspersed with 2 minute cooling periods at an output setting of 6 . The disrupted mito­ chondria were centrifuged at 20,000 x g for 10 minutes. The pellet was discarded and the supernatant was subjected to a final centrifugation at

105,000 x g for 90 minutes. The supernatant represents the mitochondrial fraction.

ii. Fetal Rat and Hamster Tissue

Pregnant Sprague Dawley rats (3-10 days) and PD-4 hamsters (8-12 days) were killed by decapitation and their uteri surgically removed. Fetuses were removed from the uterine horns, separated from their placentas, rinsed in homogenizing buffer, then homogenized in 3 volumes (w/v) of the same buffer in a Waring blender for 30 seconds. Cytosol and mitochondrial frac­ tions were prepared by the same procedure used for liver fractionation described in the previous section.

iii. HeLa, KTsv-40 and WI-38 Cells

He La (CCL-2), WI-38 and HTsv-40 cells (kindly provided by Dr. D.A. Wolff and Dr. G.E. Milo) were grown in 32 oz. prescription bottles in Eagle's or

Joklik's minimum essential medium supplemented with 10% calf serum. The cells were harvested by trypsinization just after reaching confluencey.

The cells were washed extensively by suspending in homogenizing buffer and centrifuging at 1,500 x g for 15 minutes. The washed cells were re suspended in a minimal volume of osmolysis buffer. The cells were disrupted either by sonic irradiation, as described for liver, but with an output setting of 3, or rapidly frozen and thawed 3 times in acetone-dry ice. The cell debris was sedimented by serial centrifugations for 15 minutes at 1,500 x g and 20,000 x g. The final supernatant fraction represents the whole cell extract. 52

RESULTS

1. Electrophoretic Survey of Mammalian Tissues For Deoxynucleosi.de Kinases

The electrophoretic profiles of dCyd, dThd, dAdo and dGuo phosphory- lating activities found in adult rat liver and thymus are shown in Figure 2.

Kinase activities apparently specific for all four deoxynucleosides are found in rat liver mitochondria, co-migrating at Rf 0-56, and a second dGuo kinase is seen at Rf 0.30. The cytosol fraction from rat liver appears to contain considerably higher levels of the slower-moving dGuo kinase (Rf 0.31), but barely detectable amounts of the other kinases. Mitochondria from rat thymus also exhibited all four deoxynucleoside kinases co-migrating of Rf

0.60, but in addition contained minor amounts of a dThd (Rf 0.23), dGuo

(Rf 0.33) and dCyd kinase (Rf 0.83). In rat thymus cytosol the predominant deoxynucleoside kinases were the slower migrating dThd (Rf 0.20) and dGuo kinases (Rf 0.30) and the fast-moving dCyd kinase (Rf 0.83), while the four co-migrating activities were all detectable at Rf 0.53. As shown in Figure

3 , in preparations of liver mitochondria a second dAdo phosphorylating activity was occasionally observed at Rf 0.86. This activity was also observed in the mitochondria from spleen (Figure 4) that apparently lacked the dAdo phosphorylating activity associated with the Rf 0.60 dGuo phos­ phorylat ing activity.

Adult bovine liver and calf thymus exhibited the deoxynucleoside kinase patterns shown in Figure 5. The mitochondrial fraction of liver showed dCyd and dThd kinases co-migrating to Rf 0.40 separated from apparent dAdo Figure 2. Electrophoretic Profile of Deoxynucleoside Kinases in Adult Rat Liver and Thymus. Conditions for analytical disc PAGE and prepara­ tion of subcellular fractions were as described in Methods. Enzyme assays were incubated for 18 hours at 37°C. Protein samples applied: 360-540 ug for liver and 65-75 ug for thymus. Kinase: dAdo, (A--A); dGuo, ( # - - # ) ; dCyd, (A A ) ; dThd, (O O).

53 54

15 RAT LIVER MITOCHONDRIA 10

_/ 5 \ 0 _ Sw2u I v* w^s2vSw £w 4 v2

15

10 RAT LIVER CYTOSOL

5 ■* J « \ 0 _2s2o?=£=2^icI^^£=£s2q2=^^?sliirS*»£ji2pti±liii!il=^^?i?a5

15

10 RAT THYMUS MITOCHONDRIA

5

0 _&&b$sIs£3$«^ai:E3S?5!:;s ii* I

15

RAT THYMUS CYTOSOL 10

5

0 0-1 0.2 0.3 04 0.5 0.6 07 0.8 0.9 c 1.0 Figure 3. Electrophoretic Profiles of Deoxynucleoside Kinases in Adult Rat Liver Mitochondria. Conditions for analytical disc PAGE and prepara­ tion of mitochondria were as described in Methods. Enzyme assays were incubated for 18 hours at 37°C. Protein sample applied was 1,600 pg. Kinase: dAdo, (A A); dGuo, dCyd, (A— A)i dThd (O O)

55 CPM x i ------1 ------r 9 1 MITOCHONDRIA 1 I RT LIVER RAT i i Figure 4. Electrophoretic Profile of Deoxynucleoside Kinases in Adult Rat Spleen Mitochondria. Conditions for analytical disc PAGE and preparation of mitochondria were as described in Methods. Enzyme assays were incubated for 18 hours at 37°C. The protein sample applied was 128 ug. Kinase: dAdo, (▲ dGuo, ( # ---#); dCyd, ( A — A ) ; dThd, ( O — O).

57 in ao ' I M I / RAT SPLEEN RAT SPLEEN MITOCHONDRIA Rf 05 06 07 08 Q9 10 \ i I 0 2 0 3 0 4 0 1 - 1 0 30 5 0 20 4 0 .0 1 X Wd D I Figure 5. Electrophoretic Profile of Deoxynucleoside Kinases in Adult Bovine Liver and Calf Thymus Cytosol and Mitochondrial Fractions. Con­ ditions for analytical disc PAGE and preparation of subcellular fractions were as described in Methods. Enzyme assays were incubated for 18 hours at 37*C. Protein samples applied: 1,000-1,200 ug for liver and 280-520 Mg for thymus. Kinase: dAdo, (▲— —A); dGuo, (# — — # ) ; dCyd, ( A A ) \ dThd, CO O).

59 CPM X 10' 100- 100 100 0 5 25 50 0 5 _ _ aa alvtsl tala L £ £ I O y JL 1 . 03 . 0. . 07 . 10 0.9 8 0 0.7 0.6 .5 0 0.4 0.3 0.2 01 lutbl lu| Jrtvia 1* utiat a i t t u v l w * I 1 a i v t r J | u v l l t f w t v l v l l w b t u M l t v I MITOCHONDRIA AF THYMUS CALF CALF THYMUS CALF CYTOSOL BEEFLIVER MITOCHONDRIA EF LIVERBEEF CYTOSOL * W I

60 61

and dGuo phosphorylat ing activities at Rf 0.56. The cytosol fraction from

liver.had the same four activities, but apparently in lower concentrations.

The mitochondrial fraction from calf thymus had the same dCyd/dThd (Rf

0.40) and dAdo/dGuo ( R f 0.56) activities, but in addition, a lower migra­

ting dThd kinase (R f 0.23) and rapidly co-migrating dCyd/dGuo/dAdo (R f

0.86) kinase activities were observed. In the cytosol of calf thymus the

slower moving dThd kinase (Rf 0.23) and the rapidly co-migrating dCyd/dGuo/

dAdo activities (R f 0.86) were the predominant species, with only traces

of the slower migrating dGuo kinase. None of the other activities observed

in the mitochondrial fraction were detected in the cytosol.

The mitochondrial fractions from adult hamster and mouse liver presented

analogous kinase patterns and are shown in Figures 6 and 7 respectively.

This pattern resembles that exhibited by the mitochondrial fraction of rat

liver, to the extent that two dGuo kinase activities were observed. How­ ever, the pattern of separation of the central pyrimidine and purine deoxynucleoside kinase peaks resembles that observed in the bovine mito- condrial fractions.

A whole cell sonicate of freshly confluent He La cells was also analyzed

(Figure 7), and the pattern which emerged is like that which one might expect from a whole cell sonicate of rat thymus.

2. Calf Thymus Deoxyguanosine Kinase Activities

Because of the renewed interest in purine deoxynucleoside kinases as a result of their implication in imnunodeficiency diseases, a preliminary comparative study of the cytosol and mitochondrial dGuo kinase activities was deemed appropriate. Calf thymus was chosen as the source of enzyme because of the following: (1) both cytosol and mitochondrial enzymes are Figure 6. Electrophoretic Profile of Deoxynucleoside Kinases in Adult Hamster Liver Mitochondria. Conditions for analytical disc PAGE and preparation of mitochondria were as described in Methods. Enzyme assays were incubated for 18 hours at 37 °C. The protein sample applied was 1,300 pg. Kinase: dAdo, ( ^ — A); dGuo, ( # - - # ) ; dCyd, (A—A)J dThd, (O—O).

62 ----- 1

1 MITOCHONDRIA I HAMSTER LIVER HAMSTER LIVER I t

1 r Rf ------1 ------1 03 04 05 ------1 ------t 50 100 01 X WdD n Figure 7. Electrophoretic Profile of Deoxynucleoside Kinases in Mouse Liver Mitochondria and HeLa Cells. Conditions for analytical disc PAGE and preparation of cellular extracts were as described in Methods. Enzyme assays were incubated for 18 hours at 37CC. Protein samples applied: 520 yg for mouse and 230 ug for HeLa cells. Kinase: dAdo, (A A); dGuo, ( O •); dCyd, ( A — A); dThd, (Q—O)-

64 CPM X 10' 40 20 . 02 . 0. . 06 7 08 09 l.C 0.9 8 0 .7 0 0.6 0.5 .4 0 0.3 0.2 0.1 Rf ^i-Y;S^;e5£5a^-r-?“-^§5i MITOCHONDRIA * HL SONICATE WHOLE ea CELLS HeLa MOUSE LfVER MOUSE *

k

66 present in the tissue; (2) relatively large quantities of tissues are available; (3) the cytosol enzyme had been extensively purified and characterized previously; and (4) the association of thymus with the inntune system.

a. Preliminary Separation of Cytosol and Mitochondrial Deoxyguanosine Kinase Activities'

Initial analytical disc PAGE studies revealed that the sequential differential centrifugation procedure used to prepare the two subcellular fractions was incapable of completely separating the two dGuo kinase variants. Figure 5 shows that significant contamination of the mitochondrial extract by cytosol kinase activities occurs, as well as contamination of the cytosol by mitochondrial enzyme activities. Therefore a modification of the differential centrifugation procedure was developed. In this pro­ cedure the thymus is homogenized in the same manner as described in Methods previously, followed by centrifugation at 16,700 x g for 30 minutes. The cytosol or soluble fraction is prepared by subsequent centrifugations from the supernatant fraction, as described in Methods. The pellet is resus­ pended in 3 volumes (w/v) of homogenizing buffer, then processed as described in Methods to produce the mitochondrial fraction. As shown in Figure 8, this method provides a mitochondrial fraction free of cytoplasmic dGuo kinase. However, significant amounts of mitochondrial dGuo kinase are found in the cytosol fraction, presumably due to mitochondrial breakage during tissue homogenization. Therefore an additional step which would provide complete separation was necessary in the case of cytosol.

After examining the interaction of both activities with DEAE-cellulose, a suitable procedure was found which separated the cytosol and mitochondrial dGuo kinases. This procedure is described below. Figure 8. Analytical Disc PAGE of the Calf Thymus dGuo Kinases from Cytosol and Mitochondrial Fractions Generated by Sequential Differential Centrifugation. The procedure for generation of subcellular fractions was as described in Results. Analytical disc PAGE was as described in Methods. Assay for dGuo kinase was for 5 hours at 37°C. Protein sample applied for both fractions was-500 pg.

67 nmols dGMP nmols dGMP - 0 4 - 0 6 - 0 5 10- - 0 3 70- 20 10H 5- - . 0 0 0 05 04 03 02 0.1 CYTOSOL ITOCHONDRIA M 69

The crude cytosol and mitochondrial fractions (40 ml each) were con­ centrated using anvnonium sulfate. Optimal conditions were achieved using

40 to 80% saturated aimtoniun sulfate fraction for both. The pellets pre­ cipitated with 80%-saturated anrnonium sulfate were re suspended in 15 ml of

CM buffer (50 mM Tris/HCl, pH 7.5^, 10 n*1 2-mercaptoethanol and 10% glycerol) and were subsequently dialyzed against 4 1 of the same buffer overnight in

Spectrapor Membrane No. 2. Separately, dialyzed cytosol (413 mg) and mito­ chondrial (306 mg) protein fractions were applied to a DEAE-cellulose column (2.5 x 8.5 cm) equilibriated with CM buffer. The column was eluted successively with a linear 0 to 0.3 M KC1 gradient (500 ml), then with 0.8 M KC1 (150 ml), all in CM buffer. The results of the column development are shown in Figure 9. Under these conditions the cytosol and mitochondrial dGuo kinases are readily separated. Recovery of mitochondrial dGuo kinase from the ammonium sulfate fractionation step was very poor, and this accounts for the apparent absence of mitochondrial en2yme from the cytosol in this experiment. Subsequently, the ammonium sulfate step was omitted from the purification procedure for the mitochondrial enzyme.

Electrophoresis of the predominant dGuo kinase peaks from this experiment confirmed that the mitochondrial and cytosol activities migrated solely with Rf values of 0.52 and 0.79, respectively, as in Figure 8.

b. Partial Purification of Cytosol and Mitochondrial Deoxyguanosine Kinase Activities

i. Mitochondrial Deoxyguanos ine Kinase

Studies conducted to determine the appropriate conditions for purifi­ cation of the mitochondrial dGuo kinase from calf thymus provided the

following conclusions: 1

Figure 9. Chromatography of Calf Thymus Ammonium Sulfate Fractions and DEAE-cellulose. Conditions for column were as described in Results. ( 0 # ) , dGuo kinase; (A---A). Ado kinase; (------), protein absorbance at 280 nm.

70 ENZYME ACTIVITY (UNITS/M L) 15 0 3 30- - 5 0 1.5 4 ITOCHONDRIA M ------YOS L SO CYTO 0. M C - KCI M .3 -0 0 -.MKCI C K 0-0.3M 4*— 0l8M 0l8M 4*— KCI 71 72

(1) The enzyme requires sulfhydryl protecting agents and is further

stabilized by glycerol in lengthy colunn chromatography steps.

(2) Under optimal conditions amnonium sulfate significantly inhibits

the mitochondrial enzyme (up .to 50% loss of activity).

(3) Streptomycin sulfate fractionation does not significantly enhance

or stabilize purification results of the enzyme apparently the result of

insignificant amounts of interfering nucleic acids in the mitochondrial

fraction.

(4) The enzyme binds to Blue Sepharose (CL-6B), and does so most

favorably at pH 7.5 in Tris buffer containing 5 mM MgCl2. The activity

can be desorbed from the affinity column by ATP or by raising either pH

or ionic strength. While higher recoveries are achieved by increasing pH or ionic strength, specific activities are significantly higher with

ATP elution. Optimal purification results when the Blue Sepharose CL-6B

is sequentially washed with 1.0 mM NADH and 35 mM KCI in the presence of

5 mM MgCl2, after sample loading, but before enzyme elution with 10 mM ATP minus MgCl2.

Thumus glands (632 g) were processed by the procedure described in

Methods with the modification in centrifugation described in this section

to yield 57 ml of mitochondrial extract (Fraction I).

DEAE-Cellulose Ion Exchange Chromatography

The crude mitochondrial extract was dialyzed against 4 1 of CM buffer

overnight in Spectrapor Membrane No. 2. The dialyzed mitochondrial extract was applied to DEAE-cellulose (DE*23, Whatman) column (2.5 x 8 cm) equili-

briated with CM buffer. The colunn was eluted successively with a linear

0 to 0.3 M KCI gradient (500 ml), then with 50 ml of 0.3M KCI, all in CM 73 buffer at a flow rate of 200 ml/hour and collecting S ml fractions.

Active peak fractions were pooled and concentrated by ultrafiltration

(Amicon, PM-10 membrane) to a final volume of 29 ml (Fraction II).

Sephacryl S-200 Chromatography

The concentrated DEAE-cellulose eluate was divided and applied in two cycles on a Sephacryl S-200 (Superfine, Pharmacia) column (2.5 x 69 cm) equilibrated with 100 mM Tris/HCl, pH 8.0^ buffer containing 100 mM

KCI, 10 nM 2-mercaptoethanol and 10% glycerol. As shown in Figure 10, dGuo kinase activity was eluted in a single sharp synmetrical peak. Active fractions from the two column runs were pooled and concentrated by ultra- filtration (Amicon, IM-10 membrane) to a final volume of 44.5 ml (Fraction

III).

Blue Sepharose CL-6B Affinity Chromatography

The gel filtration concentrate was dialyzed against 2 1 of Buffer A

(50 mM Tris/HCl, pH 7.5^ buffer containing 5 mM MgCl2, 10 mM 2-mercaptoe- thanol and 101 glycerol) for 10 hours with a change of dialysis buffer after

5 hours. Conductivity and pH of the protein solution were checked at the termination of dialysis to insure equilibration had been achieved. The dialyzed fraction was applied to a Blue Sepharose CL-6B (Pharmacia) column

(2.5 x 4 cm) equilibrated in Buffer A with a flow rate of 200 ml/hour.

