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H u m a n deoxycytidine kinase: The purification and characterization of the enzyme from hu m a n leukemic cells
Kim, Min-Young, Ph.D.
The Ohio State University, 1989
Copyright ©1989 by Kim, Min-Young. All rights reserved.
UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106
HUMAN DEOXYCYTIDINE KINASE:
THE PURIFICATION AND CHARACTERIZATION OF THE ENZYME
FROM HUMAN LEUKEMIC CELLS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of the Ohio State University
By Min-Young Kim, M.S.
* * * * *
The Ohio State University
1989
Dissertation Committee: Approved by Professor David H. Ives
Professor Edward J. Behrman
Professor George S. Serif
Professor Lee F. Johnson Adviser Department of Biochemistry Copyright 1989 Min-Young Kim To my parents
ii ACKNOWLEDGMENTS
I would like to express my sincerest appreciation to
my adviser, Dr. David H. Ives, for his guidance,
understanding, and support throughout my studies.
I am deeply grateful to Dr. Seiichiro Ikeda for his
support and help during this work.
I also would like to acknowledge the assistance
provided by the faculty members of the Department of
Biochemistry. Many thanks also go to the people whom I met in our laboratory.
To my parents, parents-in-law, and family, my deepest
appreciation goes to them for all their continued support
and well wishes.
Finally, I extend my love and thanks to my husband
Yoen-Seung and my daughter Heejae whose love, patience,
and encouragement made this possible.
iii VITA
March 17, 1957 Born - Seoul, Korea
1979 Bachelor of Pharmacy EWHA WOMANS' University Seoul, Korea
1987 M.S. Biochemistry The Ohio State University
1983-present Graduate Teaching / Research Associate, Department of Biochemistry The Ohio State University
PUBLICATIONS
1. Kim, M. Y., Ikeda, S., and Ives, D. H. (1988), "Affinity purification of human deoxycytidine kinase: Avoidance of structural and kinetic artifacts arising from limited proteolysis," Biochem. Biophys. Res. Commun. 156, 92-98.
2. Kim, M. Y., and Ives, D. H. (1989), "Human deoxycytidine kinase: Kinetic mechanism and End-product regulation," (submitted).
FIELDS OF STUDY
Studies in Enzymology Professor David H. Ives
Studies in Protein Biochemistry Professor Gary E. Means
Studies in Immunology Professor Richard F. Mortensen and Bruce S . Zwilling
iv TABLE OF CONTENTS
Page ACKNOWLEDGMENTS ...... iii
VITA ...... iv
LIST OF TABLES ...... vii
LIST OF FIGURES ...... viii
ABBREVIATIONS ...... xi
ABSTRACT ...... xiii
INTRODUCTION ...... 1
1. Deoxycytidine kinase ...... 1 2. Affinity media for deoxycytidine kinase . . 11 3. Multiple forms of enzymes ...... 17
EXPERIMENTAL PROCEDURES
1. Material ...... 25 2. Methods ...... 26
i. Enzyme a s s a y ...... 26 ii. Preparation of crude extracts from human leukapheresis specimens ...... 28 iii. Protein a s s a y ...... 29 iv. Polyacrylamide gel electrophoresis . . . 30 v. Molecular weight determinations.... 31 vi. Purity of reagents for kinetic experiment ...... 32 vii. Immunization of rab b i t s ...... 32 viii. Collection of s e r u m ...... 35 ix. E L I S A ...... 36 x. Western immunoblotting ...... 37 xi. Immunodetection...... 39
v TABLE OF CONTENTS (cont.)
Page RESULTS
1. dCyd kinase purified by dCp4A-Sepharose affinity chromatography ...... 42 2. Enzyme purity and subunit molecular weight 43 3. Molecular weight of native enzyme ...... 44 4. Substrate and donor specificity ...... 46 5. Conditional proteolytic modification during purification ...... 50 6. Kinetic effects of proteolysis ...... 53 7. Kinetic mechanism of dCyd kinase from T-ALL cells
i. Time-course of dCMP production .... 55 ii. Initial velocity measurements in the absence of p r o d u c t s ...... 55 iii. Initial velocity measurements in the presence of p r o d u c t s ...... 61 iv. End-product inhibition ...... 70 8. Looking for possible isozymes with new affinity media
i. dCp^-Sepharose affinity chromatography 73 ii. dAp^-Sepharose column ...... 79 iii. Purity analysis ...... 81 iv. Electrophoretic mobility ...... 83 9. Immunoassay
i. ELISA ...... 86 ii. Purification of the serum ...... 89 iii. Western immunodetection ...... 90 10. Attempt to determine the N-terminal amino acid sequence ...... 93
DISCUSSION ...... 94
APPENDIX ...... 102
BIBLIOGRAPHY ...... 112
vi LIST OF TABLES
Page Table
1. Some chemotherapeutic agents activated by dCyd kinase ...... 2
2. Substrate specificity of human dCyd kinase ...... 48
3. Phosphate donor specificity of human dCyd kinase ...... 49
4. Summary of kinetic constants for the two substrates ...... 60
5. Product inhibition constants and patterns . 68
6. End-product inhibition constants ...... 74
7. The effect of the variation of total concentrations of magnesium and ATP on the concentration of MgATP and other species at pH 7.5, with a constant 20% molar excess of total magnesium to total A T P ...... 106
8. The calculation of total concentrations of magnesium required, when the ratio of total ADP and MgADP is 1:1 and unbound Mg2+ is at a fixed concentration...... 109
9. The calculation of total concentrations of magnesium required for a minimum of 98% of the total ATP to form a complex with magnesium and for unbound Mg2 to be at a fixed concentration in the presence of A D P ...... 110
10. The calculation of total concentrations of magnesium required according to the increasing concentrations of total ATP ...... Ill
vii LIST OF FIGURES
Page Figure
1. Putative modes of binding of substrates and multisubstrate analogs at the active site of deoxynucleoside kinase ...... 15
2. Structures of affinity chromatography media . 16
3. Determination of the polypeptide molecular weight by SDS gel electrophoresis...... 45
4. Estimation of the molecular weight of the native dCyd kinase by the "Ferguson" relationship . . 47
5. SDS-polyacrylamide gel electrophoresis of dCyd kinase purified with and without protease inhibitors ...... 51
6. Non-denaturing polyacrylamide gel electrophoresis of parental and proteolyzed dCyd and dAdo kinase ...... 52
7. Lineweaver-Burk kinetics of mixture of parental and proteolyzed dCyd kinase .... 54
8. Time course of the % conversion of dCyd to product, dCMP ...... 56
9. The effect of varying dCyd concentration on the reaction rate at fixed concentrations of MgATP ...... 58
10. The effect of varying MgATP concentration on the, reaction rate at fixed concentrations of d C y d ...... 59 LIST OF FIGURES (cont.)
page Figure
11. Purity analysis of reagents by F P L C ...... 62
12. Product inhibition by ADP; the effect of varying MgATP concentration on the reaction rate, with a 2.2 uM dCyd concentration...... 64
13. Product inhibition by ADP; the effect of varying dCyd concentration on the reaction rate, with a 2.1 mM MgATP concentration ...... 65
14. Product inhibition by dCMP; the effect of varying MgATP concentration on the reaction rate, with a 2.2 uM dCyd concentration ...... 66
15. Product inhibition by dCMP; the effect of varying dCyd concentration on the reaction rate, with a 1.0 mM MgATP concentration ...... 67
16. The proposed kinetic mechanism for human dCyd kinase purified from T - A L L ...... 69
17. End-product inhibition by dCTP; the effect of varying MgATP concentration on the reaction rate, with a 2.2 uM dCyd concentration ...... 71
18. End-product inhibition by dCTP; the effect of varying dCyd concentration on the reaction rate, with a 0.12 mM MgATP concentration...... 72
19. Purification of dCyd kinase by dCp4-Sepharose affinity column ...... 76
20. Purification profile of dCyd kinase from different kinds of human leukemic cell lines and cultured human T-lymphoblasts by dCp^-Sepharose affinity column ...... 77
ix LIST OF FIGURES (cont.)