Collecting 5 ml fractions, the column was washed successively with 300 ml of equilibration buffer containing 1.0 nM NADH (Buffer B) and with 300 ml containing 35 mM KCI (Buffer C). Deoxyguanosine kinase activity was then eluted with equilibration buffer containing 10 nM ATP but lacking MgCl2

(Buffer D). Any remaining kinase activity was washed from the column with

Buffer E (50 mM Tris/HCl, pH 7.54, 10 nM 2-mercaptoethanol, 10% glycerol and 1.0 M KCI. The elution profile of dGuo kinase activity from the Blue Figure 10. Elution Profile of Calf Thymus Mitochondrial dGuo Kinase from Sephacryl S-200. Enzyme applied to the column (2.5 x 69 cm) was 10.6 mg of Fraction II. Conditions for development were as described in Results. Assay for dGuo kinase was as described in Methods for the general procedure kinase assay. (0---#), dGuo kinase; ( O O ) , *280-

74 75 280 05 40 20 FRACTION 20 25 n n WIN A1IAI1DV 3WAZN3 76

Sepharose column is shown in Figure 11. Under these conditions the major-

1. . ity of recoverable kinase activity was found in the 10 mM ATP eluate with only residual activity coming off the column with high salt. Fractions containing activity in the 10 nW ATP eluate were pooled and concentrated by ultrafiltration (Amicon, PM-10 membrane), followed by further concen­ tration and dialysis by a MicroProDiCon concentrator (Bio-Molecular Dyna­ mics) to a final volume of S.5 ml (Fraction IV).

A summary of the total purification scheme is presented in Table 5.

Fraction IV was stored in 50% glycerol at -20°C. Under these conditions the enzyme is stable for several months.

ii. Cytosol Deoxyguanosine Kinase

The cytosol dGuo phosphorylating activity was purified by a modifica­ tion of the method described for the isolation of calf thymus dCyd kinase

(83). The calcium phosphate gel step was omitted, activity was eluted from DEAE-cellulose with 0.8 M KC1, rather than with Tris, and this was diluted and applied to Blue Sepharose column as above, finally being eluted with 0.8 M KC1 in CM buffer. Details of the procedure performed by Michael

Carr in this laboratory and purification results are presented in Appendix

B.

c. Properties of Cytosol and Mitochondrial Deoxyguanosine Kinase Activities'

In the separation procedures previously described, i.e. gel electro­ phoresis and DEAE-cellulose ion exchange chromatography, the cytoplasmic dGuo kinase is invariably associated and entirely congruent with dCyd and dAdo kinase activity peaks. This, coupled with the complete absence of any other peaks of these activities in the cytosol, makes it very clear that the cytosol form described is identical with the dCyd/gGuo/dAdo kinase Figure 11. Blue Sepharose CL-6B Affinity Chromatography of Calf Thymus Mitochondrial dGuo Kinase (Fraction III). Conditions for the column (2.5 x 4 cm) and description of elution buffers A-E are described in Results. The protein sample applied was 47.2 mg of Fraction III. Assay of each fraction (5 ml) was conducted as described in Methods for the general procedure kinase assay.

77 ENZYME ACTIVITY [U /M l] 4.0 6.0 2.0 8.0 »|«- — A • 5 0 75 50 25 B -M4- 0 15 150 125 100 FRACTION 00 4 • " Table 5.

Purification of Calf Thynus Mitochondrial dGuo Kinase

Specific Volume Activity Protein Activity Yield Fold Fraction (Ml) (U/nl) (ng/nl) (U/nl) (*) Pure

I Crude Mitochondrial 57 0.89 32 0.027 100 - Extract

11 DEAE-Gellulose 29 2.4 6.5 0.369 137 12.3

111 Sephacryl S-200 44.5 1.03 1.06 0.972 66 36

IV Blue Sepharose 5.5 3.04 0.87 3.49 36 130 80 studied previously (83,87). However, the mitochondrial enzyme is a newly discovered activity. In studying some of its properties, comparative data were obtained with the cytosol enzyme under similar conditions, so that their properties could be contrasted more easily,

i. Isoelectric Focusing

Isoelectric focusing of the partially purified dGuo kinase activities in a 71 polyacrylamide gel using ampholytes producing a gradient from pH 5 to 7 also resulted in separation of the two activities. As shown in

Figure 12, the mitochondrial and cytoplasmic enzymes behave differently, with respective pi values of 5.89 and 5.41. As expected, the latter value is comparable to that reported for the calf thymus cytosol dCyd/dGuo/dAdo kinase (83).

ii. Molecular Weight Determination

The molecular weight of the mitochondrial dGuo kinase was estimated from the results of the Sephacryl S-200 purification step (Figure 10).

A molecular weight of 55,000 was calculated by the method of Laurent and

Killander (168), by comparison to a series of known protein standards

(Figure 13). This value is almost identical to that reported for the cytysol enzyme (56,000) by Durham and Ives (83).

iii. Nucleoside Specificity

The rates at which the mitochondrial and cytosol kinases phosphory- lated various nucleosides were determined at two concentrations of these substrates, 0.02 and 0.5 nW. As shown in Table 6, the mitochondrial enzyme is more specific than the cytosol variant, and appears to be strictly a purine deoxynucleoside kinase, except for its unusual ability to phos- phorylate the riboside Guo, but not Ado or Ino. The cytosol enzyme Figure 12. Isoelectric Focusing (pH 5-7) of the Calf Thymus dGuo Kinase Activities. Conditions for focusing and assay for dGuo kinase were as described in Methods. Enzyme was 65 ug Fraction IV (mito­ chondrial) and 11 pg Fraction IV (cytosol). Cytosol dGuo kinase (#~— #); mitochondrial dGuo kinase (O— O); pH (— •— . — ).

81 5 10 15 20 25 30 35 oo N GEL SLICE NUMBER Figure 13. Molecular Weight Determination of the Calf Thumus Mitochon­ drial dGuo Kinase by Gel Filtration on Sephacryl S-200. Conditions for the column were as described in Methods, except the column was 2.5 x 63 cm and calibration was limited to the proteins indicated. 98 mg of Fraction II was applied to the column. The column had a flow rate of 30 ml/hour and 11 ml fractions were collected. Assay for dGuo kinase was by the general procedure as described in Methods. The arrow indi­ cates the molecular weight of the dGuo kinase as determined by the elu­ tion volume of the enzyme.

83 06

Trypsin Inhibitor

04

Hyaluronidass BSA

02-

4.2 4.4 4.6 4J 5250 LOG M O L W T . 85

Table 6. Nucleoside Specificity of dGuo Kinase Activity From Calf Thymus. Conditions were as described in Methods for the general pro­ cedure assay of dGuo kinase, except incubation was for 15 minutes and nucleoside concentration was varied as indicated. Enzyme was 7.7 Mg of Fraction IV (mitochondria) and 1.4 ug of Fraction IV (cytosol).

Nucleotide Synthesized

Mitochondtial Cytosol

Nucleoside 0.02 mM 0.50 mM______Q.Q2 unM 0.50 mM

pmols

dGuo 60 120 29 760

Guo 3 72 <1 <1

dAdoa 8b 160b 27 120

Adoa <1 <1 <1 <1

dlno 102 240 <1 <1

Ino <1 <1 <1 <1

ara-Aa <1 <1 <1 <1

dCyd <1 <1 46 120

Cyd <1 <1 11 160

dThd <1 <1 <1 <1

Urd <1 <1 <1 <1 a70 uM Coformycin added to inhibit adenosine deaminase. bProduct was entirely dIMP; no dAMP was detected. 86 phosphorylates dCyd, dAdo and dGuo from among the deoxyribonucleosides, and also Cyd, as has been observed previously (8S, 87, 118). Since it does not phosphorylate Urd, this activity is not Urd/Cyd kinase, however (169).

Unlike the cytosol enzyme, which has some slight activity toward ara-A, the mitochondrial enzyme does not phosphorylate this analogue signifi­ cantly under these conditions.

iv. Identification of Enzyme Products

The partially purified mitochondrial kinase produced a single radio- labeled product from [3H]-dGuo, one which co-migrated with S'-dGftP upon ion exchange paper chromatography in a system which resolved both dGuo and

5'-GMP from 5'-dCJtP (Figure 14). This product was completely reconverted to dGuo after digestion with purified snake venom 5*-specific nucleotidase

(Figure 15).

As noted in Table 6, the product of dAdo phosphorylation by the par­ tially purified mitochondrial kinase was found to be entirely dIMP, des­ pite the removal of the majority of adenosine deaminase by the Blue

Sepharose CL-6B purification step and the addition of 70 wM Coformycin, a potent inhibitor of the deaminase. Similar results have been reported for a dCyd kinase preparation from calf thymus (87). The deamination was found to occur at the level of nucleoside, with no evidence for the presence of a deoxyadenylate deaminase. From this, it can be concluded that dlno is a much better substrate than dAdo, and this is supported by direct assay of dlno phosphorylation (Table 6). It is not clear why the Cofor­ mycin fails to completely inhibit the apparent residual adenosine deamin­ ase in the preparation. T have observed at least three electrophoretic variants of this activity in this tissue, similar to that found in other 4

Figure 14. Identification of the Tritium-labeled Product of the Calf Thymus Mitochondrial dGuo Kinase Reaction By Ion-Fxchange Paper Chro­ matography. Conditions of assay and paper chromatography (System 5, Table 4) were as described in Methods. 0.025 ml of the terminated reac­ tion mixture was applied to the chromatogram in 5 pi aliquots. dGuo and dOiP markers were chromatographed along with the reaction aliquot and are located in the positions shown by the arrows.

87 i------1------1------i------r GMP dGuo 25 I

20

T 15 2

I W U Figure 15. Identification of the Phosphorylated Deoxyguanosine Product of the Calf Thymus Mitochondrial dGuo Kinase Reaction. The dGuo kinase reaction was conducted using the general procedure as described in Methods. Aliquots (0.04 ml) of the terminated reaction were neutralized and 0.025 ml chromatographed using System 1, Table 4 as described in Methods before treatment (Panel A) or after incubation with 5'-nucleo­ tidase (Panel B). Conditions for the 5'nucleotidase incubation were as described in reference (170).

89 r <

o 3 _ ru

CN O 00 ,.01 x WdD 91

tissues (171-173). It is possible that there is a species of deaminase which is less strongly inhibited by Coformycin. In contrast, the cytosol

kinase preparation was completely free of deaminase contamination and

converted dAdo only to dAMP.

v. Apparent Kin for dGuo

The apparent values for dGuo were determined for the two enzymes over a 50-fold range of nucleoside concentration. A Lineweaver-Burk plot

of the results is shown in Figure 16. The K,,, values for the mitochondrial

and cytoplasmic enzymes were 6 uM and 770 uM, respectively. The latter

value is comparable to those from previous reports of dGuo phosphorylation

by dCyd kinase, with values ranging from 180 to 310 yM (84, 85) to 3 mM

(87), whereas the value obtained with the mitochondrial enzyme is very

similar to the values of 0.32 and 7.7 yM reported for porcine skin and neonatal mouse skin dGuo kinase, respectively (130, 131).

vi. Effects of NUcleotides on Enzyme Activity

The effects of various pyrimidine and purine nucleotides on the dGuo

kinase activities were tested and the results appear in Tables 7 and 8,

respectively. The mitochondrial enzyme, like the cytosol variant, is

regulated by nucleotides, but in a strikingly different fashion. The

cytosol pattern of inhibition is that of dCyd kinase (83, 87), with the

strongest inhibition produced by dCTP. In contrast, the mitochondrial

dGuo kinase is inhibited most strongly by its distal end product, dGTP,

and the structurally analogous dITP, but not at all by dCTP. Both dGDP

and dGMP are more potent inhibitors of the mitochondrial enzyme than of the

cytosol enzyme, whereas for dCDP, dADP and dAHP the reverse is true. The

mitochondrial kinase is also very strongly affected by positive modulators,

especially by dTDP which stimulates the reaction velocity over 6-fold at Figure 16. Effect of Varying dGuo Concentration on the Reaction Rate of Calf Thymus dGuo Kinase Activities. Conditions were as described in Methods for the general procedure assay of dGuo kinase, except that the dGuo concentration was varied as indicated and incubation was for 15 minutes. Velocity is expressed as nmol dGMP formed per minute. The enzymes were: 8.7 jig of Fraction IV (mitochondria) (0---#); 1,4 ug of Fraction IV (cytysol) (A A)*

92 93

• t -

CO

O x

CO

CN

CM O CO 94

Table 7. Fffects of Various Pyrimidine Nucleotides on dGuo Kinase Acti­ vities From Calf Thymus, Conditions were as described in Methods for the general procedure assay of dGuo kinase, except incubation was for 15 minutes. Enzyme was 7,7 ug of Fraction IV (mitochondria) and 1.4 ug of Fraction IV (cytosol) and nucleotides were added to assay as shown.

dGuo Kinase Relative Activity, %

Micleotide mM Cytosol Mitochondrial

None - 100a 100b

dCTP 0.1 0.1 96 1.0 0 100

dCDP 0.1 2 105 1.0 <1 146

CDP 0.1 86 107 1.0 43 143

dTTP 0.1 100 127 1.0 100 283

dTDP 0.1 86 253 1.0 49 620

UTP 0.1 100 119 1.0 108 190

UDP 0.1 81 143 1.0 35 314

UMP 0.1 95 100 1.0 92 91

NaP-P 0.1 100 100 1.0 95 121

a100% activity for cytosol activity corresponds to 38 pmols dGMP formed/ 15 minutes b100% activity for mitochondrial activity corresponds to 83 pmols dGMP formed/15 minutes. 95

Table 8. Effects of Various Purine Nucleotides on dGuo Kinase Activities From Calf Thymus. Conditions were as in Table 7.

dGuo Kinase Relative Activity, %

Nucleotide mM Cytosol Mitochondrial

None - 100 100

dCTP 0.1 89 27 1.0 37 4

dCDP 0.1 76 45 1.0 26 7

dGMP 0.1 100 90 1.0 100 67

GTP 0.1 95 100 1.0 92 88

GDP 0.1 95 105 1.0 86 121

(WP 0.1 95 102 1.0 95 107

dATP 0.1 79 94 1.0 24 70

dADP 0.1 24 100 1.0 3 100

dAMP 0.1 100 100 1.0 84 100

ADP 0.1 95 105 1.0 70 116

AMP 0.1 95 105 1.0 92 107

dITP 0.1 100 35 1.0 68 6

IMP 0.1 95 100 1.0 92 102 96 a concentration of 1.0 mM. Other nucleotides having a diketo pyrimidine base, such as UDP and dTTP, are also strongly stimulatory, whereas UDP effectively inhibits the cytosol enzyme. Both the positive and negative modulators appear to regulate the mitochondrial dGuo kinase quite speci­ fically; CDP, ADP, GDP and dADP produced little or no stimulation at

1.0 mM. 3. Purification of Deoxyguanos ine Kinase From Bovine Liver

Because of the very interesting properties exhibited by the newly dis­ covered calf thymus mitochondrial dGuo kinase, a more extensive purifica­ tion and characterization of the enzyme was desired. Due to difficulties in obtaining fresh calf thymus and small numbers of mitochondria, the enzyme was purified using bovine liver mitochondria which could be prepared rapidly from large quantities of fresh and readily available tissue.

Studies conducted to determine the appropriate conditions for purifi­ cation of the mitochondrial dGuo kinase from bovine liver provided the following conclusions:

(1) A modified version of the sequential differential centrifugation procedure for liver mitochondria production was found more appropriate.

The new procedure provided foT more efficient production of large quanti­ ties of mitochondria.

(2) The bovine liver enzyme demonstrates properties similar to those exhibited by the calf thymus enzyme, i.e. it requires sulfhydryl protecting agents, is stabilized by glycerol, is inhibited by anrnoniun sulfate and interacts similarly with DEAE-cellulose and Blue Sepharose CL-6B.

(3) Significantly higher purification is achieved with Blue Sepharose by omitting the 1.0 i«M NADH and 35 mM KC1 pre-wash and eluting the column with 1.0 mM ATP instead of 10 mM AT? to desorb the dGuo kinase. Numerous 97 attempts to use dGTP in place of ATP as an elutriant were unsuccessful.

(4) The enzyme successfully binds to ATP-agarose, dATP-agarose,

Biogel-tfT, Phenyl Sepharose CL-4B, but not to UDP-Sepharose, AdR-Sepharose,

AMP-agarose or Octyl Sepharose CL-4B.

(5) The enzyme is relatively stable to short periods of heating at

60°C in the presence of one of its substrates, ATP*Mg.

(6) Due to some lysosomal contamination of the crude mitochondrial fraction and the recent findings that liver mitochondria contain serine protease and carboxypeptidase activities (174,175), 0.05 mM PMSF and 1.0 nM EDTA were added to all buffers used in purification.

Using these findings the following procedure was found most favorable for the isolation of the bovine liver mitochondrial dGuo kinase.

All operations were performed at 4°C.

Preparation of Mitochondria

Bovine liver was obtained fresh immediately after slaughter from a local abattoir. One kilogram portions of liver were rinsed in FW buffer

(50 mM Tris/FCl, pH 7.54, 0.25 M sucrose, 50 nM KC1, 5 nM MgCl2, 10 pM

2-mercaptoethanol, 1.0 mM FDTA and 0.05 mM PMSF), cut into small pieces and homogenized in 3 1 of FM buffer in a Waring Blendor (Model CB-5) for

5 seconds at low speed followed by 15 seconds at high speed. The homo- genate was filtered through four thicknesses of cheesecloth to prepare for centrifugation. All centrifugation up to and including the 20,000 x g step was performed in a Sorvall RC2-B centrifuge using a GS-3 rotor.

Nuclei, red blood cells and cell debris were removed by centrifugation at

1,200 x g for 15 minutes. The supernatant was carefully decanted and stored temporarily. The pellet was resuspended in 1 1 of FM buffer by homogenization in a Waring blendor (Model FC-2) for 15 seconds. The 98 hotnogenate was centrifuged at 1,200 x g for IS minutes. The sediment was discarded and the supernatant added to the first low speed supernatant.

This process was repeated two more times so that a total of 3 kg of liver was processed. The pooled low speed supernatant fractions were centri­ fuged in 3 1 portions at 16,700 x g for 15 minutes to sediment mito­ chondria. The supernatant was removed along with the ''fluffy" layer above the mitochondria and discarded. The integrity and homogeneity of the mitochondria generated by this procedure were analyzed by electron microscopy and marker enzyme distribution. Electron micrographs of the

16,700 x g pellet (Plate I) show that it is enriched in mitochondria with limited contamination by lysosomes, rough endoplasmic reticulum, peroxi­ somes and other cell debris. Closer examination of the mitochondria

(Plate II) reveals a high degree of structural integrity with intact outer membranes and numerous cristae. Table 9 shows that the distribution of dGuo kinase activity in the centrifugation fractions is analogous to the two mitochondrial markers glutamate dehydrogenase and mitochondrial dThd kinase. The 16,700 x g pellet (P-16.7) contained 48% of the total dGuo kinase activity with an approximate 3-fold increase in specific activity. This compares favorably with the results exhibited by dThd kinase and glutamate dehydrogenase. Further assessment of lysosomal contamination, on the basis of the assay of acid phosphatase, revealed

19% of the total activity resided in the fraction. The microsomal and lysosomal contamination were not considered significant enough to warrant repeated washing of the mitochondria before lysis.

Preparation of the Crude Mitochondrial Extract

The mitochondria from each centrifugation were resuspended by vigorous stirring in 250 ml of CM buffer (50 n#* Tris/HCl, pH 7.54, 1° "W Plate I. Electron Micrograph of the Crude Bovine Liver Mitochondrial Fraction (P-16.7) Magnification X7.250. The 16,700 x g pellet was dehydrated, fixed in glutaraldehyde-0s04 and stained with uranyl acetate as described by O'Brien and Kalf (176). Abbreviations: M » mitochondria; Ly ■ lysosome; RER * rough endoplasmic reticulum; P * peroxisome.

99 100

W

M

3 t

i Plate II. Electron Micrograph of the Crude Bovine Liver Mitochondrial Fraction (P-16.7): Magnification X24.000. Conditions for electron microscopy were as in Plate I, Abbreviations: M = mitochondria; Ly = lysosome; RFR = rough endoplasmic reticulum.

101 102 Table 9. Distribution of Mitochondrial Marker Fnzymes and fteoxyguanosine Kinase in the Sequential Differential Centrifugation Fractions From Bovine Liver. Aliquots (0.5 ml) of each fraction were rapidly frozen and thawed, sonicated on ice for 1 minute, then centrifuged at 30,000 x g for 15 minutes. Hie supernatants were assayed for glutamate dehydrogenase, thymidine kinase, deoxyguanosine kinase and protein as described in Methods.

Glutamate dThd dGuo Dehydrogenase Kinase Kinase % % % Total Specific Total Speci fic Total Specific Fraction Activity Activity Activity Activity Activity Activity

Liver flomogenate 100 0.025 100 0.007 100 0.076

(S-1.2)j 67 0.015 67 0.008 62 0.081

(P-1.2h ND3 ND ND ND ND ND

(S-1.2)2 17 0.036 21 0.019 19 0.136

(P-1.2)2 18 0.073 18 0.013 16 0.063

S-16.7 17 0,008 26 0.004 27 0.058

P-16.7 66 0.114 51 0.020 48 0.189 aNot Determined 104

2-mercaptoethanol, 10$ glycerol, 1,0 mM I-PTA and 0.05 mM F?tSF). The pooled mitochondria (3 1) were placed in a 3 1 Frlenmeyer flask and incubated with slow stirring at 4°C overnight to allow maximum swelling and lysis. Further solubilization of dGuo kinase was performed by sonication. The lysed mitochondria were sonicated in 400 ml aliquots using a Bronwill Biosonik Model BP-1 set at maximum output with a 9 mn diameter probe. With constant stirring, the aliquots were irradiated for

1 minute intervals interspersed with 2 minute cooling periods for a total of 8 minutes. Temperature of the suspension was monitored during the pro­ cedure, and was maintained between 4 and 10°C, The rate of release of dGuo kinase, as well as of glutamate dehydrogenase and dThd kinase, from the mitochondria, by exposure to sonic radiation foT various time inter­ vals up to S minutes, is shown in Figure 17. The three enzyme activities measured in the supernatant extract rise sharply, demonstrating an 8 to

15-fold increase in activity. After additional sonication the dGuo and dThd kinases reach a plateau at 6 to 8 minutes. When lysed osmotically the dGuo kinase activity is recovered predominantly in the pellet of a

20,000 x g centrifugation (Table 10). When lysed with 1.0$ Triton X-100

(v/v) or sonication the activity is recovered predominantly in the super­ natant (Table 10). However, maximal and reproducible levels of activity are exhibited if the mitochondria are first, subjected to osomotic shock followed by sonication as described above.

Following irradiation, the sonicate was centrifuged at 20,000 x g for

15 minutes. The pellet was discarded and the supernatant was further clarified by centrifugation at 250,000 x g for 90 minutes using a Ti60 rotor in a Reckman Model L2-6SB preparative ultracentrifugc. The Figure 17. Time Dependent Release of dGuo Kinase, dThd Kinase and Gluta­ mate Dehydrogenase From Mitochondria Ry Sonication. Preparation of the crude mitochondrial fraction (P-16,7) and sonication were as described in Results. At the time intervals indicated 2 ml aliquots were removed and centrifuged at 30,000 x g for 15 minutes. The supernatants were assayed for dGuo Kinase ( # --- #) , dThd kinase (O---O) and glutamate dehydro­ genase (A A ) as described in Methods.

105 106

1200 % 0 0 Relative Relative Activity, O o O O

100 1(17

Table 10. Sedimentation of Mitochondrial dGuo Kinase From Bovine Liver After Osmotic, Triton X-100 and Sonic Lysis of Mitochondria. The P-16.7 fraction was used as the source of mitochondria. Osmotic lysis was accomplished by diluting 0.1 ml of the mitochondrial pellet 1:10 in 50 Tris/HCl buffer, pH 7.54 containing 10 mM 2-mercaptoethanol, mixing and incubating for 2 hours at 4°C. Triton X-100 lysis was accomplished - by the same procedure, except the dilution buffer contained 1% (v/v) Triton X-100. Sonication of the mitochondria was by diluting the mito­ chondria as for osmotic lysis then sonicating the suspension inmediately. Following treatment, the lysed mitochondria were centrifuged at 20,000 x g for 15 minutes. Aliquots of the supernatant and pellet fresuspended in 0.5 ml of the dilution buffer) were assayed for dGuo kinase activity as described in Methods. The results represent the average of two experiments.

Percent Lysis Fraction Total Activity

Osmotic Supernatant 11 Pellet 89

Triton X-100 Supernatant 96 Pellet 4

Sonication Supernatant 96 Pellet 4 108 supernatant fraction represents the crude mitochondrial extract (Fraction

I). This procedure invariably produced 2 1 of Fraction I, containing approximately 14,000 units of dGuo kinase activity. When stored at -20WC the enzyme remains stable for several months. Isolation of dGuo kinase from this material was accomplished by cycling from 250 to 300 ml aliquots through the six purification steps described below.

DFAF-Sephacel Chromatography

Fraction I (258 ml) was divided into two equal portions and each dia- lyzed simultaneously against 4 1 of OM buffer in Spectrapor ffembrane No.

2 for 24 hours. Conductivity and pH were monitored to insure equilibration had been achieved. The dialyzed mitochondrial extract was applied to a

DHAE-Sephacel column (2.5 x 20 cm) equilibrated with OM buffer. After the fraction had entered the bed, the column was eluted with 1 1 of OM buffer, then with a linear 0 to 0.35 M KC1 gradient (1 1) in OM buffer at a flow rate of 60 ml/hour, collecting 15 ml fractions. As shown in Figure 18, dGuo kinase activity eluted from the column at approximately 0.1 M KC1 as a single sharp peak. Active fractions were pooled to yield 246 ml of

Fraction II.

Thermodenaturation at 60°C

Fraction II was placed into a 250 ml Frlenmeyer flask, then ATP‘Mg was added to give a final concentration of 10 m'h The flask was sealed, then incubated in a 37*C water bath until the temperature of the protein solu­ tion reached 37“C. Then the flask was placed into a 60°C water bath and incubated 5 minutes with constant stirring. The flask was then placed into an ice bath, and incubated with constant stirring until the temperature of the protein solution was less than 10oC, Sediment was removed by centri­ fugation at 20,000 x g for 30 minutes. The supernatant (Fraction III) was Fraction 18. Elution Profile of Bovine Liver Mitochondrial dGuo Kinase From DFAF-Sephacel. Conditions for column were as described in Results. Deoxyguanosine kinase C#” *0) was assayed as described in Methods, except the preincubation was omitted. Protein CO O) was measured by absorbance at 280 nm. KC1 concentration (------) was monitored by measurement of conductivity,

109 o Gn O u P M GJ 280 r o P kcT* fO KJ O * o — . IO G J O O O O ENZYME ACTIVITY [UNITS/ML] O ro o Oi ND o O oo O O FRACTION on Ill concentrated via ultrafiltration (Amicon, YM-10 membrane) to a final volume of 14 ml.

Sephacryl S-200 Chromatography

The Fraction III concentrate was applied to a Sephacryl S-200 (Super­ fine) column (2.5 x 145 cm) equilibrated with 100 mM Tris/HCl, pH 8.O4 buffer containing 100 mM KC1, 10 mM 2-mercaptoethanol, 1.0 mM EFTTA, 0.05 mM FHSF and 10? glycerol. The column was eluted with the same buffer at a flow rate of 30 ml/hour, collecting 12 ml fractions. The dGuo kinase elution profile appeared as a single sharp peak (Figure 19). Active fractions were pooled and concentrated by ultrafiltration (Amicon, YM-10 membrane) to a final volume of 17 ml (Fraction IV).

Blue Sepharose CL-6B Affinity Chromatography

Fraction IV was dialyzed against 4 1 of 50 mM Tris/HCl, pH 7.3^ buffer containing 10 mM 2-mercaptoethanol, 10% glycerol, 10 mM MgCl2 and 0.05 mM

ET1SF (Buffer A) for 12 hours in Spectrapor No. 2 membrane. Following dia­ lysis, the fraction was applied to a Blue-Sepharose CL-6B column (2.5 x 11 cm) equilibrated with Buffer A. The column was washed with 600 ml of

Buffer A, then dGuo kinase was eluted from the column with 1.2 1 of 50 itM Tris/HCl, pH 7.34 buffer containing 10 m M 2-mercaptoethanol, 10% gly­ cerol, 0.05 mM FHSF, 1.0 mM FETA and 1.0 mM ATP (Buffer B). Exhaustive elution of the column was required for maximum recovery (50%), since con- * siderable trailing of activity results at this ATP concentration. Evidence of trailing is observed in Figure 20, which shows the elution profile of dGuo kinase from a Blue Sepharose column under similar conditions, but where half the normal volume of Buffer B was used to elute the activity.

After washing with 20 column volumes of Ruffer B, activity remained on the column and could be recovered by further elution with Buffer C (Buffer Figure 19. Flution Profile of Bovine Liver Mitochondrial dCuo Kinase From Sephacryl S-200. Conditions for the column were as described in Results. Deoxyguanosine fcinase (+ was assayed as described in Methods, except the preincubation was omitted. Protein (O ----- O ) was measured by the method of Bradford as described in Methods.

112 ENZYME ACTIVITY [ u /ml]

—• K> W K tft I------1------1------1------1------r

PROTIEN (MG/ML] Figure 20. F.lution Profile of Bovine Liver Mitochondrial dCuo Kinase From Blue Sepharose CL-6R. Conditions for the column were as described in Results. Buffers were: (A) , 50 mM Tris/HCl, pH 7.34, 10 mM VgCl2 , 10 nM 2-mercaptoethanol, 10S glycerol and 50 uM PMSF; (B) 50 mM Tris/ HC1, pH 7 .3 4 , 10 mM 2-mercaptoethanol, 10% glycerol, 50 wM PMSF, 1.0 11M EDTA and 1.0 mM ATP; (C), Buffer B, except contains 10 #1 ATP. Assay for dGuo kinase was as described in Kfethods, except preincubation was omitted.

114 115

O

CQ FRACTION

O

CO CN (TW /n) AllAliDV 3WAZN3 116

B containing 10 nfrt ATP). Nevertheless, elution of dGuo kinase from the

Blue Sepharose column is achieved with 1.0 mM ATP; this elution effect seems to be specific since the enzyme is not eluted with buffers containing

KC1 of similar ionic strength. Numerous attempts to use dGTP in place of

ATP as an elutriant were unsuccessful.

The 1.0 mM ATP eluate was concentrated by ultrafiltration (Amicon,

YM-10 membrane) to a final volume of 17.5 ml (Fraction V).

Sephacryl S-200 Chromatography

Fraction V was applied to the same Sephacryl S-200 (Superfine) column described above, and the column was eluted under the same conditions.

Active fractions were collected and concentrated as described previously to a final volume of 11.1 ml (Fraction VI). Fnzyme prepared in this manner was used in many of the characterization studies because of the relatively large quantity of enzyme present and because the enzyme remained stable during extended periods of incubation at 4°C.

Preparative Pise PAGE

Additional purification of the dGuo kinase was achieved using prepara­ tive disc PAGE. Two polyacrylamide slab gels were prepared as described in *tethods for the analytical disc PAGE system, except the formation of channels in the stacking gel was omitted. A 2.0 ml aliquot of Fraction

VI was diluted with 0.8 ml of the sample buffer as described in Methods, 0 then 1.4 ml of the mixture was applied to the top of each gel. Electro­ phoresis was conducted as described for the analytical system, except the

Ortec double slab buffer chamber and power supply were used. Following electrophoresis, the gels iNfere removed from the glass cells and the dGuo kinase activity located by cutting a 0.25 cm wide slice from each side of the slab. One of the gel slices was stained for protein and the other 117 assayed for dGuo kinase as described in Methods. Once the activity was located, a 4 mm wide band containing activity was cut from the gel. The two horizontal slices were minced into small pieces, placed in 5.0 ml of

OM buffer containing 1.0 mM ATP, and incubated at 4°C overnight. Elution of the gel was repeated two more times but with 2 hour incubation periods.

The eluates were pooled and concentrated via a MicroProDiCon to a final volume of 1.8 ml. The entire Fraction VI was processed in this manner to yield 8.9 ml of Fraction VII. The enzyme fraction was stored in 50% gly­ cerol at -20°C. After two months under these conditions this enzyme pre­ paration retained 80% of its activity. However, at 4°C the preparation lost 50% of its activity after one hour.

A summary of the purification scheme is presented in Table 11. Deoxy- guanosine kinase was purified, 1,857-fold from bovine liver, tinder optimal assay conditions the average specific activity of the final fraction was

141 units/mg protein. Electrophoresis of the native enzyme (Fraction VII) by disc PAGE, using 7% acrylamide in the separating gel, demonstrated a single band of protein migrating congruently with dGuo kinase activity

(Figure 21). Similar results were observed when the enzyme preparation was subjected to isoelectric focusing (pH 5-7), continuous PAGE at pH 7.0 and disc PAGE using 5% or 9% acrylamide in the separating gel. However, after disc PAGE using acrylamide concentrations of 11% or higher in the separating gel, the dGuo kinase activity was observed to migrate with a very faintly stained protein band moving slightly faster than a second more prominent band. This suggests that the dGuo kinase activity in the preparation represents only a small portion of the total protein. .Several attempts were made using alternate chromatographic steps to remove the Table 11.

Purification of Bovine Liver Mitochondria] dGuo Kinase.

Specific Vo lime Activity Protein Activity Yield Fold Fraction (ml) (U/ml) (mg/ml) (N/mg) (4) Pure

I Crude Mitochondrial 258 7.0 40 0.175 100 - Extract

11 DLAL-Sephacel 246 5.8 8.9 0.652 80 4.1

III Thermodenaturat ion 253 4.7 2.8 1.679 66 9.6

IV Sephacryl S-200 17 62.3 10 6.230 60 35.6

V Blue Sepharose 17.5 31.8 0.63 50.47 31 288

VI Sephacryl S-200 11.1 48.8 0.64 76,25 30 436

VII Preparative Oise PAGF. 8.9 7.9 0.056 141.1 4 806a

aTliis represents a 1,857-fold purification from the Crude Liver Ibroogenate Figure 21. Analytical Disc PAGE of Bovine Liver Mitochondrial dGuo Kinase. Conditions were as described in Methods. Enzyme was 7 ug of Fraction VII. The top panel shows the gel stained with Coomassie Brilliant Blue; direc­ tion of migration was left to right. The bottom panel shows the enzymatic activity in slices of a duplicate gel.

119 nmols d G M P

OZI 121

contaminating protein from the enzyne, but with unsatisfactory results.

Preparative disc PAGE using 12% acrylamide in the separating gel was found unsatisfactory because of low yields and inability to excise the en2vme completely from the contaminating protein.

Additional evidence for inhomogeneity was observed when Fraction VII was subjected to SDS electrophoresis. As shown in Figure 22, most of the protein was separated into two components, with average molecular weights of 32,300 and 34,700 (Figure 23). Whether or not one of these components is the dGuo kinase could not be determined. As a result, a reasonably accurate determination of enzyme purity was impossible.

4. Properties of Peoxyguano sine Kinase From Bovine Liver

a. Frizyme Assay

Both the calf thymus and bovine liver mitochondrial dGuo kinase exhibit hysteretic behavior as defined by Frieden (136). Lfnder routine assay con­ ditions where the reaction is initiated by adding enzyme, the formation of dGMP as a function of time undergoes an initial lag period before the steady-state rate of phosphorylation is attained. The lag was exhibited by both enzymes at all levels of purification.