Page Figure
21. dAp^-Sepharose chromatography of crude extract of CML cells ...... 80
22. SDS-polyacrylamide gel electrophoresis of dCyd kinase purified from different human leukemic cell lines ...... 82
23. Electrophoretic mobility profiles of dCyd kinase purified from CML, AML cell lines and comparison with the enzyme from T-ALL cells in one gel . 84
24. Electrophoretic mobility profiles of dCyd kinase purified from MOLT-4, B-CLL, and B-ALL cell lines and comparison with the enzyme from T-ALL cells in one gel ...... 85
25. Determination of the titer, by ELISA, of crude antiserum raised by the disk implantation method ...... 87
26. Determination of the titer, by ELISA, of crude antiserum raised by the nitrocellulose power method ...... 88
27. SDS-polyacrylamide gel electrophoresis of purified IgG preparations ...... 91
28. Western immunodetection of dCyd kinase . . . 92
29. Equations relating the various forms of ATP and magnesium complexes ...... 103
x ABBREVIATION
ADA Adenosine deaminase
Ado Adenosine
ADP Adenosine 5' -diphosphate
AIDS Acquired Immunodeficiency Syndrome
ALL Acute lymphoblastic leukemia
AML Acute myeloblastic leukemia
AMP Adenosine 5' -monophosphate
Ara-A 9-p-D-arabinofuranosyladenine
2-F-ara-A 9-|3-D-arabinofuranosyl-2- fluoroadenine
Ara-C 1-0-D-arabinofuranosylcytosine
ATP Adenosine 5' -triphosphate
BSA Bovine serum albumin
Cyd Cytidine
CML Chronic myelocytic leukemia
CLL Chronic lymphocytic leukemia dAdo 2 ’ -deoxyadenosine dATP 2' -deoxyadenosine 5' -triphosphate dAp4 Deoxyadenosine 5'-tetraphosphate dCF Deoxycoformycin dCMP 2' -deoxycytidine 5' -monophosphate dCTP 2' -deoxycytidine 5' -triphosphate dCyd 2' -deoxycytidine dcLAdo 2', 3'-dideoxyadenosine ddCyd 2', 3 ’-dideoxycytidine dGuo 2* -deoxyguanosine dGTP 2' -deoxyguanosine 5' -triphosphate
DNA Deoxyribonucleic acid dCp4A Deoxycytidine 5'-adenosine 5'''-
p^,p^ -tetraphosphate dCp4 Deoxycytidine 5' -tetraphosphate dNTP Deoxynucleoside 5 1 -triphosphate dThd 2' -deoxythymidine
DTE Dithioerythritol
EDTA Ethylenediamintetraacetate
FPLC Fast protein liquid chromatography
Guo Guanosine
HIV Human Immunodeficiency Virus
PMSF Phenylmethylsulfonyl fluoride
RNA Ribonucleic acid
Tris Tris-(hydroxymethyl)-aminomethane
TSK T-lymphoblast-specific nucleoside kinase ABSTRACT
HUMAN DEOXYCYTIDINE KINASE:
PURIFICATION AND CHARACTERIZATION OF THE ENZYME
FROM HUMAN LEUKEMIC CELLS
By
Min-Young Kim
The Ohio State University, 1989
Professor David H. Ives, Adviser
Homogeneous human deoxycytidine kinase was purified from acute leukemic T-lymphoblasts by affinity chromatography with the multisubstrate analog, deoxycytidine 5’-adenosine 5'''-p^,p^-tetraphosphate
(dCp4A). Starting with an ammonium sulfate fraction, purification was achieved in one step, with the kinase being eluted from the column by the end product inhibitor, dCTP. Chromatography of extract treated with protease inhibitors yielded a monomeric polypeptide, inasmuch as the relative molecular mass of the native protein, 59,300,
xiii is comparable to the value of 52,000 from sodium dodecyl
sulfate polyacrylamide gel electrophoresis. When purified
without protease inhibitors the enzyme. exhibited
fragments, suggesting that proteolytic cleavage of the
parental polypeptide had occurred during affinity
chromatography. Both the parental and proteolyzed enzymes
phosphorylated deoxyadenosine and deoxyguanosine, as well
as deoxycytidine. However, the proteolyzed enzyme had an
increased apparent for dCyd. In consequence of this, a
mixture of the two forms yielded bimodal kinetic plots,
whereas linear kinetic was produced by each form alone.
Kinetic properties of the parental purified dCyd
kinase were investigated. The results of steady state
initial-rate kinetic analysis and product inhibition
studies indicate that substrate binding follows an ordered
sequential pathway, with the ATP being the first substrate
to bind and dCMP the last product to dissociate. At
subsaturating substrate concentrations, only dCMP produced competitive inhibition, against ATP, while against varied deoxycytidine dCMP exhibited mixed-type inhibition. ADP produced noncompetitive inhibition against either
substrate. The limiting K^s for deoxycytidine and MgATP were 0.94 uM and 30 uM, respectively.
The end-product inhibitor dCTP exhibited competitive
inhibition against varied ATP, with a dissociation
xiv constant estimated to be 0.7 uM when extrapolated to zero
ATP concentration. dCTP was purely noncompetitive against
varied deoxycytidine. Based on these kinetic results, and
on the strong and specific inhibition by dCTP, it is
proposed that this end-product functions as
a multisubstrate analog, with its deoxycytidine moiety
binding to the nucleoside site, and its triphosphate group
overlapping and binding to the phosphate-donor site of the
enzyme.
Based on its structure, the new affinity column deoxycytidine 5'-tetraphosphate-Sepharose (dCp4-Sepharose) was synthesized and dCyd kinase was purified in one step from a variety of spontaneous human leukemic cell
lines (AML, CML, B-ALL, B-CLL, T-ALL), as well as from cultured T-lymphoblast cells (MOLT-4).
Enzyme purified from B-CLL, AML, CML cell lines yielded one major band that has the same Rf as the enzyme from T-ALL (52 Kd) by SDS-polyacrylamide gel electrophoresis. On the other hand, B-ALL and MOLT-4 cell showed a low molecular weight band only (30 Kd). However, the electrophoretic mobilities in 12% non-denaturing gels were identical for the dCyd kinase from all different kinds of leukemic cell lines, except that the B-ALL, B-CLL and MOLT-4 cell preparations had an extra minor peak, all at the same position. dAdo and dCyd phosphorylating
xv activities co-migrated indicating that these activities are all associated with the same protein.
Preliminary western immunoassay of T-ALL crude extract with polyclonal antibody raised by the disk implantation method revealed a single sharp band of immunoreactive material at 50 Kd, a thin band at 30 Kd and a heavy band at 60 Kd, which suggests the possibility that the 30Kd band might be a cross-reactive dCyd kinase isoenzyme or proteolyzed product.
xvi INTRODUCTION
1. Deoxycytidine kinase
The nucleoside kinases are an important group of intracellular enzymes which phosphorylate nucleoside analogs active as anticancer and antiviral drugs (1,2), as well as endogenous nucleosides. The deoxynucleoside kinases contribute to the effectiveness of important deoxynucleoside analogs used as chemotherapeutic agents by phosphorylation. Table 1 shows some of chemotherapeutic agents activated by deoxycytidine kinase
(dCyd kinase).
dCyd kinase is the pyrimidine salvage pathway enzyme which catalyzes the phosphorylation of deoxycytidine
(dCyd) to deoxycytidine 5 '-phosphate (dCMP) in the presence of a nucleoside triphosphate as phosphate donor.
Recently, particular attention has been focused upon the phosphorylation of the antiviral agent, 2',3'- dideoxynucleoside, a compound active against human immunodeficiency virus ( HIV ). HIV is now recognized as
1 2
Table 1
Some chemotherapeutic agents activated by dCyd kinase
Cytosine arabinoside (Cytarabine)
5-fluoro deoxycytidine
Adenine arabinoside (Vidarabine)
2-fluoroadenine arabinoside
2 1, 3'-dideoxy cytidine
6-Thioguanine deoxyriboside
Guanine arabinoside
Deoxycoformycin (indirect activation) the etiological agent of acquired immunodeficiency syndrome (AIDS) (3-5). In AIDS and its preceding lymphadenopathy syndrome, there is detectable virus replication as determined by the presence of particulate reverse transcriptase activity (3).
It was reported that 2',3'-dideoxypurine and
-pyrimidine nucleosides inhibited the replication of HIV
(6). The major antiviral effect of these analogs is thought to result from direct inhibition of virally encoded reverse transcriptase (RNA-directed DNA polymerase) by the triphosphate derivatives, or from chain termination of proviral DNA synthesis caused by incorporated analog (6-9), or perhaps from both effects.
Mitsuya et al. (10) showed that all four 2', 3 * — dideoxynucleoside triphosphates were incorporated into elongating DNA chains by HIV reverse transcriptase, leading to inhibition of both viral DNA synthesis and subsequent messenger RNA expression. Thus an understanding of the metabolic pathways by which dideoxynucleosides are converted into their active metabolites is important to the exploitation of these compounds in the treatment of AIDS.
Ullman, et al. (11) have described the cytotoxic effects and the metabolism of 2',3'-dideoxycytidine
(ddCyd) in a human T lymphoblast cell line (CCRF-CEM) and 4
in several clonal derivatives which are genetically
deficient either in their ability to transport
nucleosides, or in dCyd kinase activity. The results
implicated the plasma membrane nucleoside transport system
and the intracellular dCyd kinase as essential
determinants in the initiation of ddCyd metabolism in
human T cells. The conclusion that dCyd kinase is
responsible for the initiation of ddCyd metabolism is
consistent with the studies of Cooney, et al. (12), who
demonstrated that dCyd could abolish ddCyd phosphorylation
in human ATH8 cells, and that a dCyd kinase-deficient
derivative of the non-thymic p388 cell line did not
effectively metabolize ddCyd.
Starnes and Cheng (9) also showed that ddCyd was the
most potent of the compounds tested, as it completely
blocked the viral cytopathic effect at concentrations
greater than 0.5 uM, a dosage which was at least 10-fold
less than that which inhibited cell growth in the absence
of virus. Studies with human Cyd-dCyd deaminase showed
that ddCyd was not significantly susceptible to deamination, a major pathway for inactivation of l-(3-D-
arabinofuranosyl cytosine in humans (13). They also
showed that ddCyd was a poor substrate for both human cytoplasmic and mitochondrial dCyd kinase compared with dCyd. In addition to a high Kjj, value, the maximum rate of ddCyd phosphorylation was significantly less than that for dCyd with both enzymes. There is no evidence that
HIV induces any unique nucleoside metabolizing enzymes; therefore it is likely that one of these kinases is responsible for phosphorylation of ddCyd in HIV-infected cells.
Studies with extracts of human thymus revealed that at least two enzymes, dCyd kinase and Ado kinase have the capacity to catalyze the phosphorylation of 2', 3' - dideoxyadenosine (ddAdo) to the 5'-ddATP, but the relative contribution of these two enzyme pathways to ddAdo metabolism was not investigated (14). The pathways of ddAdo metabolism were investigated by Johnson et al. (15) with use of the human T-lymphoid cell line CCRF-CEM which is deficient in either dCyd kinase or Ado kinase activity, or both. Their findings indicated that ddAdo activation in human T-lymphoblasts could occur by either of the following pathways: directly, by phosphorylation to ddAMP by the action of either dCyd kinase or Ado kinase and, indirectly, through deamination to ddlno to ddIMP and reamination to ddAMP in a reaction catalyzed by adenylosuccinate synthetase and lyase. Activation of ddAdo through the direct route, however, was apparent only in the presence of a protective concentration of the adenosine deaminase inhibitor, 2'-deoxycoformycin. In noninhibited cells the indirect route is the major pathway
for the activation of ddAdo to ddATP in T-lymphoid cells.
One of the most widely used deoxynucleoside analog
antimetabolites for the treatment of human acute leukemias
is l-|3-D-Arabinofuranosylcytosine (ara-C) (16,17).
Metabolic phosphorylation of ara-C to its triphosphate
derivative, ara-CTP, is also required for its biological
action in vivo , and dCyd kinase is responsible for
activating ara-C to its monophosphate ara-CMP (18,19).
Deoxycytidine, the natural substrate, has a higher
affinity for dCyd kinase than ara-C (19-21). Resistance
of tumor cell lines to the growth-inhibiting effects of
ara-C has been associated with a decreased level of dCyd
kinase (19,22), with-a reduced level of intracellular
accumulation of ara-CTP, and a low level of incorporation
of ara-C into DNA (23). But, ara-C resistant cells had no
significant reduction of thymidine incorporation into DNA
(23). The role of dCyd kinase in the antitumor activity of the dCyd and ara-C analogs was further assessed by kinetic studies with dCyd kinase extracted from L1210/0 cells. All dCyd and ara-C analogs caused a competitive inhibition of dCyd kinase, the most potent inhibitor being
5-fluoro-dCyd (24).
dCyd kinase, unlike Ado kinase , is found primarily in lymphoid tissues in mouse (21) and man (25,26), a finding that has led to the suggestion that this distribution may be responsible for the selective
lymphotoxicity seen in adenosine deaminase (ADA) deficiency (25,26). When ADA deficiency occurs, the accumulated dAdo is phosphorylated to produce toxic levels of dATP (27,28). When adenine arabinoside is accumulated intracellularly, there is an increase of adenine arabinoside triphosphate levels (29,30).