The time dependence of dGuo phosphorylation as a function of bovine liver dGuo kinase concentration is presented in Figure 24. All of the time course curves show an initial lag period followed by an increase in rate until a maximal velocity is reached. The value of the lag time is deter­ mined by extrapolating the linear portion of the time curve back to the time axis. From this it can be seen that the lag t^ values range from 2 to 5 minutes. The dependence of the lag time on enzyme concentration can be more clearly observed from the data replotted in Figure 25. Over the Figure 22. Photometric scan of an SDS PAGF Gel of Bovine Liver dGuo Kinase (Fraction VII). Conditions for electrophoresis and staining were as described in Methods. Fnzyme was 6 yg of Fraction VII. The gel was scanned at 1.0 cm/minute using a Gilford Model 240 Spectrophotometer equipped with a Linear Transport Model 2410. .05

A 560 0.1 O BOTTOM TOP 123 Figure 23. ftolecular Weight Determination of the Two Major Protein Components in Fraction VII By SDS PAGF. Conditions for electrophoresis were as descrihed in Methods. The arrows indicate the Rf values of the major protein components.

124 12S

4.7

• Ovalbumin 4.6 • GPDH

4.5

. Chymotrypsinogen •'

4.3 ApoferrMin •- M yoglobin 4.2

0 0.20.4 0.6 0.8 1.0 Figure 24. Reaction Time Course of Bovine Liver Mitochondrial dCuo Kinase: Effect of Varying Fnzyme Concentration on Length of Lag Period. Conditions were as described in Methods, except preincubation was omitted and the reaction was initiated by the addition of en2yme (Fraction VI) giving a final concentration of 53.5 ug/ml (O) . 27 Mg/ml (•) • 13.5 Mg/ml (A), and 11 ug/ml (A) in the assay.

126 nmols dGMP 14 18 16 4 A ] 6 8 5 O 5 20 15 JO 5 0 MINUTES 0 3 50 127 Figure 25. Effect of Varying Enzyme Concentration on the Length of the Lag Period. These data are replotted from Figure 24.

128 to LAG TIME (MIN) LAGTIME K C/1 O N oo «o Ch o o CO o o o [E]o (jjg/ML) 130

5-fold range of dGuo kinase concentration tested, it can be seen that as enzyme concentration increases, the lag time decreases. This relation­

ship is characteristic of enzymes demonstrating an alteration in aggre- gational state, i.e. a transition occurs during the assay period from a

less catalytically active form to one that is more active (136). This effect is presumably induced by one or more components in the assay mix­ ture. As shown in Figure 26, the lag period could be completely eliminated by preincubating the dGuo kinase with 16 mM ATP or 16 mM ATP-Mg at 37 *C

for 30 minutes. However, preincubation with ATP*Mg substantially reduced the reaction velocity. Preincubation of the enzyme with 0.13 mfi dGuo or

16 nM MgCl2 did not eliminate the lag period. Preincubation with MgCl2 did, however, reduce the reaction velocity to a level similar to that observed after preincubation with ATP*Mg. While ATP preincubation under these conditions eliminated the lag, it did not alter the steady-state rate of phosphorylation of dGuo as seen by comparing the linear portion of the non-preincubated and ATP-preincubated curves.

With ATP preincubation the phosphorylation of dGuo by the bovine liver dGuo kinase was linear up to 301 conversion of the substrate to dG*fP, and for at least 60 minutes at 37°C (Figure 26). Similar results were obtained after incubation for 30 minutes at 37°C whether utilizing the standard

DE-81 disk assay or by measuring product isolated from the assay by paper chromatography (Figure 27), In addition, the rate of formation of dGf^tP by dGuo kinase was linear with respect to enzyme when the concentration was varied over 6-fold (Figure 28).

The purified bovine liver mitochondrial dGuo kinase produced a single radiolabeled product from [3Hl-dGuo, one which co-migrated with 5*-dG',P Figure 26. Effect of Preincubating the Bovine Liver Mitochondrial dGuo Kinase With Various Components of the Assay Mixture on the Reaction Time Course. Prior to assay the enzyme (3.2 ug of Fraction VI) was incubated at 37°C for 30 minutes in 95 mM sodium acetate buffer, pH 537, 13 mM DTE, 0.675 (v/v) Triton X-100 and where indicated 16 mM ATP, 16 mft MgCl2 , 16 itM ATP-Mg or 0.13 mM [3Hl-dGuo (0.1-0.5 uCi/assay) in a total volume of 0.15 ml. The assay ivas initiated by the addition of the absent component(s) to bring the final volume to 0.2 ml and concentration of components to that described in Methods. The final MgCl2 concentration was 10 mM. At the time intervals indicated 0.02 ml aliquots of the reaction mixture were removed and placed in 0.12 ml of 0.1 N formic acid to terminate the reaction. These aliquots were then processed as separate assays as described in *fethods.

131 nmols dGMP (a) U i O V| T “T T 132 Figure 27. Reaction Time Course of Bovine Liver dGuo Kinase: Comparison of DE-81 Desk Assay Method With Chromatographic Quantitation of Product. Assay was with 0.56 iig of Fraction VI as described in Methods with the following modifications. Two identical assays were performed each with a final volume of 0.2 ml. At the time intervals shown 0.02 ml aliquots were removed, and the reaction was terminated by addition of 0.12 ml of 0,1 N formic acid. The aliquots from one reaction were processed by the DE-81 disk method (O) and the second set by paper chromatographic separation of dGuo from dGffP (System 1, 'table 4) (A)* 134 3 0 20

15 MINUTES

dWOP spiuu Figure 28. Dependence of Bovine Liver Mitochondrial dGuo Kinase Activity on Enzyme Concentration. Assay conditions were as described in Methods» except the concentration of enzyme (Fraction VI) was varied as indicated and incubation was for 15 minutes.

135 O' CO nmoles dGMP/15min

PROTEIN (JJ9> 9£I 137 in an ion exchange paper chromatography system which separated 5*-Off* from 5'-dCMP (Figure 29). This product was completely reconverted to dGuo upon digestion with purified venom 5'-specific nucleotidase but not by digestion with 3*-specific nucleotidase from rye grass (Figure 30).

Preliminary investigations conducted to determine the nature of the phosphorylation reaction provided the following results (Table 12):

(1) The reaction would not proceed in the absence of ATP or MgCl2.

(2) TTie conversion of 0.1 mM [3H]-dGuo is relatively insensitive to the addition of 0.5 mM guanine, adenine or hypoxanthine.

(3) Omission of DTE from the reaction resulted in a lower velocity and the addition of Hg+2 to the assay in the absence of ETE inhibited the reaction.

Hicse findings, plus the fact that the product of the phosphorylation reaction is 5*-dCMP and not 5'-GMP, indicate the reaction is mediated by a sulfhydryl sensitive kinase, and not by the sequential actions of a nucleoside phosphorylase and a phosphoribosyl transferase.

b. Analysis of the Hysteretic Effect

The activation of the catalytic process by ATP was found to be dependent upon the temperature, time and ATP concentration used in preincubation.

Figure 31 shows that incubation with 16 mM ATP at 4°C for up to 60 minutes did not remove the lag. However, after 30 minutes at 37°C the lag was * eliminated, and the enzyme remained in the activated state even after an additional 60 minutes incubation at 40C. However, when ATP is removed from the activated form of the enzyme by rapidly passing the preincubated preparation through a Sephadex G-25 column, the enzyme reverts back to the inactivated state (data not shown). Figure 32 reveals that when the enzyme Figure 29. Identification of the Tritium-labeled Product of the Bovine Liver dGuo Kinase Reaction By Ton Exchange Paper Chromatography. Con­ ditions of assay and paper chromatography (system 5, Table 4) were as described in Methods. 0.025 ml of the terminated reaction mixture was applied to the chromatogram in 5 pi aliquots. dGuo and dGMP markers were chromatographed along with the reaction aliquot and are located in the positions shown by the arrows.

138 GMP dGMP dGuo

* <

0.2 Figure 30. Identification of the Phosphorylated Deoxyguanosine Product of the Bovine Liver dGuo Kinase Reaction. The dGuo kinase reaction was conducted as described in Methods. Aliquots (0.04 ml) of the terminated reaction were neutralized and 0.025 ml chromatographed using system 1, Table 4 as described in Methods, after no treatment (Panel A), incuba­ tion with 3f-nucleotidase (Panel B) or incubation with S’-nucleotidase (Panel C). Conditions for the nucleotidase incubations were as described in references (170,177). dGuo and dGMP markers were chromatographed along with the reaction aliquot and are located in the positions shown by the arrows.

140 141

dSJo

dGMP

0.6 0.7 0.S 0.9 1.0 142

Table 12. Properties of the Bovine Liver Mitochondrial dGuo Kinase Reaction. The complete reaction mixture was as described in Methods. Deletions and additions to the assay were as indicated. Enzyme was 0.11 ug of Fraction VI.

Conditions Relative Activity, %

Complete 100a

minus ATP 0

minus MgCl2 0

minus DTE 76

minus DTE plus 0.1 mM HgCl2 69

minus DTE plus 1.0 mM HgCl2 0

plus 0.5 mM Guanine 114

plus 0.5 mM Adenine 101

plus 0.5 mM Hypoxanthine 103

a0.92 nmols dGMP formed/30 minutes Figure 31. Reaction Time Course of Bovine Liver Mitochondrial dGuo Kinase: Temperature Dependence of the Lag Phenomenon. Conditions were as indicated in Methods, except: enzyme was preincubated with ATP at 37*C for 30 minutes (O); enzyme was preincubated with ATP at 37°C for 30 minutes followed by incubation at 4°C for 60 minutes (®); enzyme was preincubated with AtP at 40C for 30 minutes (A); enzyme was preincubated with ATP at 4°C for 60 minutes (A)- Enzyme was 3.2 yg of Fraction VI.

143 nmols dGMP MINUTES 0 3 0 5 144 Figure 32. Reaction Time Course of Bovine Liver Mitochondrial dGuo Kinase: Effect of Varying Preincubation Time With ATP on the Length of the Lag Period. Conditions were as described in Methods except preincubation of the enzyme with 16 mM ATP was for 60 minutes (Q), 30 minutes (A)» IS minutes (A). 5 minutes (Q) and none (O)* Enzyme was 3.2 Mg of Fraction VI.

145 nmols dGMP 147 is incubated with 16 mM ATP at 37°C from 5 to 60 minutes, optimal activa­ tion occurs after 30 minutes. When the enzyme was preincubated at 37°C for 30 minutes and the ATP concentration varied from 0.1 to 10 mM, it was determined that 1.0 mM ATP, under these conditions, was sufficient to bring about complete activation (Figure 33).

The catalytic activation process is apparently mediated by ATP and not by some degradative product of ATP. Assay for ATPase activity revealed the complete absence of this activity in the enzyme preparation (Fraction

VI and VII). A more sensitive analysis of the integrity of the ATP, after incubation for 30 minutes at 37 °C, was conducted using both carrier and carrier-free {3H]-ATP. After standard preincubation, the depro- teinized solution was subjected to ion exchange paper chromatography and paper electrophoresis. As shown in Figure 34 and 35, no evidence for

ATP hydrolysis products could be observed, even after incubation with 32 Mg of Fraction VI which is 10 times the amount used under normal assay con­ ditions (such as in Figure 26). Comparison of these results with controls

(incubation mixture less protein) indicated no significant change in ATP concentration had occurred.

A series of studies were conducted in which dGuo kinase was preincu­ bated with various ligands in order to determine if any other metabolites might bring about activation. A summary of the results of this study is * presented in Table 13. Besides ATP and ATP*Mg, both dGTP and CTP eliminate or significantly decrease the lag period, suggesting the effect may be a generalized nucleotide triphosphate effect (Figure 36). As expected, ADP,

AMP and cAMP had no effect on the lag period. Surprisingly, neither the neither the ATP analogue AMPPCP (Figure 37) nor the product of the reaction, Figure 33. Reaction Time Course of Bovine Liver Mitochondrial dGuo Kinase: Effect of Varying ATP Concentration in the Preincubation Mix­ ture on the Lag Period. Conditions were as indicated in Methods, except the concentration of ATP in the preincubation mixture was varied as follows: 0.1 mM (#); 1.0 mM CO); 5.0 itM (A); 10 mM (A)* Fnzyme was 3.2 vg of Fraction VI.

148 nmols dG M P 5 0 5 0 2 15 10 / / MINUTES 0 3

0 4 149 Figure 34. Analysis of ATP By Ion Exchange Paper Chromatography After Incubation With Bovine Liver dGuo Kinase. Enzyme (2.8 ug, Fraction VI) was incubated with 95 rnM sodium acetate buffer, pH 537, 13 nW DTE, 0.671 (v/v) Triton X-100 and 13 mM [3H]-ATP (0.1 yCi/assay) or carrier-free in a total volume of 0.15 ml for 30 minutes at 37°C. After incubation a 0.02 ml aliquot was removed and placed in 0.12 ml of 0.1 N formic acid and mixed. After centrifugation at 1,500 x g for 10 minutes 0.01 ml of the supernatant was chromatographed, descending on DE-81 ion-exchange paper in 0.1 M ammonium formate, pH 3.1. After development the chroma­ togram was dried and processed as described in Methods.

Figure 35. Analysis of ATP By Paper Electrophoresis After Incubation With Bovine Liver dGuo Kinase. Incubation was as described in Figure 30, except 32 ug of Fraction VI was used with 16 nM [3H]-ATP (0.3 uCi/assay). After termination of incubation, 0.01 ml of the supernatant was placed on a 18 x 58 cm strip of Orange Ribbon paper and electrophoresis was per­ formed in 0.1 M anmonium formate, pH 2.7 at 2 kV (2 mA) for 1 hour. After the run the paper was dried and processed as described in Methods.

150 1S1

ATPAMP

CARRIER

ATP ADP AMP

U 5 CARRIER- FREE

0.1 0.2 0.3 0.4 0-5 0.6 0.7 0.9

IS

16

14

12

10

8

6

4

2 152

Table 13. Sunmary of Results From Bovine Liver Mitochondrial dGuo Kinase Reaction Time Courses: Effect of Preincubation of the Enzyme With Various Agents. Conditions were as described in Methods, except additions to the preincubation mixture were as indicated.

V£ Addition Lag Observed Relative to Control, °a

None Yes 100

ATP (5 mM) No 100

ATP*Mg (20 mM) No 70 dGuo (0.2 mM) Yes 100

MgCl2 (5 mM) Yes 72 dCMP (1 mM) Yes 100

ADP (1 mM) Yes 100

AMP (1 mM) Yes 100 cAMP (1,5 mM) Yes 100, 100

CTP (5 mM) Yesb 100 dGTP (0.01 m ) No 48

AMPPCP (5,16 mM) Yes 90, 75 dTDP (1 mM) Yes 4

CaCl2 (5 mM) Yes 65

EETA (1,5 i*l) Yes 100, 100

Vf corresponds to maximal velocity after lag period; control corresponds to reaction where enzyme was preincubated 30 minutes in buffer only blag period was significantly reduced Figure 36. Reaction Time Course of Bovine Liver Mitothondrial dGuo Kinase: Effect of Preincubation of Enzyme With Various Ligands. Con­ ditions were as described in Methods, except enzyme was 4.S pg of Fraction VI and the preincubation mixture contained 5 mM ATP (#), 5 mM CTP (A), 1 mM d(M> (A), 0.01 mM dGTP (Q) and none (Q) •

153 154

5

4

3 1 dGTP

0 MINUTES Figure 37. Reaction Time Course of Bovine Liver Mitochondrial dGuo Kinase: Effect of Preincubation of Enzyme With AMPPCP. Conditions were as described in Methods, except 30 mM MgCl2 was used in the assay and pre­ incubation, in addition to buffer, contained 5 mM ATP (A), 16 mM ATP (A), S mM AMPPCP (□), 16 mM AMPPCP (0) and none (Q).

155 10 15 20 25 30 MINUTES 157 dCMP, (Figure 36), affected the lag period to any great extent.

The observations that (1) the lag time is inversely proportional to the dGuo kinase concentration, (2) preincubation of the enzyme with ATP eliminates the lag period, and (3) removal of ATP from preincubated enzyme by gel filtration restores the lag period, strongly suggest that dGuo kinase must be activated by ATP and this activation process must include a change in aggregational state of dGuo kinase. Observations by others with other enzyme systems with similar properties suggest that conformational change alone is unlikely (136,142). Therefore, gel fil­ tration and glycerol gradient sedimentation studies in the presence or absence of ATP were conducted to determine if an alteration in the size of the dGuo kinase occurred after preincubation with ATP. The elution position of activated and non-activated dGuo kinase from a Sephacryl S-200 column (1.5 x 65 cm) at 4°C and 22*C in the absence and presence of 5 mM ATP did not change (data not shown). Similarly, the sedimentation pat­ tern of the activated or non-activated enzyme in 10-30% glycerol gradients in the absence or presence of 5 nM ATP did not change significantly

(Figure 38). As shown in Figure 38, the enzyme sedimented as a single symmetrical peak under all conditions tested, with peak fractions located no more than one fraction apart. The results of these studies seem to argue that the mechanism for the hysteretic effect does not involve a stable change in the aggregational state of the enzyme, at least at 4° or at room temperature.

c. ATP-Mg Optima

The effect of varying the MgCl2 concentration on the bovine liver mito­ chondrial dGuo kinase activity at fixed ATP concentrations is shown in Figure 38. Glycerol Gradient Centrifugation of Bovine Liver Mitochon­ drial dGuo Kinase. Preparation of gradients, centrifugation and assay were as described in Methods. Fraction VI protein (32 yg) was incu­ bated in 100 mM Tris/HCl, pH 7.3^, 10 mM 2-mercaptoethanol with or without 5 mM ATP for 30 minutes at 37°C. After cooling the treated protein solutions (0.1 ml) were applied to glycerol gradients (10-30%, v/v) containing 100 mM Tris/Hcl, pH 7.34, 1° nM 2-mercaptoethanol, with or without 5 mM ATP. Specific conditions: enzyme incubated without ATP, sedimented in gradient without ATP (O) and with ATP ( ^ ) ; enzyme incubated with ATP, sedimented in gradient without ATP (#) and with ATP (A)* Ovalbumin and BSA (2 mg/ml) were applied in 0.1 ml to separate gradients in the same buffer and gradients without ATP. Their posi­ tion in the gradient was monitored by absorbance at 280 nm.