The isolation of kinase-deficient mutant lymphoid cell lines has greatly clarified understanding of the enzymatic basis for purine deoxynucleoside phosphorylation. The impetus for such studies has been the suspected roles of dATP and dGTP accumulation in contributing, respectively, to the combined immune defect in ADA deficiency (25,27), and to the selective T-cell dysfunction in purine nucleoside phosphorylase deficiency (40).
The enzymes responsible for the phosphorylation of dAdo and ara-A were studied using mutant cell lines deficient in Ado kinase and dCyd kinase. These studies indicated that both Ado kinase and dCyd kinase have a role in adenosine analog phosphorylation (31-33).
For the treatment of lymphoid cancer, a potent ADA inhibitor, deoxycoformycin (dCF) has been used (34,35).
In determining the effectiveness of ADA inhibition as a treatment for lymphocytic leukemia, the most important
factor is selective toxicity, which is due primarily to
the expansion of the intracellular pool of dATP derived
from dAdo (25,27). Of the various types of ALL, T-ALL has been most responsive to treatment with dCF, and when incubated with dCF and dAdo in vitro , T-lymphoblasts accumulate large amounts of dATP and were killed (36). In contrast, cultured B-lymphoblastoid cell lines accumulate much less dATP from dAdo than do T-cell lines, and they are more resistant to dAdo toxicity (25,37,38). Different rates of dATP accumulation could result from different levels of enzymes that phosphorylate dAdo or degrade dAdo nucleotides. The levels of total dAdo phosphorylating activities, and of the two specific enzymes capable of catalyzing this reaction, dCyd kinase and Ado kinase, differ by no more than 2- to 4- fold in cytoplasmic extracts of a number of T- and B- cell lines (37,39).
Hershfield, et al. (39) isolated from the CEM human
T-lymphoblastoid cell line mutants that are deficient in either Ado kinase, dCyd kinase, or both activities, and observed that dAdo was toxic at concentrations below 100 uM, and concluded that both dCyd kinase and Ado kinase contribute to its toxicity by phosphorylating it.
Carson, et al.(41) designed experiments to determine if dAdo analogs which are not substrates for ADA might similarly be toxic toward lymphocytes and lymphoid tumors.
Two such compounds, 2-chloro dAdo and 2-fluoro dAdo were
converted to the respective triphosphates by intact human
lymphoblasts, with resulting inhibition of DNA synthesis.
Human B lymphoblasts deficient in dCyd kinase, but not in
Ado kinase, were resistant to the growth inhibitory
effects of the deoxynucleosides and failed to form
detectable 2-fluoro-dATP. The addition of dCyd to the
culture medium inhibited analog nucleotide formation and permitted the cells to grow normally. These results
strongly suggested that the toxicity of the dAdo analogs
required phosphorylation by dCyd kinase (41). However,
there is controversy over the identity of the nucleoside kinases that are responsible for intracellular phosphorylation of dAdo in ADA deficiency and of dGuo in purine nucleoside phosphorylation deficiency. To distinguish the nucleoside kinases present in T and B
lymphoblastoid cells, Osborne (42) coupled discontinuous
PAGE with autoradiography and showed that dCyd kinase, dAdo kinase and Ado kinase are all present in both T and B lymphoblasts. While Ado kinase is expressed at nearly equal levels in B and T cells, the deoxynucleoside kinases are expressed at much lower levels in B cells than in T cells. The reduced expression of dCyd kinase and dAdo kinase in B lymphoblasts may account for the lower 10
accumulation of deoxypurine nucleotides in B cells as
compared with T cells (42).
Mandelbaum, et al. (43) measured radioactive DNA
after incubation of normal and B leukemic peripheral
mononuclear cells from c-ALL and CLL patients with
labeled dThd and dCyd, and showed that for dThd,
incorporation into DNA was similar in normal and c-ALL
cells but lower in B-CLL cells, and that for dCyd,
incorporation was highest in c-ALL and lowest in CLL cells. These results also indicated that the dThd kinase and dCyd kinase are expressed at lower levels in B-CLL than c-ALL and normal cells.
Iizasa and Carson (44) reported that dividing human B
lymphoblasts, but not T lymphoblasts, release substantial amounts of dCyd into the medium, and have an active dCyd- dCMP interconversion cycle. Exogenous dCyd has been shown to protect T cells from the toxic effects of exogenous dAdo and dGuo (25). (Whether or not human lymphocytes are exposed normally to exogenous dCyd is not known.) Thus, the differential rate of dCyd release by T and B lymphocytes may affect the sensitivities of the two cell types to the growth inhibitory effects of exogenous deoxynucleosides (44).
Yamada, et al. (45) suggested that a T-lymphoblast- specific nucleoside kinase (TSK) might be a key enzyme in 11 different dysfunctions between T cells and B cells in
adenosine deaminase and purine nucleoside phosphorylase deficiencies. The phosphorylation rates of dAdo and dGuo in TSK containing T-lymphoblasts must be higher than the rates in B-lymphoblasts.
Based upon the kinetic properties ( apparent and
VmaX/Km) of dCyd kinase, Sarup and Fridland (46) suggested that dCyd kinase is potentially capable of functioning as a highly efficient salvage mechanism for deoxynucleosides and is capable of responding to a wide range of intracellular oscillations in dCyd and ara-C concentrations.
2. Affinity media for deoxynucleoside kinase
Because both bacterial and animal deoxynucleoside kinases comprise only a small fraction of total cellular protein, the synthesis of suitable affinity chromatography media was critical to the development of studies of these enzymes.
Deoxynucleosides linked to Sepharose through the 3'- hydroxyls, or through various positions on the purine or pyrimidine bases, were developed. dCyd, dAdo and dGuo 3'- derivatives were prepared and the interactions of the soluble or immobilized derivatives were studied with a variety of deoxynucleoside metabolizing enzymes ( 47 ). 12
Carbonyldiimidazole was chosen as the means of attaching
spacer arms to Sepharose because the linkage is both
stable and nonionic. The coupling of dCyd-, dAdo-, of dGuo-3'-(4-aminophenyl phosphate) to the carbonyl- derivatized Sepharose was almost stoichiometric. Using the dGuo-3'-(4-aminophenyl phosphate)-Sepharose affinity medium, mitochondrial dGuo kinase was purified in one step and characterized by Park and Ives (48).
Although deoxynucleoside kinases from other sources were purified successfully on some of these media, none of these 3'-hydroxyl-linked media retained the bacterial or mammalian cytosol deoxynucleoside kinases effectively
(47). In each instance, the attachment of a linker group to this position of a deoxynucleoside drastically weakened its binding by an enzyme.
Since nearly every known deoxynucleoside kinase utilizes a ternary enzyme complex between the enzyme and its ATP and deoxynucleoside substrates in the reaction mechanism, linking these substrates into multisubstrate analogs would create more tightly bound affinity ligands which might be linked to Sepharose through the relatively nonspecific "ATP end" of the molecule.
A series of multisubstrate-type inhibitors of the deoxynucleoside kinases were synthesized and used to construct media for affinity chromatography. dNp4A 13
(deoxynucleoside 5'-adenosine 5'''-p^,p^-tetraphosphate)
were synthesized and coupled to Sepharose and each dNp4A proved to be very useful as an affinity ligand for
resolving and purifying closely related kinase species
(49). These media have been found to be metabolically
stable, even in the presence of crude extracts, and are chemically stable at all but extreme pH values. Since these affinity ligands bind more selectively and tightly
to the active sites of deoxynucleoside kinases than either
ATP or deoxynucleosides, neither substrate alone is a suitable eluent for one of these affinity columns, but are moderately effective when used in combination. These bisubstrate affinity media were found capable of retaining not only the bacterial dCyd kinase (49) but also human cytosol dCyd kinase (50).
It was proposed by Ikeda and Ives that the deoxynucleoside triphosphate end-products (dNTP) also behave as bisubstrate inhibitors for many kinases (51), which led to their use in eluting enzyme from these affinity columns. The mode of binding of natural dNTP end products to the active sites of their target deoxynucleoside kinase from Lactobacillus acidophilus was investigated (51). It was found that natural dNTP bind more tightly (K^ = 0.4-3 uM) to the active sites of the corresponding bacterial kinase than do the dNp4A 14 bisubstrate analogues (K^ = 1.4-9.2 uM). It was proposed that the deoxynucleoside moiety fits at the deoxynucleoside binding site, determining the inhibition specificity, whereas the triphosphate group interacts with the ATP binding site, enabling the molecule to bridge these two subsites as a multisubstrate analog (51). These multiple binding determinants reinforce the affinity for the molecule, making it a potent end-product inhibitor.
Putative modes of binding of substrates and multisubstrate analogs at the active site of deoxynucleoside kinase are illustrated in Figure 1.
It was noticed that the bulky adenosine portion of dNp4A did not fit optimally at the ATP binding site, perhaps even interfering with proper alignment of the phosphate group within the ATP site. On the basis of these observations, new affinity media were constructed in which the elements of dATP or dCTP were linked to
Sepharose through the terminal phosphate, inserting an extra phosphate group between the dNTP and a hexyl group to replace the fourth negative charge lost in the covalent attachment of dNTP (52). The resulting ligand appeared to provide the basis for the ideal affinity medium for purifying dCyd kinase. Figure 2 shows structures of various affinity chromatography media developed in our laboratory. 15
Figure 1. Putative modes of binding of substrates and multisubstrate analogs at the active site of deoxynucleoside kinase (51). i, substrates (deoxynucleoside and MgATP) ; ii, dNp4A ; iii, dNpjA ; and iv, dNTP. Symbol : A, adenine ; B, any base ; R, ribose ; dR, deoxyribose ; and p, phosphate group. 16
h o c h2/ o . b A.
/ \ ? H S epharose - o - c - n h - c h 2- c h 2- n h - c - c h 2- c h 2-c-nh - NH i= NH V / / 7 7 / / / / 7 c. Sepharose B -NH-fCH^-O-P-O-P-O-P-O-P-O-i O l Sepharose o' °' o' o' Figure 2. Structures of affinity chromatography media. A. Structure of dN-3 1 -4-aminophenyl phosphate-Sepharose (47) B. Structure of dNp^A-Sepharose (49) C. Structure of dNp^-Sepharose (52) ( B may be bases adenine, cytosine, guanine, thymine) 17 Triphosphate end products (dNTP) and synthetic bisubstrate analogs (dNp4 A) exhibited identical modes of binding, and also proved to be useful tools for distinguishing kinetic mechanisms of kinases which follow a sequential pathway. For a random Bi Bi kinetic mechanism, dNTP is competitive vs. either substrate since it overlaps both substrate binding sites. For a kinase which follows an ordered Bi Bi kinetic pathway, dNTP competes only with the first substrate to bind and is noncompetitive with the second (51). It remains to be seen whether the mechanism of inhibition observed with the Lactobacillus enzyme is applicable to the majority of mammalian kinases, as well. 3. Multiple forms of enzymes The first generally accepted descriptive term for the existence of different molecular forms of proteins with the same enzymatic specificity was introduced by Markert and Miller (53), who coined the word "isozymes (or isoenzymes)" to describe this phenomenon. The earliest uses of the term isoenzymes were without implications as to the reasons for the existence of the multiple enzyme forms so described, although the problems posed for genetics by the multiplicity of proteins with a common activity were soon recognized (53). However, as the nature of some multiple forms of enzymes became clearer through genetic and structural studies, it became possible to define isoenzymes in terms of their genetic origins. According to the current recommendations of the Commission on Biological Nomenclature of IUPAC-IUB (54), isoenzymes are defined as multiple molecular forms of an enzyme occurring within a single species, as a result of the presence of more than one structual gene. The multiple genes may be due to the presence of multiple gene loci or of multiple alleles. Also included in this definition of isoenzymes are multiple forms of enzymes which arise by the association of protein subunits that are themselves products of distinct structural genes. Variant forms of enzymes which originate by post- translational modifications of a single polypeptide chain, as in the conversion of inactive precursors of proteolytic enzymes to their active forms, are not regarded as isoenzymes, nor are the covalently-modified (e.g. phosphorylated or dephosphorylated) or conformationally- different forms in which certain enzymes may exist, and through which regulation of their activities is effected (54). The definition of isoenzymes as the products of distinct structural genes implies that those multiple enzyme forms which fall within its scope will differ, to a 19 greater or lesser extent, in their amino acid sequences. In turn, these differences in primary structure will also entail greater or lesser differences in the higher levels of protein structure. The groups of genes which determine the structures of families of isoenzymes can represent several different phenomena (55): i) the existence of multiple gene loci, ii) the occurrence, as the result of mutation, of pairs of unlike genes (alleles) at the same locus, or, iii) the modification of structures or expression of genes in somatic cells, e.g. as an accompaniment of malignant transformation. In addition, active molecules of oligomeric enzymes may arise by the association of similar but non-identical subunits, either in vivo or in vitro. When the different subunits are the products of separate structural genes, the hybrid molecules thus formed are themselves included in the formal definition of isoenzymes. The different subunits concerned may be the products of separate gene loci or of allelic genes at the same locus (56). Although successful attempts have been made to demonstrate multiple forms of an enzyme, it is necessary to consider the possibilities that such observations are the result of artifacts of the experimental techniques employed, or of proteolytic degradation of an enzyme, or 20 of ill-defined phenomena such as aggregation or association of a