158 ACTIVITY CU/ML) 2 TOP BSA A O BOTTOM ^

6s+s6=Aa4=4 159 160

Figure 39, When the concentration of either ATP or MgCl2 greatly exceeds

that of the other, an inhibitory effect is observed. At 5, 10, 15 or 20 mM ATP maximal velocity accurs when the ratio of ATP to Mg+2 is about 1:3.

In all cases, except for 15 mM, as the ATP concentration increased so did

the peak activity. For routine assays of the dGuo kinase 10 mM ATP and

30 nM MgCl2 were used in the reaction mixture. However, for extended time courses (60 minutes) the MgCl2 concentration was decreased to 10 mM, since at higher concentrations an inhibitory effect on the enzyme was observed after 30 minutes reaction time.

d. Phosphate Donor Specificity

Hie relative effectiveness of various ribo- and deoxyribonucleoside triphosphates to function as phosphate donors in the bovine liver dGuo kinase reaction is listed in Table 14. Maximal rate of reaction was obtained with ATP, with CTP and dITP being the only other effective donors, exhibiting 61$ and 45$ of the activity of ATP, respectively. Unlike other deoxynucleoside kinases which demonstrate a broad specificity with regard to phosphate donor (Table

3), the mitochondrial dGuo kinase is relatively specific, with nucleotides

such as GTP, dATP and UTP showing very little activity. dGTP and dITP are not phosphate donors to dGuo since they are both potent inhibitors of the mitochondrial dGuo kinase. Table 14 also shows the inability of AMP, ADP

and sodium pyrophosphate to serve as donors, confirming that phosphoryla-

4 tion is mediated by a kinase. .

e. Divalent Cation Specificity

The mitochondrial dGuo kinase was able to utilize *(n+^ and Ca+2 as

cations in partial replacement of Mg+2 (Table 15). However, Fe+2, Co+^,

Zn+2 and Cu+2, which are cations usually found to most effectively replace

Mg+2 for other deoxynucleoside kinases, were ineffective with the Figure 39. Effect of MgCl2 Concentration on the Bovine Liver dGuo Kinase Activity at Several Fixed ATP Concentrations. Assay conditions were as described in Methods, except the ATP and MgCl2 concentrations were varied as indicated. Enzyme was 1.3 ug of Fraction VI.

161 r

[ATP]1! (mM) I 20 1

A 15

• 5 1 1 0 1

o 10 20 30 40 50 60 MgCI2 [mM] 163

S Table 14. Phosphate Donor Specificity of the Bovine Liver Mitochondrial dGuo Kinase. Assay conditions were as described in Methods, except that preincubation was omitted and where indicated other nucleotides were substituted for ATP (10 mM). Enzyme was 1.1 ug of Fraction VII.

Donor Relative Activity, %

ATP 100a

CTP 61

dTTP 45

dCTP 29

UTP 24

I TP 21

GTP 10

dATP 5 dGTP 1

dITP X 1

AMP Z1

ADP ..'1

Sodium pyrophosphate 1

a100% activity corresponds to 3.2 nmols dGMP fonned/30 minutes 164

Table 15. Divalent Cation Specificity of the Bovine Liver Mitochondrial dGuo Kinase. Assay conditions were as described in Methods, except where indicated MgCl? (30 mM) was replaced by other divalent cations. Enzyme was 1.3 ug of Fraction VI.

Metal Relative Activity, %

MgCl2 100

MgS04 76

MnCl2 62

MnSo^ 31

CaCl 2 44

FeS04 5

FeCl2 0

ZnS04 0

CoS04 0

CuSo4 0

CuCl2 0

a100% activity corresponds to 0.75 nmols dCMP formed/ 30 minutes 165 mitochondrial dGuo kinase (54,83,87). A comparison of the chloride and sulfate salts of Mg+2 and Mn+2 demonstrated that for both cations the chloride salt form was significantly more active.

f . Phosphate Acceptor Specificity

The ability of the purified bovine liver dGuo kinase to utilize var­

ious nucleosides and deoxynucleosides as phosphate acceptors was inves­ tigated at two concentrations, 0.02 mM and 0.5 mM. The results are shown

in Table 16. Analogous to the results observed for the calf thymus mito­ chondrial dGuo kinase the bovine liver mitochondrial enzyme is very

specific, phosphorylating dGuo and dlno exclusively. In contrast, the purified bovine liver enzyme is unable to phosphorylate Guo. This high degree of specificity for dGuo resembles that reported for dGuo kinase

activity isolated from porcine and neonatal mouse skin (130,131).

In the case of crude bovine liver mitochondrial extracts, the dAdo

kinase activity which co-migrated with dGuo kinase activity after analy­

tical disc PAGE (Figure 5) was separated from the dGuo kinase after the

DEAE-Sephacel chromatography step. The observations that (1) Ado kinase

(known to phosphorylate dAdo) also co-migrates with these activities after

analytical disc PAGE, and (2) Ado kinase activity is largely removed by

the DEAE-Sephacel chromatography step suggest that the phosphorylation of

dAdo in these disc PAGE studies is mediated by Ado kinase.

Efforts to determine if the enzyme's deoxynucleoside specificity

changed, i.e. ability to phosphorylate dAdo, in the presence of the effec­

tors dGTP anddTPP failed to demonstrate any alteration in acceptor

specificty.

Because dlno is not a naturally occurring deoxynucleoside its phos­

phorylation by the mitochondrial enzyme is thought to be due to its 166

Table 16. Nucleoside Specificity of the Bovine Liver Mitochondrial dGuo Kinase. Assay conditions were as described in Methods, except various nucleosides were substituted for dGuo at 0.02 nW and 0.50 mM as indicated. Enzyme was 1.1 ug of Fraction VII.

Nucleotide Synthesized Nucleoside 0.02 nM 0.50 mM

pmols

dGuo 429 3,320

dlno 25 980

dAdo 0 0

dCyd 0 0

dThd 0 0

Guo 0 0

Ino 0 0

Ado 0 0

Cyd 0 0

Urd 0 0 167 structural similarity to dGuo. Thus the enzyme's ability to phosphorylate dGuo and dlno but not Guo or Tno indicates the importance of the deoxyri- bose moiety in substrate recognition. However, its ability to phosphory­ late dGuo and dlno but not dAdo indicates a strict requirement for a keto group at the purine C-6 position, but a lack of specificity at the purine

C-2 position.

g. pH Optimum

The effect of pH on the rate of bovine liver mitochondrial dGuo kinase was measured over a pH range of 4.1 to 11.5. As shown in Figure 40, the enzyme exhibits a sharp well defined optimum at pH 5.0. When assayed at pH values above or below the optimum a precipitous decline in activity is observed. This is in contrast with other deoxynucleoside kinases which generally exhibit broad plateaus of activity with optimum values ranging from 6 to 10 (83,87,119). Nevertheless, this value, is in good agreement with the pH optimum of 5.2 reported for the neonatal mouse skin dGuo kinase (131).

h. Effect of Triton X-100

When Triton X-100 is added to the dGuo kinase assay mixture a signi­ ficant increase in reaction rate results (Figure 41). When Triton X-100 was added to the reaction mixture at concentrations ranging from 0.01% to 3.0% (v/v), a marked increase in kinase activity was observed with maximal stimulation occurring at 0.1%. Preincubation of the dGuo kinase

(Fraction VI) with Triton X-100 at 37°C produced a similar level of stimulation as observed when the detergent was added directly to the assay

(Figure 42). As shown in Figure 42, while the Triton X-100 increases maximal velocity its presence did not eliminate the lag period observed in the reaction time course, whether preincubated with the enzyme or added Figure 40. Effect of pH on the Activity of the dGuo Kinase From Bovine Liver Mitochondria. All buffers were made to a constant ionic strength. Buffers used were as follows: pH 4.1 - 5.8, sodium acetate; pH 5.6 - 7.2, KH2P04/Na2HF04 ; pH 7.3 - 8.5, Tris/HCl; pH 9.0 * 11.4, sodium glycinate. Enzyme was 2.6 pg of Fraction VII and assay was as described in Methods, except the final buffer ionic strength was 0.05 and the reaction was terminated by heating for 2 minutes in 100°C water bath. The pH value of reactions was measured after a parallel control reac­ tion (less [3H]-dGuo) was terminated.

168 TT

0 0— o— o 169 8.0 9.0 10.0 11.0 pH Figure 41. Effect of Varying Concentrations of Triton X-100 on the Bovine Liver Mitochondrial dGuo Kinase. Assay was as described in Methods, except the concentration of Triton X-100 was varied in the reaction as indicated. Enzyme was 1.3 ag of Fraction VI.

170 171

160

140

TOO*

5 15 2 0 25 30 PERCENT TRHON X-100, (% ) Figure 42. Reaction Time Course of Bovine Liver Mitochondrial dGuo Kinase: Effect of Enzyme Preincubation at 4°C and 37°C, With or Without Triton X-100. Enzyme was 1.6 ug of Fraction VI and assay was as described in Methods, except: reaction was initiated by addition of enzyme preincubated at 48C for 30 minutes with 0.5" Triton X-100 Cv/v) ( O ) ; reaction was initiated by addition of enzyme preincubated at 4°C for 30 minutes without Triton X-100 CO) i reaction was initiated by addition of enzyme preincubated at 37°C for 30 minutes with 0.5% Triton X -100 Cv/v) added at the start of the assay CA); reaction was initiated by addition of enzyme preincubated at 37“C for 30 minutes without Triton X-100 in the assay CA); enzyme was incubated with 0.67% Triton X-100 Cv/v) for 60 minutes at 37°C and assay was initiated by the addition of ATP-Mg CO)'

172 nmols dGMP MINUTES 0 3 0 4 0 5 0 6 173 174

at the start of the assay.

In addition, the presence of Triton X-100 was attributed to increased

stability of the partially purified dGuo kinase. In one experiment dGuo kinase (Fraction VI) was inactivated over 90% upon incubation at a constant temperature of 37°C in 285 mM sodium acetate buffer pH 5*037 for 18 hours in the absence of Triton X-100. Incubation of the enzyme under the same conditions except with the addition of 0.5% Triton

■X-100 (v/v) decreased the rate of inactivation over 40%.

i. Molecular Weight Determination

i. Glycerol Gradient Centrifugation

The sedimentation pattern of the purified bovine liver mitochondrial dGuo kinase obtained by centrifugation in 10-30% glycerol gradients has been described previously (Figure 38). The position of dGuo kinase, rela­ tive to BSA and ovalbumin, after centrifugation is also shown in Figure

38. Using the method described by Martin and Ames (162), a molecular weight of 55,100 was calculated.

ii. Sephacryl S-200 Gel Filtration

The molecular weight of the purified mitochondrial dGuo kinase was estimated by gel filtration as described in Methods. By comparing the elution volume of the bovine liver dGuo kinase with that of other known protein standards, a native molecular weight was calculated to be 55,000

(Figure 43).

iii. Polyacrylamide Gel Electrophoresis

The molecular weight of the bovine liver mitochondrial dGuo kinase was determined by the method of Hedrick and Smith (179) which is based on the relationship between electrophoretic mobility and protein size defined by Ferguson (180). Molecular weight standards and dGuo kinase Figure 43. Molecular Weight Determination of the Bovine Liver Mitochon­ drial dGuo Kinase by Gel Filtration on Sephacryl S-200. 0.2 ml of Fraction V was applied to the column. The column was run at a flow rate of 15 ml/hour and 3.8 ml fractions were collected. All other conditions for the column, calibration and assay of dGuo kinase were as described in Methods. Hie arrow indicates the molecular weight of the dGuo kinase as determined by the elution volume of the enzyme.

175 11 (j

M yeglobm VyTrypim InWbitof

0 .5

0 .4

0.2

4 .0 5 .0 5 . 5 LOG MOLWT. 177 were subjected to electrophoresis as described in Methods using gel con­ centrations of 5, 7, 9 and 11? acrylamide. The Rf of each standard and dGuo kinase were then plotted against the gel concentration, and the slope of the resulting lines calculated. The slopes of the standards determined in this manner were then plotted against their respective molecular weights with the results presented in Figure 44. The molecu­ lar weight calculated for dGuo kinase from this plot is 6S,000 daltons.

The reason for the significantly higher molecular weight value deter­ mined by disc PAGE is not fully understood. Nevertheless, the values calculated from gel filtration and glycerol gradient centrifugation are, within the range of experimental error, identical. The value of 55,000 daltons is in agreement with the molecular weight value calculated by gel filtration for the calf thymus mitochondrial dGuo kinase (Figure 13).

j . Heat Lability

The time dependent heat inactivation of the partially purified bovine liver mitochondrial dGuo kinase at 37®, SO® and 65“C is presented in

Figure 45. After 30 minutes at 37®C the enzyme has lost negligible acti­ vity while at 50®C and 65®C the enzyme has lost 38? and 92? of its initial activity. A semi-log replot of the time dependent heat inactivation of the dGuo kinase at 50“C reveals the inactivation is a first order decay process (Figure 46). In the presence of 10-mM ATP the first order inac­ tivation of the enzyme at 65®C is significantly decreased, with the dGuo kinase retaining 11? activity after 30 minutes incubation. As shown in

Figure 47, in addition to ATP, dTDP, ATP-Mg and dGTP decrease the time dependent heat inactivation of the dGuo kinase at 65®C. In contrast, at

a concentration of 2 mM, dGuo or dlno had little or no effect on the

rate of heat inactivation. Of all the ligands tested the most efficient Figure 44. Molecular Weight Determination of the Bovine Liver Mitochon­ drial dGuo Kinase Using Disc PAGF.. Conditions for gel preparation and electrophoresis were as described in Methods for analytical disc PAGE, except separating gel acrylamide concentrations used were 5%, 7%, 91 and 11%. Position of protein standards and dGuo kinase in gels were monitored by staining and assay of activity, respectively, as described in Methods.

178 NEGATIVE SLOPE 10 15 • ^ Ovalbumin bsa 10

WT. 1CT x . T .W L O M DH 20 hsKrbe a PhospKorybse

179 Figure 45. Effect of Temperature on the Stability of Bovine Liver Mito­ chondrial dGuo Kinase, Enzyme samples (0.05 ml) were placed in sealed conical plastic vials and heated in a water bath at the temperature indicated for the time periods shown. Following heating, the samples were quenched on ice, centrifuged at 1,500 x g for 15 minutes, then an aliquot of the supernatant was removed and assayed for dGuo kinase activity. Assay was as described in Methods. Enzyme was Fraction IV (7.8 mg/ml) which had not been subjected to thermodenaturation at 60°C.

180 RELATIVE ACTIVITY. % o

• >

CnO

181 Figure 46. Heat Inactivation of Bovine Liver dGuo Kinase at 50°C and in the Presence of ATP at 65". Conditions were as in Figure 45, except the samples heated at 65°C contained 10 mM ATP.

182 C C 5 RELATIVE ACTIVITY RELATIVE % LOG 10 O) o £81 MINUTES Figure 47. Stability of Bovine Liver Mitochondrial dGuo Kinase at 65*C In the Presence of Various Ligands. Conditions were as in Figure 45, except the enzyme samples were heated at 65°C with the following addi­ tions: none (Q); 10 mM ATP (A); 10 mM MgCl2 (□)*. 2 mM dGuo (O); 2 mM dINO (A); 2 mM dTDP (□); 2 mM ATP (<>); 2 mM ATP* Mg ( + ); 0.03 mM dCTP ( ^ ) .

184

186 protecting agent was the distal end-product dGTP. After 30 minutes incu­ bation at 65°C in the presence of 0.03 mM dGTP, the dGuo kinase retained

76% of its initial activity. This finding is analogous to that found

for calf thymus dCyd kinase where substantial stabilization was achieved by its distal end-product dCTP (83).

k. Isoelectric Focusing

Isoelectric focusing of the crude bovine liver mitochondrial dGuo kinase (Fraction I) using broad pH range empholytes (pH 3.5-10) produced a major peak of activity at pH 5.6 (Figure 48). Focusing of the purified dGuo kinase using narrow pH range ampholytes (pH 5-7) produced a single

symmetrical peak at pH 5.75 (Figure 49). This value is very similar to

that found for the calf thymus mitochondrial dGuo kinase of pH 5.89

(Figure 12).

1. Effect of Nucleotides of Enzyme Activity

Various nucleoside mono-, di- and triphosphates were examined to deter­ mine their effect on the mitochondrial dGuo kinase reaction rate, and the

results are presented in Tables 17, 18 and 19, respectively. Similar to

the calf thymus mitochondrial dGuo kinase, the bovine liver dGuo kinase is most strongly inhibited by dGTP and dITP, and to a lesser extent by dGDP

and dGMP. The bovine liver dGuo kinase when assayed at pH 5.0, is inhibi­

ted by dTDP and UPP. In addition, both dTTP and UTP are inhibitory. Thus

the positive modulation pattern exhibited by the calf thymus enzyme, when

assayed at pH 8.0, for nucleotides with diketo pyridmine bases is a pat-

term of negative modulation for the bovine liver enzyme at the lower pH.

When the bovine liver dGuo kinase was assayed in the presence of 1.0 mM

dTDP at pH 8.0, the reaction velocity was stimulated almost 3-fold. Figure 48. Isoelectric Focusing (pH 3.5-10) of Bovine Liver Mitochondrial dGuo Kinase. Conditions for focusing were as described in Methods. Enzyme source was 240 ug of Fraction I. Assay was by the general pro­ cedure for dGuo kinase, except 0.5 M Tris/HCl, pH 8.O37 was used as buffer.