single enzyme with other components. A partial list of the cells from which dCyd kinase has been isolated and studied includes: calf thymus (20,57-60), murine neoplasms (61,62), blast cells of acute myelocytic human leukemia (63), Lactobacillus acidophilus R-26 (64,65), human leukemic T-lymphoid and myeloid cells (46), MOLT 4F T cell extract (45) and human leukemic spleen (66,67). Considerable variability between species and tissues in the properties of dCyd kinase, and in the deoxyribonucleoside salvage metabolism in general, has been observed. The enzyme from calf thymus has a broad substrate specificity (57,58). However dCyd kinase purified from a murine neoplasm P815, either sensitive or resistant to ara-C, did not phosphorylate purine deoxynucleosides (61). Human cytoplasmic and mitochondrial dCyd kinases from blast cells of AML patients have been purified and compared. Both purified isozymes have similar molecular weights, activation energies and both catalyze the reaction by a sequential mechanism. These two isozymes differ with respect to their substrate specificities and their sensitivity to inhibition. With cytoplasmic dCyd kinase, Cyd and ara-C can both serve as substrates. On the other hand, the 21 mitochondrial enzyme phosphorylated dThd but,not Cyd or ara-C (63). Gower, et al.(6 8 ) reported the isolation of both mitochondrial and cytoplasmic enzymes from calf thymus that catalyzed the phosphorylation of dGuo. The cytoplasmic enzyme apparently is the nonspecific enzyme catalyzing the conversion of dGuo, dAdo, dCyd and Cyd to their corresponding nucleotides. The mitochondrial enzyme does not phosphorylate dCyd , Cyd or dAdo. Fabianowska-Majewska and Greger (69) reported that rat liver mitochondria contain dThd, dCyd, dAdo, and dGuo kinase activities. However few details regarding these activities were given. Yamada, et al.(45) identified a T-lymphoblast- specific nucleoside kinase (TSK), partially purified from human MOLT 4F T cell extract, that phosphorylated dCyd, dAdo, dGuo and ara-C, similar to dCyd kinase. They compared this TSK fraction with retained dCyd kinase activity; TSK has a much smaller Km for dAdo, and a higher efficiency ratio for dAdo than for dCyd. Its molecular weight was only 26,000 and it exhibited a very alkaline pi (8.2). Schrecker (19) suggested that the phosphorylation of dCyd and dGuo was mediated by the same enzyme in cell-free extracts of mouse L1210 cells. There is evidence that 22 certain deoxyribonucleosides or analogs may be substrates for more than one kinase. For example, the phosphorylation of 9-(3-D-arabinofuranosyl-2-fluoroadenine (2-F-ara-A) is mediated primarily by dCyd kinase (70,71), and that of 9-0-D-arabinofuranosyladenine (ara-A) by both Ado kinase and dCyd kinase(32). These adenosine analogs, ara-A, 2-F-ara-A are inhibitors of DNA synthesis in cell culture and in vivo , and they have antileukemic activity in mice (72). Previous work has established that at least two enzymes, Ado kinase and dCyd kinase are capable of phosphorylating dAdo, although less efficiently than they phosphorylate the substrates for which they are named (58,59,73). Sarup and Fridland (46) also showed that in human lymphoblasts and myeloblasts, purine deoxynucleosides and their analogs were phosphorylated by dCyd kinase, Ado kinase, and dGuo kinase. dCyd kinase was found to be the major purine phosphorylating activity in both cultured and noncultured T lymphoblasts and in myeloblasts. This dCyd kinase from either lymphoid or myeloid cells showed broad substrate specificity and dCyd was the preferred nucleoside substrate. The enzyme exhibited substrate activation with both pyrimidine and purine deoxyribonucleosides, suggesting that there is more than 23 one substrate binding site on the kinase. Hurley, et al. (74) found human placental dCyd kinase activity associated with dAdo or dGuo phosphorylating activities, but no dCyd kinase activity capable of using both purine substrates. Furthermore, an apparently homogenous murine dCyd kinase unable to phosphorylate purine deoxyribonucleosides has been described (61,62). Recently Datta, et al.(75) purified dCyd kinase from human T-lymphoblasts and the molecular weight was 60,000 and the O stokes radius was 32 A. The enzyme was a dimer, and the subunit molecular weight was 30,500. dGuo and dAdo and Cyd phosphorylating activities copurified with dCyd kinase Habteyesus and Eriksson (76) reported that changes in dCyd kinase activity during the cell cycle were not accompanied by changes in purine deoxynucleoside kinase activity. Bohman and Eriksson (67) purified dCyd kinase from human leukemic spleen. Only one form of dCyd kinase activity was found, and the subunit molecular weight was 30,000. The same enzyme molecule phosphorylated dCyd, dAdo and dGuo. The apparent molecular weight of the active enzyme was 60,000. The enzyme activity was strongly inhibited by dCTP, which was noncompetitive towards dCyd and competitive with ATP-MgC^. 24 Purified dCyd kinase, obtained from mouse sarcoma cells (61,62) differs in several respects from the human enzyme. Therefore, there are apparently considerable species variations, among mammalian cells, in the structure and function of dCyd kinase, and it is also unclear how many multispecific kinases are expressed in human cytosol Progress towards understanding the mechanisms of regulation of human deoxynucleoside kinases has been impeded by the bi-modal or non-linear kinetic patterns sometimes obtained when nucleoside or ATP is varied (39,46). Such, results mimic those seen earlier with animal cell preparations (58,60). Typically, double reciprocal plots have yielded two slopes, from which two KjpS have been derived. It has not been clear whether this phenomenon is due to cooperative behavior or to a mixture of enzyme forms. We demonstrated that this kinetic phenomenon can be produced experimentally with a heterogeneous mixture containing native and proteolytically-degraded enzyme (50). Many different properties and variations for the dCyd kinase have been described and not all of these conflicting results can be equally correct. It is quite possible that the enzymes isolated and characterized from other sources may be a modified version of the native enzyme, with corresponding changes in their properties. EXPERIMENTAL PROCEDURES 1. Materials Human leukemic lymphocytes collected by leukapheresis were provided by the Tumor Procurement Service of the OSU Comprehensive Cancer Center as coded specimens. MOLT 4 cells (cultured human T-lymphoblasts) were provided by the Cell Culture Laboratory of the OSU Comprehensive Cancer Center. Nucleoside and nucleotides were obtained from Sigma, Boehringer Mannheim, P-L Biochemicals, Plenum, United States Biochemicals or Calbiochem. Tritiated nucleosides were supplied from ICN . The affinity ligand, dCp^A, was synthesized in our laboratory. Adipic acid dihydrazide-Agarose purchased from Sigma was coupled to periodate-oxidized affinity ligand to make the affinity column (49). All protease inhibitors were purchased from Sigma. Chemicals for polyacrylamide gel electrophoresis were supplied by Bio-Rad Laboratories and Serva. Bradford 25 26 reagent was obtained from Bio-Rad Laboratories and Deoxycoformycin was a gift from the National Cancer Institute. Nitrocellulose membrane was purchased from Schleicher & Schuell. The immunoassay kit (biotinylated alkaline phosphatase) was obtained from Vector Laboratories. 