187 187

Figure 48. Isoelectric Focusing CpH 3.5-10) of Bovine Liver Mitochondrial dGuo Kinase. Conditions for focusing were as described in Methods. Enzyme source was 240 ug of Fraction I. Assay was by the general pro­ cedure for dGuo kinase, except 0.5 M Tris/HCl, pH 8.037 was used as buffer. •s 10 15 ”20 '25 30 35 SLICE NUMBER Figure 49. Isoelectric Focusing (pH 5-7) of Bovine Liver Mitochondrial dGuo Kinase. Conditions for focusing were as described in Methods. Enzyme source was 13 ug of Fraction VII. Assay was as described in Methods, except 10 mM MgCl2 and 0.5% Triton X-100 were used with preincubation omitted.

189 r ------1

------1 pH ------1 nmols dGMP nmols ------1 N> CJ ^ ------i P .O P Q r r 061 SLICE NUMBER 191

Table 17. Effects of Various Nucleoside Monophosphates on Bovine Liver Mitochondrial dGuo Kinase. Assay conditions were as described in Methods. Enzyme was 0.56 ug of Fraction VII and nucleotides were added to assay as indicated.

dGuo Kinase Nucleotide mM Relative Activity, %

None - 100

d(M> 0.1 93 1.0 75 0.1 97 1.0 92

dCMP 0.1 98 1.0 99

CMP 0.1 99 1.0 88 dAMP 0.1 99 1.0 97

AMP 0.1 91 1.0 91 dIMP 0.1 101 1.0 102 IMP 0.1 101 1.0 98 dTOP 0.1 95 1.0 93

UMP 0.1 94 1.0 84 192

Table 18. Effects of Various Nucleoside Diphosphates on-Bovine Liver Mitochondrial dGuo Kinase. Conditions were as in Table 17.

dGuo Kinase Nucleotide irM Relative Activity, '%

None “ 100

dGDP 0.1 30 1.0 4

GDP 0.1 92 1.0 59

dCDP 0.1 91 1.0 70

CDP 0.1 89 1.0 62

dADP 0.1 97 1.0 72

ADP 0.1 94 1.0 76

IDP 0.1 94 1.0 61

dTDP 0.1 13 1.0 10

UDP 0.1 10 1.0 8 193

Table 19. Effects of Various Nucleoside Triphosphates on Bovine Liver Mitochondrial dGuo Kinase. Conditions were as in Table 17.

dGuo Kinase Nucleotide nW Relative Activity, %

None - 100a

dGTP 0.1 2 1.0 <1

GTP 0.1 91 1.0 54

dCTP 0.1 96 1.0 82

CTP 0.1 97 1.0 93

dATP 0.1 80 1.0 34

dITP 0.1 8 1.0 <1

ITP 0.1 95 1.0 69

dTTP 0.1 51 1.0 40 i UTP 0.1 28 1.0 21

0 a100$ activity corresponds to 1.75 nmols dGMP formed/30 minutes 194

Therefore, for the mitochondrial dGuo kinase the regulatory effect of

dTDP is controlled by pH.

m. Preliminary Kinetic Analysis

Initial velocity kinetic experiments using the non-activated form of the bovine liver dGuo kinase to determine the effect of varying dGuo concentration on the reaction rate demonstrated bimodal kinetics (Figure

50). When the dGuo concentration was varied over a 100-fold range the

Lineweaver-Burk plot showed a bimodal character that gave two apparent

Kjh values corresponding to two ranges of dGuo concentration; *5 x

10-^ M for the higher range and 7.3 x 10"5 M for the lower range.

However, when the activated form of the dGuo kinase was used the

Lineweaver-Burk plot demonstrated linear kinetics with an apparent ^

for dGuo of 4.5 x 10“5 M (Figure 51). This value is significantly higher than the value of 6.0 x 10“6 M obtained for the calf thymus mito­ chondrial dGuo Kinase (Figure 16). The difference in the values may reflect the different conditions under which the mitochondrial enzymes

from thymus and liver were assayed and/or may indicate they are differ­ ent enzyme species. In support of the former possibility, when the apparent % for the bovine liver enzyme was determined using the routine assay without Triton X-100, a value of 2.3 x 10~5 M was calculated.

Thus Triton X-100 increases the 1^ for dGuo, as well as the maximal velocity.

The effect of dlno on the rate of reaction using the activated form of the bovine liver enzyme and in the presence of varying concentrations of dGuo is also shown in Figure 51. The pattern of competitive inhi­ bition by dlno against dGuo suggests that both compete for a coninon site Figure 50. Effect of Varying dGuo Concentration on the Reaction Rate of Bovine Liver dGuo Kinase. Conditions were as described in Methods, except preincubation was omitted and assay was for 15 minutes. Enzyme was 0.56 wg of Fraction VII.

195 196

as

20 40 60 80 100

[dGuo x lO^M] Figure 51. Effect of Varying dGuo Concentration on the Reaction Rate of Bovine Liver dGuo Kinase; Inhibition by dlno. Conditions were as described in Methods, except assay was for 15 minutes. Enzyme was 0.56 pg of Fraction VII.

197 198

2.0 [dlno]:

a 0.50mM

1.0 * 0.2 5m M a 0.10 mM 0.5 o None

[dGuox 10‘3M] 199 on the enzyme. By replotting the slopes versus the dlno concentration an apparent of 2.5 x 10“4 M is calculated (Figure 52).

The effect of dGTP on the reaction rate, using the activated form of the enzyme, in the presence of varying concentrations of dGuo and at saturating ATP*Mg, is shown in Figure 53. The Lineweaver-Burk plot of the data shows the inhibition produced is noncompetitive with respect to the phosphate acceptor.

Kinetic data for ATP represent steady state velocities, i.e. they were obtained after complete disappearance of the initial lag period discussed previously. Velocities were linear with enzyme concentration and were always determined in 0.5 mM dGuo, a saturating concentration for this substrate. As shown in Figure 54, the inhibition by dGTP is competitive with respect to the phosphate donor. The apparent for

ATP, calculated from the control line, is approximately 1.5 x 10~3 M.

The apparent for dGTP calculated from a replot of the slopes versus dGTP concentrations is 1.2 x 10~7 M (Figure 5S). Figure 52. Replot of Data From Figure 51, Slopes Versus dlno Concentration,

200 SLOPE (x 10'3) O 00 o K 3 — r~ T" ~T"

p to 3 o p GJ

P. 201 In Figure 53. Effect of Varying dGuo Concentration on the Reaction Rate of the Bovine Liver dGuo Kinase; Inhibition by dGTP. Conditions were as described in Methods, except assay was for 15 minutes. Enzyme was 0.56 pg of Fraction VII.

202 [dGTP]:

A JOjuM

1 juM None Figure 54. Effect of Varying ATP Concentration on the Reaction Rate of the Bovine Liver dGuo Kinase; Inhibition by dGTP. Conditions were as described in Methods, except the preincubation was omitted, 0.5 irM dGuo was used in the assay, and assay time was for 30 minutes. Enzyme was 0.56 ug of Fraction VII..

204 20S

[dGTP]* 0.3 pM

None

2 4 6 8 10

'[ATP X 10'3M] Figure 55. Replot of Data From Figure 55, Slopes Versus dGTP Concentrat ion.

206 207

g 0.3 02

Q1 , 0.2 0.3 [dGTP x 106Ml DISCUSSION

The electrophoretic patterns of pyrimidine deoxynucleoside kinase activities of all tissues analyzed conformed very closely to the results of others [56,69,96). The analytical disc PAGE results indi­ cate that purine deoxynucleoside kinase isozymes also occur in mam­ malian cells and can be distinguished by their subcellular location and electrophoretic mobilities. All tissues analyzed, whether prolifera­ ting or non-proliferating, contained unique co-migrating dGuo/dAdo phosphorylating activities in the mitochondria. While in rat tissues and HeLa cells these activities co-migrated with the mitochondrial dCyd/ dThd kinase activities, in bovine, hamster and mouse tissues they were separated. In rat and bovine tissues this dAdo phosphorylating activity is separated from the dGuo kinase activity by DEAE-cellulose chromato­ graphy and is associated with the Ado kinase activity peak. Subsequent analytical disc PAGE studies of the crude bovine liver mitochondrial extract Tevealed that Ado kinase co-migrates with the mitochondrial dGuo/ dAdo activities. While direct assay of both the calf thymus and bovine liver DEAE-cellulose fractions reveals dAdo kinase activity, it was determined that the product of the reaction was dIMP. Evidence was found that indicates that in both cases this apparent activity is the result of the sequential action of adenosine deaminase and the mitochon­ drial dGuo kinase, which has the ability to phosphorylate dlno. There­ fore, from these results it appears unlikely that mitochondria contain

208 209 a unique dAdo kinase activity. Nevertheless, in mitochondrial frac­ tions of rat liver and spleen, occasionally a second dAdo phosphory­ lating activity was observed, separated from all other deoxynucleoside kinases (Figures 3 and 4). Because of its infrequent appearance in electrophorograms, attempts to further characterize the activity were difficult. Additional evidence for the existence of a liable dAdo kinase in mammalian tissues has come from studies with calf thymus.

Investigators in this laboratory, as well as another have reported the occurrence of such an activity in extracts of calf thymus (Dr. Sue-May

Wang and Dr. John Durham, personal communications). Thus, whether this activity is a very liable mitochondrial enzyme or an artifact remains to be determined.

In electrophorograms of rat (Figure 2), hamster (Figure 6), and mouse (Figure 7) tissues, as well as HeLa cells (Figure 7) a second dGuo kinase isozyme was found in the cytosol of both non-proliferating and proliferating tissues. Further attempts to purify this activity were also hampered by stability problems. In most cases studied, mito­ chondrial kinases could be detected in the cytosol, and vice versa, probably due to some mitochondrial rupture during tissue homogenization, and imbibation or transport of cytosol enzymes into mitochondria. This may also account for some, if not all of the adenosine kinase activity found associated with mitochondria in these studies.

In a proliferating bovine tissue (calf thymus) both dAdo and dGuo kinases were observed in the cytoplasm, having identical electrophore­ tic mobilities with a dominant dCyd kinase, idiereas all three of these cytosol activities were completely absent from non-proliferating adult 210 bovine liver (Figure 5). This constitutes a major difference between the bovine and rat enzymes, since in rat thymus the cytosol dGuo kinase was electrophoretically separate from the rapidly migrating dCyd kinase, which therefore was free of purine specificity. Thus, the reported separation of dCyd kinase from dGuo kinase, using extracts of P815 mouse ascites neoplasms (91-93) is not surprising, if it can be assumed that proliferating mouse cells exhibit a kinase profile similar to that seen in rat thymus. In addition, to what extent the mitochondrial activities are involved in these reports, as well as others from porcine and neo­ natal mouse skin (130,131) remains unclear.

A comparative analysis of the partially purified calf thymus cytosol and mitochondrial dGuo phosphorylating activities revealed significant differences between the two enzymes, confirming the initial electro­ phoretic data which indicated the existence of two distinct species of dGuo kinase in the tissue. Besides differences in electrophoretic mobility and subcellular location, the study revealed differences in chromatographic behavior on DEAE-cellulose and Blue Sepharose CL-6B.

These differences were employed successfully to yield partially purified cytosol and mitochondrial enzyme fractions free from contamination by the other isozyme. When assayed under identical conditions, i.e. opti­ mal conditions described for the cytosol enzyme (83), additional differ­ ences were observed to include nucleoside specificity, apparent for dGuo, isoelectric pH and nucleotide modulation.

The observations that (1) the cytosol dGuo kinase activity was invariably associated with dCyd and dAdo kinase activities during puri­ fication, (2) the cytosol enzyme was inhibited strongly by dCTTP, dCDP and to a lesser extent by UDP and dATP and (3) the cytosol enzyme had an apparent K,,, (770 M) two orders of magnitude higher than the mito­ chondrial dGuo kinase (6 ft) indicate that this is the broadly specific dCyd/dAdo/dGuo kinase isolated and characterized by others (83-85,87).

Under phsiological conditions the cytosol enzyme would not be expected to phosphorylate dGuo to any great extent, whereas the mitochondrial enzyme would. The narrow nucleoside specificity of the newly discovered mitochondrial enzyme and its regulation by dGTP argue strongly that the enzyme’s primary physiological role is to function in the mitochondrial dGuo salvage pathway. The pattern of positive modulation exhibited by the mito­ chondrial enzyme for dTDP is qualitatively similar to that exhibited by mammalian ribonucleotide reductase, the alternative means of deoxynucleotide synthesis, where dTDP or dTTP strongly stimulates GDP reduction as part of a complex system of control regulating the activity and specificity of the reductase (180).

Additional evidence for the subcellular location of the enzyme came from work with bovine liver. The enzyme is enriched almost 3-fold by isolation of mitochondria (Table 9). The enzyme appears to be membrane bound since osmotic shock alone releases only 113 of the total activity, but lysis by sonication or Triton X-100 releases 963 of the total activity

(Table 10). These results are very similar to those found for the mito- * chondrial dThd kinase isolated from LMTK* cells (53).

Isolation of the dGuo kinase from the bovine liver mitochondria resulted in an additional 806-fold purification, making the final puri­ fication from the liver tissue 1,857-fold. This exceeds the purifica­ tion of the most recent report of a dGuo kinase from porcine skin (130).

However, the preparation was not homogeneous as demonstrated by SDS 212 disc PACE (Figure 22) and disc PAGE using multiple porosities. Two prominent protein bands are observed when the preparative gel electro­ phoresis fraction (Fraction VII) is subjected to SDS disc PAGE. While the molecular weight of these bands (32,300 and 34,700) approximate half the molecular weight of the native enzyme (55,000-65,000) as determined by gel electrophoresis, gel filtration and glycerol gradient centrifu­ gation, it is not known whether or not either of these protein bands represent the subunits of the dGuo kinase.

Purification of both the calf thymus and bovine liver mitochondrial dGuo kinase utilized a Blue Sepharose CL-6B column. This gel has been successfully used in the purification of several dehydrogenases and kinases possessing the "dinucleotide fold," due to a structural simi­ larity between the chromophore (Cibacron Blue F3GA) and NAD (181,182).

The fact that dGuo kinase was eluted from this column by using 1.0 irM

ATP, but not KC1 of similar ionic strength, suggests that the binding of the enzyme may be specific and due perhaps to the presence of this dinucleotide fold in its tertiary structure.

The enzyme exhibits classical hysteretic behavior with a lag period having a t% ranging from 2 to 5 minutes. The lag can be eliminated by prior incubation with ATP, as well as dGTP and CTP (Table 13). The transition or catalytic activation process that occurs during the assay without prior incubation of the enzyme appears to be induced by ATP or

ATP*Mg. It would appear that the activation is brought about by the binding of the substrate to the protein at a nucleotide triphosphate binding site, most likely the phosphate donor site. This is suggested since CTP acts as a relatively good donor (Table 14) and dGTP, while not 213 a donor, is a competitive inhibitor of the enzyme with respect to ATP

(Figure 54). The inability of the ATP analogue AMPPCP to significantly alter the lag period at concentrations of 5 and 16 mM may indicate that binding alone may not be the complete triggering mechanism for activation, if it can be assured that AMPPCP binds to the site with proper alignment and has a dissociation constant comparable to ATP.

The length of the lag and the fact that the length of the lag period is inversely proportional to enzyme concentration argue that a slow transition in aggregational state of the enzyme occurs during the initial period of assay rather than a slow conformational transition from a less catalytically active species (T confonner) to a more catalytically active form (R confoimer) (136). However, using gel filtration and glycerol gradient centrifugation no evidence for changes in size of the protein could be detected. These results would argue that the mechanism for the hysteretic effect does not involve a change in aggregational state of the enzyme and if aggregation does not occur, the physical change in the protein would then most likely involve conformational alteration alone. However, since these procedures require several hours to complete, and since the stability of the activated dGuo kinase under the analytical conditions used is not known for time periods longer than

60 minutes, the possibility exists that the activated form relaxed back to the non-activated form during the analytical procedures. If this reversal occurred rapidly enough then neither of these methods would have been able to detect differences in the protein's size due to the activa­ tion process. Therefore, change in aggregational state cannot be ruled out by the results described above. A more thorough understanding of 214 the activated form of the enzyme needs to be obtained before further attempts can be made to detect changes in molecular size of the enzyme. Once this information is acquired then an experimental system can be designed to detect size changes which will provide conditions that will be certain to maintain the activated state.

The possibility of a chemical modification of the enzyme such as phosphorylation or adenylylation being the mechanism of activation appears unlikely. To date those enzymes which undergo such an activa­ tion process, mediated by a second enzyme, require a divalent cation in addition to an adenylate or phosphate donor. No such requirement is observed for the activation of the dGuo kinase. In addition, there appears to be no significant qualitative or quantitative change in the

ATP during the preincubation period (Figure 34 and 35). However, if chemical modification of this nature were the case then it should be relatively easy to detect by attempting to label the enzyme with either

[^H]-ATP or Y-labeled [32P)-ATP.

The mitochondrial dGuo kinase resembles very closely the dGuo kinase activities reported from neonatal mouse skin and procine skin (130,

131). The bovine liver enzyme exhibits a very sharp pH rate profile, with an optimum at pH 5.0, resembling the neonatal mouse skin dGuo kinase (131). The enzyme is very specific for dcoxynucleosides, phos- 0 phorylating only dGuo and the structural analogue dlno (Table 16).

Kinetic evidence suggests that dlno is phosphorylated at the same site as dGuo, since dlno was found to competitively inhibit the enzyme with respect to dGuo with an apparent of 2.5 x 10“5 M (Figure 51). The irtitochondrial enzyme is relatively specific for phosphate donor 215

with ATP, CTP and dTTP being the only effective donors ( T a b l e 14). The apparent Km value for ATP was calculated to be 1.5 x 10-3 M which is relatively high (Figure 54). In terms of molecular weight, the mito­ chondrial dGuo kinase demonstrated a weight of 55,000 to 65,000 which falls within the range reported for calf thymus dCyd kinase (83,118) and the procine skin dGuo kinase (130). The bovine liver enzyme is protected from thermodenaturation by ATP, dTDP and dGTP, with the end- product inhibitor being the most efficient protecting agent (Figure

47). Little or no protection was afforded the enzyme by either of its deoxynucleoside substrates. This pattern of thermodenaturation pro­ tection very closely resembles that reported for the calf thymus dCyd kinase, where dCTP was the most efficient protecting agent (83).

In terms of nucleotide regulation the stimulatory effect produced by dTDP, as well as other structurally similar nucleotides on the calf thymus enzyme appears to be regulated by pH. Assuming the thymus and liver mitochondrial enzymes are identical proteins, when assayed at its optimum pH of 5.0, the mitochondrial dGuo kinase is inhibited by dTDP.

What significance, if any, this pH dependent regulation plays in vivo is uncertain. Nevertheless, at either pH the enzyme's distal end-product dGTP remains a potent inhibitor (Tables 8 and 19). Similar to what was found for the calf thymus dCyd kinase,, the end-product inhibitor is * competitive with respect to the phosphate donor (ATP but noncompetitive with respect to the deoxynucleoside acceptor (dGuo) (86,118). The apparent Ki calculated for dGTP was 1.2 x 10-7 M (Figure 55). However, this pattern of inhibition is in contrast to that observed foT the dGuo kinase from porcine skin where dCTP was a competitive inhibitor with respect to dGuo, and in the case of the neonatal mouse skin dGuo kinase, dGTP was a competitive inhibitor with respect to both dGuo and ATP 216

(130,131).

The bimodal kinetics exhibited by the nonactivated bovine liver dGuo kinase supports the hysteretic data which indicates the presence of two kinetically different species of the kinase. From Figure 50 it can be seen that two apparent Km values for dGuo are indicated, one for the higher range (“5 x 10“^ M) and one for the lower range (7.3 x

10"5 M). When the activated form of the enzyme is used, linear kinetics are observed, indicating a shift to a single catalytic species with a lower apparent of 4.5 x 10~^ M (Figure 51). This kinetic behavior was presumably not observed for the calf thymus enzyme because the concentration range of dGuo did not extend into the higher concentration range (Figure 16).

The mitochondrial dGuo kinase appears to be a highly regulated enzyme, both in terms of its hysteretic effects and nucleotide modula­ tion. While the mitochondrial pyrimidine deoxynucleoside kinase is sub­ ject to end product inhibition by dCTP and dTTP, there has been no evi­ dence to suggest that the enzyme exhibits hysteretic behavior (54,55,90).

It remains to be learned if mitochondrial dGuo kinase can also con­ tribute to the cytosol deoxynucleotide pools which serve nuclear DNA synthessis. Gudas, et al. (114) have demonstrated that mutants of S49 lymphoma cells lacking cytoplasmic dCyd/dGuo kinase activities had greatly reduced pools of dGuo nucleotides. However, this may not nece­ ssarily mean that the mitochondrial enzyme does not contribute to the cytosol pools, since its stringent end-pToduct inhibition would be ex­ pected to result in cessation of nucleotide synthesis when the dGTP concentration is relatively low. At the sanfe time, the very low apparent 217

Kpj of the mitochondrial enzyme would enable it to function efficiently at low substrate concentrations, ensuring the rapid flux of deoxynu- cleotide through a relatively small pool.

Evidence from isolated liver mitochondria indicates that deoxynu­ cleoside triphosphates are capable of being transported into mito- condria and incorporated into mitochondrial DNA (183,184). However, the data show very low levels of incorporation of labeled exogenous dGTP entering mitochondrial DNA. In rat, control experiments demonstrated that dGTP is rapidly broken down in mitochondria, which accounts for the particularly low incorporation of dGTP relative to dCTP, dATP and dTTP (183). This hydrolysis proceeds to the nucleoside level and is relatively specific for dGTP, since only slight breakdown of dCTP, dATP and dTTP were detected. Whether or not this instability is present under in vivo conditions is not known, thus its physiological signifi­ cance is uncertain.

The presence of a dGuo kinase in mitochondria is analogous to the mitochondrial pyrimidine deoxynucleoside kinase, suggesting its primary role is to provide deoxyguanosine nucleotides to the mitochondrial dGTP pool which serves mitochondrial DNA synthesis (53,80). Whether or not a mitochondrial dAdo kinase activity exists is unclear at this point.

However, logically one would predict that such an activity exists simply * because the organelle containes a kinase for the other three naturally ocurring deoxynucleosides. It would appear that if it is present the activity is a very labile species, and that overcoming the stability problem is necessary before its identification and characterization can be accomplished. APPENDIX

A. Electrophoretic Survey of Various Mammalian Tissues For Pyrimidine Deoxynucleoside Kinasc¥

Hie electrophoretic profiles of dCyd and dThd kinase activies found

in cytosol and mitochondrial fractions of whole fetal rat are shown in

Figure 56, The cytosol and mitochondria contain electrophoretically distinct dThd kinases with Rf values of 0.23 and 0.61 respectively.

Similarly, two dCyd kinases are found, a cytosol (Rf 0.93) and a mito­ chondrial activity co-migrating with the mitochondrial dThd kinase (Rf

0.61).

Mitochondria isolated from a variety of adult rat tissues contain the co-migrating dThd/dCyd kinase with intermediate Rf values ranging from

0.41 and 0.45. Electrophoretic results of the mitochondrial dThd/dCyd kinase from liver, kidney and spleen are shown in Figure 57 and from brain and testis in Figure 58.

Cytosol fractions from whole fetal hamster, mouse spleen and liver presented analogous profiles to that observed in fetal rat. As shown

in Figures 59 to 61, these cytosol fractions contain a slowly migrating dThd kinase (Rf 0.68-0,73).

Whole cell electrophoretic profiles of dCyd and dThd kinases from hamster

tumor cells transformed with SV-40 virus 0!Tsv_4p) and human fetal lung cells

(WI-38) are shown in Figure 62 and 63, respectively. The profiles are identical

in that they both contain distinct cytosol dThd and dCyd kinases at Rf values 0.20 and 0.60 for the hamster cells and 0.28 and 0.68 for the human cells. The

218 Figure 56. Electrophoretic Profile of dThd and dCyd Kinases in Whole Fetal Rat Cytosol and Mitochondrial Cell Frations. Conditions for analytical disc PAGE and preparation of subcellular fractions were as described in Methods. Enzyme assays were for 17-19 hours at 37“C. Cell fraction: (A---A); cytosol; (O---O) , mitochondria.

219 CPMx 10■ 01 2 0.3 0.4 0 3 . 0 .2 0 0.1 ) -s^ . —c>— G-is.^a.o— g - o - a . o* \ ^ R 5 0. * 0. 9 1.0 .9 0 .8 0 0*7 .6 0 -5 0 DEOXYCYTIDINE KINASE THYMIDINE KINASE . . . . _ E A RATFETAL E A RATFETAL __»0

v^aj^0OiO i *v*^QaOjO^-0*O

a - a * a 220 -< i Figure 57. Electrophoretic Profile of dThd and dCyd Kinases in Mito­ chondria Isolated From Adult Rat Liver, Kidney and Spleen. Conditions for analytical continuous PAGE and preparation of mitochondria were as described in Methods. Enzyme assays were for 16. hours at 37°C. Kinase: (O-- O) , dThd; (A---A), dCyd.

221 KIDNEY

Q-Q.Q-O.Q

SPLEEN

"ol 03 03 04 05 06 07 08 09 to Figure 58. Electrophoretic Profile of dThd and dCyd Kinases in Mito­ chondria Isolated From Adult Rat Brain and Testis, Conditions for analytical continuous PAGE and preparation of mitochondria were as described in Methods. Enzyme assays were for 16 hours at 37°C. Kinase: (O CO, dThd; (A A ) , dCyd.

223 0 1 6 0 8*0 1 0 9 0 9 0 7'0 C O 7/0 VO 0 7-v-v-v-v-v- *^.,0*0— O-O— O-O-o-O'

02 S I 1 S 3 1

5 ^ - 3-3-3:3--,3TS-'S‘i01S—3— S*S—3 * 3 — 3 .01 x WdD x .01

0€

07

0 9

t-zz Figure 59. Electrophoretic Profile of dThd and dCyd Kinases In Cytosol of Whole Fetal PD-4 Hamster Tissue. Conditions for analytical continuous PAGE and preparation of cytosol were as described in Methods. Enzyme assays were for 18 hours at 37°C. Kinase: (O 0)» dlhd; (A-— A), dCyd.

225 CPM x 10-5

911 Figure 60. Electrophoretic Profile of dThd and dCyd Kinases In Cytosol of C3H Mouse Spleen. Conditions for analytical continuous PAGE and preparation of cytosol were as described in Methods. Enzyme assays were for 18 hours at 37°C. Kinase: (O-- O) » dThd; ( A --- A ) » dCyd.

227 228

LU LU QL CO

o

o

CO

CN

to CN -01 x WdD Figure 61. Electrophoretic Profile of dlhd and dCyd Kinases In Cytosol of C3H Mouse Liver. Conditions for analytical continuous PAGE and preparation of cytosol were as described in Methods. Enzyme assays were for 18 hours at 37°C. Kinase: CO-- O) » dThd; (A---A) » dCyd.

229 CN MOUSE UVER CYTOSOL c-ol O G * * o m CN 'CO > C K CO o o o o CN o 230 Figure 62. Electrophoretic Profile of dThd and dCyd Kinases In a Whole Cell Extract of HTsv_4o Cells. Conditions for analytical continuous PAGE and preparation of whole cell extract were as described in Methods. Enzyme assays were for 15 hours at 37°C. Kinase: ( O — O). dThd; ( A A), dCyd.

231 CPM x 1CT3 o O

p

vi s

H P Q CELLS

o•I

o

1

233 i------1------1------r

W I-38 CELLS WHOLE CELL

: h

i \i 0- O-O-O-KK)— L0— 0-0—0-0 0.1 0.2 03 04 0.6 07 0.8 09 1.0 23S mitochondrial co-migrating dThd/dCyd kinase activity migrated with an intermediate Rf of 0.43 in hamster and 0.48 in human cells.

B. Purification of the Cytosol Deoxyguanosine Fhosphorylating Activity frrom Calf Thymus

Hie isolation procedure described below provided a 100-fold purifi­ cation of the cytosol dGuo phosphorylating activity, free from mito­ chondrial dGuo kinase as demonstrated by analytical disc PAGE.

A crude soluble or cytosol fraction (6.9 mg) was prepared from 632 g of thymus glands as described in Methods (Fraction I).

Ammonium Sulfate Fractionation

Solid ammonium sulfate (226 g) was slowly added with stirring to

Fraction I over a 10 minute period to bring the solution to 40% satura­ tion. After additional stirring for 30 minutes, the mixture was centri­ fuged at 20,000 x g for 20 minutes. The small white pellet was discarded and the supernatant was brought to 80% saturation by adding an additional

271 g of ammonium sulfate in the manner described above. After centri­ fugation the supernatant was discarded and the pellet resuspended in

50 ml of 0M buffer (Fraction II).

DEAE-Cellulose Chromatography

Fraction II was dialyzed in Spectrapor Membrane No. 2 against 2 1 of

CM buffer overnight. Dialysis buffer was changed and dialysis continued for an additional 24 hours. A portion of the dialyzed Fraction II (688 mg) was applied to a DEAE-cellulose column (2.5 x 4.9 cm) equilibrated with CM buffer. The column was eluted successively with a linear 0 to

0.3 M KC1 gradient (300 ml), then with 0.8 M KC1 (100 ml), all in CM buffer. Deoxyguanosine kinase activity in the 0.8 M KC1 was was collected and concentrated by ultrafiltration (Amicon, H4-10 membrane) to a final 236 volume of 9.5 ml (Fraction m ) .

Blue Sepharose CL-6B Chromatography

Fraction III was diluted 1:1 in OM buffer, then applied to a Blue

Sepharose CL-6B column (3.5 x 4.9 cm) equilibrated in CM buffer. The column was washed successively with 250 ml of CM buffer containing 1.0 mM NAEH, a linear 0 to 0.3 M KC1 gradient (350 ml) in OM buffer, 0.8

M KC1 in 0M buffer (100 ml), followed by 10 mM ATP in 0M buffer (210 ml).

Deoxyguanosine kinase activity in the 0.8 M KC1 eluate was collected and concentrated by ultrafiltration (Amicon, PM-10 membrane) to a final volume of 9 ml (Fraction IV) .

Fraction IV was dialyzed against 0M buffer as described above. The fraction was concentrated further by a MicroProDiCon concentrator, then stored in 50% glycerol at -20°C. Under these conditions the enzyme was stable for several weeks. BIBLIOGRAPHY

1. Markert, C.L., and M011er, F. C1959) Proc. Natl. Acad. Sci. U.S.A. 45, 753-763.

2. Enzyme Nomenclature (1972) Recommendations of the International Union of Pure and Applied Chemistry and the International Union of Biochemistry, Eslsevier Publishing Company, Amsterdam, pp. 23-25.

3. Noltmann, E.A (1975) in "Isozymes," Market, C.L., ed., Volume I, Academic Press, New York, pp. 451-470.

4. Cahn, R.D., Kaplan, N.O., Levine, L., and Zwilling, E. (1962) Science 136, 962-969.

5. Nance, W.E., Claflin, A., and Smithies, 0. (1963) Science 142, 1075-1077.

6. Wilson, A.C., Cahn, R.D., and Kaplan, N.O. (1963) Nature, Lond. 197, 331-334.

7. Markert, C.L. (1968) Ann. N.Y. Acad. Sci. 151, 14-40.

8. Zinkham, W.H. (1968) Ann. N.Y. Acad. Sci. 151, 598-610.

9. Markert, C.L., and Apella, E. (1963) Ann. N.Y. Acad. Sci. 103, 915-929.

10. Plagemann, P.G.W., Gregory, K.F., and Wroblewski, F. (1960) J. Biol. Chem. 235, 2288-2292.

11. Pesce, A., Fondy, T.P., Stolzenbach, F., Castillo, F., and Kaplan, N.O. (1966) J. Biol. Chem. 242, 2151-2156.

12. Flores, R. (1979) TIBS 4, N32-N33.

13. Ibsen, K.H., and Trippet, P. (1972) Biochemistry 11_, 4442-4450.

14. Ibsen, K.H., and Krueger, E. (1973) Arch. Biochem. Biophys. 157, 509-513.

15. Ibsen, K.H., Trippet, P., and Basabe, J. (1975) in "Isozymes," Markert, C.L., ed., Volume I. Academic Press, New York, pp. 543-559.

237 238

16. Ibsen, K.H., Schiller, K.W., and Hoas, T.A. (1971) J. Biol. Chem. 246, 1233-1240.

17. Horecker, B.L. (1975) in "Isozymes," Markert, C.L., ed., Volume I, Academic Press, New York, pp. 11-38.

18. Lebherz, H.G., and Rutter, W.J. (1979) Biochemistry 8^, 109-121.

19. Blostein, R., and Rutter, W.J. (1963) J. Biol. Chem. 258, 3280- 3285.

20. Gracy, R.W., Lacko, A.G., and Horecker, B.L. (1979) J. Biol. Chem. 244, 3913-3919.

21. Penhoet, E., Rajkumar, T., and Rutter, W.J. (1966) Proc. Natl. Acad. Sci. U.S.A. 56, 1275-1282.

22. Lai, C.Y., Chen, C., and Horecker, B.L. (1970) Biochem. Biophys. Res. Common. 40^, 461-468.

23. Susor, W.A., Kochman, M., and Rutter, W.J. (1979) Science 165, 1260-1262.

24. Koida, M., Lai, C.Y., and Horecker, B.L. (1969) Arch. Biochem. Biophys. 134, 623-631.

25. Eppenberger, H.M., Eppenberger, M., Richterich, R., and Aebi, H. (1964) Develop. Biol. 10, 1-16.

26. Jacobus, W.E., and Lehninger, A.L. (1973) J. Biol. Chem. 248, 4803-4810.

27. Dawson, D.M., EppenbergeT, H.M., and Kaplan, N.O. (1965) Biochem. Biophys. Res. Conmain. 21_, 346-353.

28. Dawson, D.M., Eppenberger, H.M., and Kaplan, N.O. (1967) J. Biol. Chem. 242, 210-217.

29. Eppenberger, H.M., Dawson, D.M., and Kaplan, N.O. (1967) J. Biol. Chem. 242, 204-209.

30. Roberts, R., and Grace, A.M. (1980) J. Biol. Chem. 255, 2870-2877.

31. Turner, D.C., Maier, V., and Eppenberger, H.M. (1974) Develop. Biol. 36, 63-89.

32. Hall, N., Addis, P., and DeLuca, M. (1977) Biochem. Biophys. Res. Comnun. 76, 950-956. 239

33. Kraml, J . , Koldovsky, 0., Heringova, A., Jirsova, V., and Kacl, K. (1970) in "Enzymes and Isozymes, Structure, Properties and Function," Federation of European Biochemical Societies Fifth Meeting, Sugar, D., ed., Volume 18, Academic Press, New York, pp. 227-239.

34. Norden, A.G.W., Tennaut,' L.L., and O'Brien, J.S. (1974) J. Biol. Chem. 249, 7969-7976.

35. Shapira, E., David, A., DeGregorio, R., and Nadler, H.L. (1976) Enzyme 21, 332-341.

36. Lusis, A.J., Breen, G.A.M., and Paigen, K. (1977) J. Biol. Chem. 252, 4613-4618.

37. Lowenstein, J.M., and Smith, S.R. (1962) Biochem. Biophys. Acta 56, 385-387.

38. Plaut, G.W.E. (1963) in "The Enzymes," Volume VII, Boyer, P.D., Lardy, H., and Myrbock, K., eds., Academic Press, New York, pp. 105-124.