2. Methods i. Enzyme assay Deoxynucleoside kinase activities were assayed using the disk anion-exchange method as described by Ives (77). The final concentrations of dCyd kinase assay components were: ATP, 10 mM; MgCl2, 12 mM; Tris-HCl (pH 7.5 at 37°C), 0.1 M; dCyd, 20 uM; [^H]dCyd (0.5 uCi/assay); 5% glycerol and dithioerythritol(DTE), 2 mM. The total assay volume was 80 ul, and the temperature of incubation was 37°C. The reaction was carried out for 30 minutes and stopped by putting the assay vials in the boiling waterbath for 2 minutes, followed by the addition of 0 . 2 ml of distilled H20 to each assay vial. In kinetic experiments, the reaction was stopped by adding 0.2 ml of 0.1N formic acid to each assay vial. 2 0 ul aliquots of diluted reaction mixture were spotted on 10 in x 10 mm squares of Whatman DE-81 anion exchange paper. These were washed free of unreacted substrate with circulating water for 30 minutes and the paper disks were dried. The disks were then placed „ into scintillation vials, eluted with 0 . 2 ml of HCl/KCl to extract radioactive nucleotide products, and the radioactivity was counted in the continuous phase of a Triton/Toluene liquid scintillation cocktail. The dpm retained on the ion-exchange disk were directly proportional to the enzyme activity in the assay mixture. Scintillation cocktail was prepared with 16.5 g of RPI preblend 2a60 (91% PPO and 9% bis-MSB), 2L of toluene and 1 L of Triton X-100. The kinase activity was calculated as follows: % conversion = (cpm of reaction - cpm in washed blank) x 1 0 0 of substrate cpm in unwashed control Enzyme Activity = nucleoside (nmole)/ tube x % conversion (nmole/min/ml) 100 x _ 1 ____ x 1 0 0 0 ul of enzyme 30 min. 20 ul One enzyme activity unit is defined as that amount of enzyme which catalyzes the formation of 1 nmole of deoxynucleotide per minute. 28 ii. Preparation of crude extracts from human leukapheresis specimens Human leukemic lymphocytes concentrated by leukapheresis were supplied by the Tissue Procurement Service of the OSU Comprehensive Cancer Center. Counts of the % of blast cells, and immunological tests determining T- or B- cell origin of cells expressing lymphoid antigens were done by the OSU cellular Immunology Laboratory. All the steps in the preparation of crude extracts and enzyme purification were done at 4°C. Human leukemic cells were centrifuged at 4,000 x g for 15 minutes in the Sorvall rotor. The supernatant serum was aspirated and discarded. The red cells were lysed by hypotonic shock by adding distilled water to the cell pellet and stirring for 2 0 seconds in a centrifuge bottle followed immediately by addition of equal volumes of twice-isotonic Seligmann balanced salt solution (SBSS) to restore isotonicity. 1 L of SBSS contained NaCl, 7.650 g; KCl, 0.200 g; Na-acetate, H 2 O 0.050 g; KH2 PO4 , 0.100 g; D-glucose 1.000 g; NaHCC^, 0.700 g; and ascorbic acid, 0.003 g . This solution also contained 0.05 % EDTA and 2 mM DTE. The cells were centrifuged at 4,000 x g for 10 minutes, and, after aspirating the supernatant, the hypotonic lysis was repeated. The white cells were collected by centrifugation and were suspended in isotonic 29 SBSS. Then the suspended white cells were sonicated in an ice bath with the micro probe of a Branson sonicator on a 2 0 % duty cycle (pulsed) and a power setting of 6 . The particulate fractions were sedimented at 20,000 x g for 2 0 minutes, and the supernant fraction was stored at -80°C. For purification of enzyme, 10% streptomycin sulfate solution (pH 7.0) was added to the crude extract drop by drop to a final ratio of 1 g of streptomycin sulfate per g of protein. After the suspension was centrifuged, the supernatant portion was fractionated with ammonium sulfate (enzyme grade) , which was added to the supernatant liquid as the solid, little by little, to 30% of saturation. After centrifugation at 20,000 x g, additional ammonium sulfate was added to the resulting supernatant fraction, raising the level of saturation to about 63%. The precipitate was collected by centrifugation and dissolved in 20 mM Tris buffer (pH 8.0, at 4°C) containing 20% glycerol, 2 mM DTE and 2 mM EDTA. The ammonium sulfate fractions were stored at -80°C. iii. Protein assay The protein concentration of the enzyme was measured by the Bradford method (78). Bovine plasma gamma globulin was used as a standard protein. Concentrated Bio-Rad dye 30 reagent (0 . 2 ml) was added to 0 . 8 ml of protein solution. The absorbance was measured at 595 run and the protein concentration was determined by interpolation from a standard curve. iv. Polyacrylamide gel electrophoresis Discontinuous non-denaturing polyacrylamide gel electrophoresis, in gels ranging from 8 to 15% acrylamide, was run according to the procedure of Laemmli, but without SDS (79), using the BIO-RAD model 360 mini vertical slab cell. The stacking gel was 4% and these gels were run in a non-denaturing mode at 4°C. Sodium Dodecyl Sulfate (SDS) polyacrylamide gel electrophoresis was run by the Laemmli method. (79) with 4% stacking gel (in 0.125 M Tris-HCl, pH 6 .8 ) and 12% separating gel (in 0.375 M Tris-HCl, pH 8 .8 ). Sample buffer containing SDS and 8 mM DTE was used to denature proteins by heating at 95°C for 4 minutes. SDS gel electrophoresis was run at room temperature. To see the protein band, after development, the gel was placed in fixative for at least 30 minutes on a shaker- table. A solution of 40% methanol/ 10% acetic acid was used as a fixative. The gel was then stained with 0.1% Coomassie Brilliant Blue R-250 in fixative and destained with 1 0 % methanol/ 7.5% acetic acid to remove the 31 background. With this procedure microgram-amounts of proteins could be detected in the bands. To identify which protein band contained the enzyme activity in non-denaturing gels, an unstained parallel channel was cut into 2 mm slices, soaked in assay mixture overnight, and the enzyme activity assayed (6 8 ). v. Molecular weight determinations The relative molecular mass (Mr ) of the native enzyme was determined by sedimentation equilibrium in a Beckman Airfuge (30° angle rotor) by the Pollet method ( 80 ), modified as described previously ( 81 ). The air-supply temperature was maintained at 4°C, and the initial sample density was 1.025 g/ml. Either bovine serum albumin or Dextran T-40 was added (5 mg/ml) to the dilute dCyd kinase samples to stabilize the density gradients, and glycerol was added to standard tubes to equal its concentration in the diluted kinase samples (6.7%). Ten successive 10 ul samples were withdrawn from the meniscus of each tube and assayed for enzyme activity or protein content. Molecular weights were also estimated by the observing the effect of polyacrylamide gel concentration (8 , 10, 12 and 15%) on the electrophoretic mobilities of native proteins, as described by Hedrick and Smith (82). Standard proteins included beta-lactoglobulin, carbonic 32 anhydrase, ovalbumin and bovine serum albumin. The slope of log Rf vs. gel concentration was determined for each protein, and molecular weights of unknown proteins were estimated by interpolation on a plot of slope vs. molecular weight. vi. Purity of reagents for kinetic experiment The purity of the ADP used for kinetics was checked by using the Mono-Q column on the Pharmacia FPLC (commercial ADP is generally contaminated with ATP). ADP or ATP (0.05 umole in 500 ul) was loaded onto the column equilibrated with 0.01 N HC1. After washing with 2.0 ml of