39. Plaut, G.W.E., and Aogaichi, T. (1967) Biochem. Biophys. Res. Comnun. 28, 628-634.

40. Atkinson, D.E. (1968) Biochemistry 7_t 4030-4034.

41. Colman, R.F. (1968) J. Biol. Chem. 243, 2454-2464.

42. Islam, M. , Bell, J.L., and Baron, D.N. (1972) Biochem. J. 129, 1003-1011.

43. Carlier, M . , and Pantaloni, D. (1973) Eur. J. Biochem. 37, 341-354.

44. Lowenstein, J.M. (1961) J. Biol. Chem. 236, 1217-1219.

45. Baker, W.W., and Newburgh, R.W. (1963) Biochem. J. 85?, 510-515.

46. Holmes, E.W., Jr., Wyngaarden, J.B., and Kelley, W.N. (1975) in "Isozymes," Markert, C.L., ed., Volume II, Academic Press, New York, pp. 425-437.

47. Caskey, C.T., Ashton, D.M., and Wyngaarden, J.B. (1964) J. Biol. Chem. 239, 2570-2S79.

48. Rowe, P.B., Coleman, M.D., and Wyngaarden, J.B. (1970) Bio­ chemistry £, 1498-1505.

49. Holmes, E.W., McDonald, J.A., McCord, J.M., Wyngaarden, J.B. , and Kelley, W.N. (1973) J. Biol. Chem. 248, 144-150. 240

50. Holmes, E.W., Wyngaarden, J.B., and Kelley, W.N. (1973] J. Biol, them. 248, 6035-6040.

51. Clayton, D.A., and Teplitz, R.J. (1972] J. Cell Sci. 10, 487-493.

52. Kit, S., and Minekawa, Y. (1972] Cancer Res. 32^ 2277-2288.

53. Berk, A.J., and Clayton, D.A. (1973] J. Biol. Chem. 248, 2722- 2729.

54. Lee, L-S., and Cheng, Y-C. (1975] J. Biol. Chem. 251, 2600- 2604.

55. Lee, L-S., and Cheng, Y-C. (1976] Biochemistry 15^ 3686-3690.

56. Adler, R. , and McAuslan, B.R. (1974) Cell 2_, 113-117.

57. Bollum, F.J., and Potter, V.R. (1959) Cancer Res. 1£, 561-565.

58. Masui, H., and Garren, L.D. (1971) J. Biol. Chem. 246, 5407- 5413.

59. Durham, J.P., Galanti, N. (1974) J. Biol. Chem. 249, 1806, 1813.

60. Munch-Petersen, B., and Tyrsted, G. (1977) Biochem. Biophys. Acta 478, 364-375.

61. Kit, S., Piekarski, L.J., and Dubbs, D.R. (1963) J. Mol. Biol. 6, 22-33.

62. Green, M., Pina, M., and Chagoya, V. (1964) J. Biol. Chem. 259, 1188-1197.

63. Kit, S., and Dubbs, D.R. (1963) Biochem. Biophys. Res. Conmun. U , 5S-59-

64. Nohara, H., and Kaplan, A.S. (1963) Biochem. Biophys. Res. Conmun. 12, 189-193.

65. Sheinin, R. (1966) Virology 28, 47-55.

66. Bull, D.L., Taylor, A.T., Austin, D.M., and Jones, C.W. (1974) Virology 57, 279-284.

67. Kit, S., Leung, W-C., Jorgensen, G.N., Trkula, D., and Dubbs, D.R. (1975) Progr. med. Virol. 21, 13-34.

68. Okuda, H., Arima, T., Hashimoto, T., and Fujii, S. (1972) Cancer Res. 32, 791-794. 241

69. Taylor, A.T., Stafford, M.A., and Jones, O.W. [1972) J. Biol. Chem. 247, 1930-1935.

70. Stafford, M.A., and Jones, O.W. (1972) Biochem. Biophys. Acta. 277, 439-442.

71. Salser, J.S., and Balis, M.E. (1976) Nature, Lond. 260, 261- 262.

72. Adelstein, S.J., Baldwin, C., and Kohn, H.I. (1971) Develop. Biol. 26, 537-546.

73. Kit, S., and Leung, W-C (1974) J. Cell Biol. 61, 35-44.

74. Willecke, K. , Reber, T., Kucherlapati, R.S., and Ruddle, F.H. (1976) Birth Defects 12_, 252-255.

75. Elsevier, S.M., Kucherlapati, R.S., Nichols, E.A., Creagan, R.P., Giles, R.E., Ruddle, F.H., Willecke, K., and McDougall, J.K. (1974) Nature, Lond. 251, 633-655.

76. Kit, S., Dubbs, D.R., and Frearson, P.M. (1966) Int. J. Cancer 1, 19-30. ”

77. Kit, S., Dubbs, D.R., Piekarski, L.J., and Hsu, T.C. (1963) Exp. Cell Res. 31, 297-313.

78. Kit, S., Leung, W-C., and Trkula, D. (1973) Arch. Biochem. Biophys. 158, 503-513.

79. Clayton, D.A., and Teplitz, R.L. (1972) J. Cell Sci. 10, 487- 493.

80. Bogenhagen, D., and Clayton, D.A. (1976) J. Biol. Chem. 251, 2938-2944.

81. Bosmann, H.B. (1971) J. Biol. Chem. 246, 3817-3820.

82. Koch, J., and Stokstad, E.L.R. (1967) Eur. J. Biochem. 3, 1-6.

83. Durham, J.P., and Ives, D.H. (1970) J. Biol. Chem. 245, 2276- 2284.

84. Ives, D.H., and Durham, J.P. (1970) J. Biol. Chem. 245, 2285- 2294.

85. Krenitsky, T.A., Tuttle, J.V,, Koszalka, G.W., Chen, I.S., Beacham, L.M., III, Rideout, J.L., and Elion, G.B. (1976) J. Biol. Chem. 251, 4055-4061. 242

86. Momparler, R.L., and Fischer, G.A. (1968) J. Biol. Chem. 243, 4298-4804.

87. Kozai, Y., Sonoda, S., Kobayashi, S., and Sugino, Y. (1972) J. Biochem. 71_, 485-496.

88. Kessel, D. (1968) J. Biol Chem. 243, 4739-4744.

89. Coleman, C.N., Stoller, R.G., Drake, J.C., and Chabner, E.A. (1975) Blood 46, 791-803.

90. Cheng, Y-C., Domin, B., and Lee, L-S. (1977) Biochem. Biophys. Acta 481, 481-492.

91. Meyers, M.B., and Kreis, W. (1976) Arch. Biochem. Biophys. 177, 10-15.

92. Meyers, M.B., and Kreis, W. (1978) Cancer Res. 38, 1099-1104.

93. Meyers, M.B., and Kreis, W. (1978) Cancer Res. 38, 1105-1112.

94. DeSaint-Vincent, B.R., and Buttin, G. (1973) Eur. J. Biochem. 37, 481-488.

95. Leung, W-C., Dubbs, D.R., Trkula, D., and Kit, S. (1975) J. Virol. 16, 486-497.

96. Postel, E.H., and Levine, A.J. (1975) Virology 63^, 404-420.

97. Durham, J.P., and Ives, D.H. (1979) Mol. Pharmacol. 5_, 358-375.

98. Momparler, R.L. (1974) Cancer Res. 3£, 1775-1787.

99. Cohen, S.S. (1976) Med. Biol. 54, 299-326.

100. Diana, G.D., and Pancic, F. (1976) Angew. Chem. Int. Ed. Engl. 15, 410-416.

101. Kulikowski, T., Zawadzki, 2., Shugar, D., Descamps, J., and De Clercq, E. (1979) J. Med. Chem. 22, 647-653.

102. Maugh, T.H., II (1979) Science 192, 128-132.

103. Giblett, E.R., Anderson, J.E., Cohen, F., Pollara, B., and Meuwissen, H.J. (1972) Lancet ii, 1067-1069.

104. Giblett, E.R., Anmann, A.J., Wara, D.W., Sandman, R., and Diamond, L.K. (1975) Lancet i, 1010-1013. 243

105. Coleman, M.S., Donofrio, J., Hutton, J. J., Hahn, L., Daoud, A., Lampkin, B., and Dyminski, J. (1978) J. Biol. Chem. 253, 1619- 1626.

106. Cohen, A.R., Hirschhom, R., Horowitz, S.D., Rubinstein, A., Polmar, S.H., Hong, R., and Martin, D.W., Jr. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 472-476.

107. Cohen, A., Gudas, L.J., Ammann, A.J., Staal, G.E., and Martin, D.W., Jr (1978) J. Clin. Invest. 61, 1405-1409.

108. Cohen, A., Doyle, D., Martin, D.W., Jr., and Ammann, A.J. (1976) N. Engl. J. Med. 295, 1449-1454.

109. Donofrio, J., Coleman, M.S., Hutton, J.J., Dauod, A., Lampkin, B., and Dyminski, J. (1978) J. Clin. Invest. 62^, 884-887.

110. Ullman, B., Gudas, L.J., Cohen, A., and Martin, D.W., Jr., (1978) Cell 14, 365-375.

111. Carson, D.A., Kaye, J., and Seegmiller, J.E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5677-5681.

112. Mitchell, B.S., Mejias, E., Daddona, P.E., and Kelley, W.N. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5011-5014.

113. Chan, T. (1978) Cell IA, 523-530.

114. Gudas, L.J., Ullman, B., Cohen, A., and Martin, D.W., Jr. (1978) Cell W , 531-538.

115. Wilson, J.M., Mitchell, B.S., Daddona, P.E., and Kelley, W.N. (1979) J. Clin. Invest. 64, 1475-1484.

116. Hershfield, M.S. (1979) J. Biol. Chem. 254, 22-2S.

117. Krygier, V., and Momparler, R.J. (1968) Biochem. Biophys. Acta 161, 578-580.

118. Krygier, V., and Momparler, R.L. (1971) J. Biol. Chem. 246, 2745-2751.

119. Krygier, V., and Momparler, R.L. (1971) J. Biol. Chem. 246, 2752-2757.

120. Ives, D.H., and Wang, S-M. (1978) in "Methods in Enzymology," Hoffee, P.A., and Jones, M.E., eds., Volume LI, Academic Press, New York, 337-345. 244

121. Schrecker, A.W. (1970) Cancer Res. 30^, 632-641.

122. Streeter, D.G., Si.wjn, L.N., Robins, R.K., and Miller, J.P. (1974) Biochemistry 1J5, 4543-4549.

123. Lindberg, B., fCLenow, H., and Hansen, K. (1967) J. Biol. Chem. 242, 350-356. --

124. Andres, C.M., and Fox, I.H. (1979) J. Biol. Chem. 254, 11388- 11393.

125. Miller, R.L., Adamczyk, D.L., and Miller, W.H. (1979) J. Biol. Chem. 254, 2339-234S.

126. Miller, R.L., Adamczyk, D.L., Miller, W.H., Koszalka, G.W., Rideout, J.L., Beacham, L.M., III, Chao, E.Y., Haggerty, J.J., Krenitsky, T.A., and Elion, G.B. (1979) J. Biol. Chem. 254, 2346-2352. --

127. Chang, C-H, Brockman, R.W., and Bennett, L.L., Jr. (1980) J. Biol. Chem. 255, 2366-2371.

128. Nakai, Y., and LePage, G.A. (1972) Cancer Res. 32, 2445-2451.

129. Baxter, A., Carswell, L.M., and Durham, J.P. (1977) Biochem. Soc. Trans. £, 979-981.

130. Green, F.J., and Lewis, R.A. (1979) Biochem. J. 185, 547-553.

131. Barker, J., and Lewis, R. (1979) Xlth International Congress of Biochemistry, Abstract No. 04-7-S95.

132. Monod, J., Wyman, J., and Changeux, J.P. (1965) J. Mol. Biol. 12, 88-118.

133. Koshland, D.E., Jr., Nemethy, G., and Filmer, D. (1966) Bio­ chemistry 5^ 365-385.

134. Friden, C. (1967) J. Biol. Chem. 242, 4045-4049.

135. Nichol, L.W., Jackson, W.J.H., and Winzor, D.J. (1967) Biochemistry 6, 2449-2455.

136. Friden, C. (1970) J. Biol. Chem. 245, 5788-5799.

137. Tarmy, E.M., and Kaplan, N.O. (1968) J. Biol. Chem. 243, 2587-2596.

138. Kirschner, K. (1971) J. Mol. Biol. 58, 51-56. 245

139. Ashby, B., and Friden, C. (1978) J. Biol. Chem. 253, 8728-8735.

140. Meyer, L.J., and Becker, M.A. (1977) J. Biol. Chem. 252, 3919- 3925.

141. Misset, 0., Brouwer, M., and Robillard, G.T. (1980) Biochemistry 19, 883-890.

142. Wohl, R.C., and Markus, G. (1972) J. Biol. Chem. 247, 5785-5792.

143. Vagelos, P.R., Alberts, A.W., and Martin, D.B. (1963) J. Biol. Chem. 238, 533-540.

144. Duncan, B.K., Diamond, G.R., Bessman, M.J. (1972) J. Biol. Chem. 247, 8136-8138.

145. Wang, C-T., and Weissman, B. (1971) Biochemistry 10, 1067-1072.

146. Kane, J.F., Holmes, W.M., Smiley, K.L., Jr., and Jensen, R.H. (1973) J. Bact. 113, 224-229.

147. Tourian, A. (1971) Biochem. Biophys. Acta 242, 345-354.

148. Vanquickenbome, A., and Phillips, A.T. (1968) J. Biol. Chem. 243, 1312-1319.

149. Metzger, B., Helmreich, E., and Glaser, L. (1967) Proc. Natl. Acad. Sci. U.S.A. 57, 994-1001.

150. Fink, K., and Adams, W.S. (1966) J. Chromatog. 22_, 118-129.

151. DuTham, J.P., and Ives, D.H. (1971) Biochem. Biophys. Acta 228, 9-25.------

152. CRC Handbook of Biochemistry, 2nd edition, 1970.

153. Ives, D.H., Durham, J.P., and Tucker, V.S. (1969) Anal. Biochem. 28, 192-205. —

154. Coleman, M.S., and Hutton, J.J. (1975) Biochem. Med. 13, 46-55.

155. DiPietro, D.L., and Zengerle, F.S. (1967) J. Biol. Chem. 242, 3391- 3396. ---

156. Pourgeois, R., Satre, M., and Vignais, P.V. (1978) Biochemistry 17, 3018-3023. —

157. King, E.J. (1932) Biochem. J. 26, 292-297. 246

158. Laemmli, U.K., and Havre, M. (1973) J. Mol. Biol. 80, 575-599.

159. Holbrook, I.B., and Leaver, A.G. (1976) Anal. Biochem. 75, 634-636.

160. Deibel, M.R., Jr., and Ives, D.H. (1977) J. Biol. Chem. 252, 8235-8239.

161. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412.

162. Martin, R.H., and Ames, B.N. (1961) J. Biol. Chem. 236, 1372- 1379.

163. Geiger, P.J., and Bessman, S.P. (1972) Anal. Biochem. 49, 467- 473.

164. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254.

165. Bio-Rad Laboratories, "Bio-Rad Protein Assay," Instruction Manual (1979)

166. Tyler, D.D., and Gonze, J. (1967) in 'Methods in Enzymology," Estabrook, R.W., and Pullman, M.E., eds., Volume X, Academic Press, New York, pp. 101-103.

167. Beattie, D.S. (1968) Biochem. Biophys. Res. Conmun. 31^, 901-907.

168. Laurent, T.C., and Killander, J. (1964) J. Chromatog. 14, 317- 330.

169. Cihak, H., and Rada, B. (1976) Neoplasma 23, 233-257.

170. Heppel, L.A., and Hilmoe, R.J. (1969) in "Methods in Enzymology," Colowick, S.P., and Kaplan, N.O., eds., Volume II, Academic Press, New York, pp. 546-549.

171. Edwards, Y.H., Hopkinson, D.A., and Harris, H. (1971) Ann. Hum. Genet. 15, 207-219.

172. Hirschhom, R., Levytska, V., Pollara, B., and Meuwissen, H. (1973) Nature New Biol. 246, 200-202.

173. Hirschhorn, R. (1977) Fed. Proc. 36, 2166-2170.

174. Banno, Y., Morris, H.P., and Katunuma, N. (1978) J. Biochem. 83, 1545-1554.

175. Haas, R., and Heinrich, P.C. (1979) Eur. J. Biochem. 96, 9-15.

176. O'Brien, T.W., and Kalf, G.F. (1967) J. Biol. Chem. 242, 2172- 2179. --- 247

177. Shuster, L., and Kaplan, N.O. (1969) in "Methods in Enzymology," Colcwick, S.P., and Kaplan, N.O., eds., Volume XI, Academic Press, New York, p. SSI.

178. Hedrick, J.L., and Smith, A.J. (1978) Arch. Biochem. Biophvs. 126, 155-164.

179. Ferguson, K.A. (1964) Metabolism 13^ 985-998.

180. Moore, E.C., and Hurlburt, R.B. (1966) J. Biol. Chem. 241, 4802- 4809.

181. Thompson, S.T., and Stellwagon, E, (1976) Proc. Natl. Acad. Sci, U.S.A. 73, 361-365.

182. Wilson, J.E. (1976) Biochem. Biophys. Res. Comnun. 72_, 816-823.

183. Parsons, P., and Simpson, M.V. (1973) J. Biol. Chem. 248, 1912-1919.

184. Ter Schegget, J., and Borst, P. (1971) Biochem. Biophys. Acta 246, 239-248.