equilibrated buffer, the ADP was eluted with a 20 ml linear gradient of NaCl (0 - 0.5 M) dissolved in 0.01 N HCl, pumping at the rate of 0.5 ml/min. vii. Immunization of rabbits Disk implantation method— New Zealand white rabbits (3-4 Kg) were allowed to stabilize for 3-4 days after arrival. Prior to immunization, the animal were bled for preimmune serum by means of a longitudinal cut on the marginal ear vein. The rabbit was weighed and the thigh was shaved with electric clippers. Nitrocellulose disks prepared with a 1 -hole punch were sterilized in a petri dish under UV light for 30 min. Using sterile micropipet tips, each disk was moistened with 15 ul of solution containing 0 .2 -0 .3 ug of antigen protein sterilized through a small disposable Durapore (Millipore) membrane filter. The dish was covered, and the disks were allowed to dry for an hour or two. The rabbit was anesthetized with Ketamine-HCl (40 mg/Kg) and Romphan (5 mg/Kg) administered intramuscularly. Iodine tincture was painted on the shaved area. Five longitudinal 1 cm incisions were made through the skin and then into the thigh muscle of one leg. To each nitrocellulose disk 25 ul of Freund's complete adjuvant was applied and immediately two disks were implanted in each incision in the muscle. (Any bleeding should be controlled by pressure with a sterile surgical sponge). A total of 2 ug of antigen protein was used for the first immunization of each rabbit. The muscle was then closed by suturing with Chromic 1 sterile gut (starting at the center of the wound), followed by stitches in the skin with surgical silk. Sterile penicillin (150,000 units) was administered by injection into the muscle of the other hind leg. As a further precaution against infection, Tetracycline (1.3 mg/ml) was added to the drinking water. After three weeks the animal was given a smaller booster dose of antigen. The animal was anesthetized as before, and two small incisions were made in the other thigh muscle. Freund's incomplete 34 adjuvant (25 ul) was applied to each nitrocellulose disk and the two disks were placed in each incision. These disks should carry a total of no more than 1 ug of antigen protein. The wounds were closed and the animal was maintained as before. Three days after the booster immunization, the animal was bled from the ear vein, repeating at 7-10 day intervals. After four collections of immune serum, a second booster immunization was performed as described before, with 1 ug of antigen protein and the animal was bled and killed by total exsanguination via cardiac puncture while under Ketamine anesthesia. Nitrocellulose powder method— The nitrocellulose adsorbent was first finely divided in a small blender, in an aqueous suspension. Larger particles were allowed to settle out, and only the milky suspension of the smallest particulates were decanted and washed by centrifugation. At 4°C, about 20 ug of homogenous enzyme protein was allowed to adsorb onto 130 ul of the packed nitrocellulose powder equilibrated with 15 mM potassium phosphate (pH 8.0), containing 20% glycerol. After resuspending continuously with a vortex mixer overnight, the nitrocellulose and its adsorbed protein was collected by centrifugation. The supernatant solution should contain less than 1% of the original activity. The pellet was 35 suspended in equal amount of Freund's complete adjuvant, and the mixture was thoroughly emulsified by vortex mixing. With a 21 ga. needle, the emulsion was injected into multiple sites along the back muscle of a New Zealand rabbit. Three weeks after the initial challenge, a booster dose (10 ug of protein on 50 ul of nitrocellulose), emulsified in Freund's incomplete adjuvant, was injected intramuscularly. Four weeks later, a second booster was administered subcutaneously at various sites on the back. Blood sample were collected from the ear vein until a suitable titer was reached, then the animal was exsanguinated under anesthesia. viii. Collection of serum Blood was collected by direct opening of an ear vein or by heart puncture. After obtaining blood, the serum was separated (83). The tube was covered and allowed to stand for one hour at room temperature or in the 37°C incubator for clot formation. The clot was separated from the wall of the tube with a wooden applicator stick and the tube of blood covered with parafilm was allowed to stand at 4°C for 24 hours. The serum was removed into clean centrifuge tubes and centrifuged at 1,000 x g for 30 minutes at about 4°C. A small amount of hemolysis often occurred, but in general a trace of hemoglobin was of 36 little significance. However, when the centrifuged serum was not clear, which was usually caused by lipid, centrifugation was repeated or the serum was filtered through filter paper. The supernatant serum was distributed into small vials and stored at -80°C. ix. ELISA (Enzyme-Linked-Immunosorbent-Assay) A solution containing purified dCyd kinase (antigen) in coating buffer(1.59 g Na2 CC>3 , 2.93 g NaHC03 in 1 L ddH2 0 , pH 9.6) was prepared and 100 ul each of this solution was added to the wells of an Immulon II microtiter plate. Blank wells for controls were filled with coating buffer without antigen. The plate was parafilmed and incubated overnight at 37°C in humid chamber. The plate was washed by flicking the plate rapidly over the sink to remove the solution and 2 0 0 ul of 0.01 M Phosphate buffer(PB) was added. A blocking solution containing 1% BSA in PB was added and the plate was incubated at room temperature for 1 hour. The plate was washed three times with 0.01 M PBS-Tween (PBS containing 0.05% Tween 20 — 8 g of NaCl and 0.5 ml of Tween 20 to 1 L of 0.01 M PB, pH 7.4). 100 ul of diluted serum samples were added to the appropriate wells, including a set of positive controls, (serum containing antibody) and normal serum control (preimmune serum). 37 Dilutions of serum started at 1/100 in PBS-Tween, and the plate was set up so that A1 was blank which can be read automatically by ELISA plate reader. The plate was incubated for 1 to 2 hours at room temperature. The plates could be incubated overnight at 4°C if the antibody concentration was low but this also increased the background. The plate was washed three times with PBS- Tween. 100 ul of a 1/1000 dilution of the peroxidase conjugated goat anti-rabbit IgG in PBS-Tween was added and incubated for 1 to 2 hours at room temperature. The plate was washed three times with PBS-Tween. 100 ul of the peroxidase substrate solution was added to each well. The substrate solution contained 4 mg of o-phenylene diamine and 10 ul of 30% H 2 0 2 in 10 ml of Phosphate citrate buffer— 24.3 ml of 0.1 M citric acid, 25.7 ml of 0.2 M Na 2 HP04 in 100 ml of ddH2 0, pH 5. The reaction was stopped at 15 to 30 min. by adding 50 ul of 1.25 M H2 S04 to each well. If the reactions were rapid, the 15 min incubation was used, and if they were less rapid the 30 min incubation was used. The A4 9 0 of each well was read on the ELISA plate reader. x. Western immunoblotting Electrophoretic transfer of proteins from a polyacrylamide gel to a nitrocellulose membrane was 38 performed by the modified method of Towbin et al. (84). SDS-PAGE was carried out with the mini vertical slab-cell using prestained marker proteins, which are useful in observing the completeness of electrotransfer. After electrophoresis was done, the stacking gel was trimmed off and a "Trans-Unit" was assembled as described in the Janssen instructions (85). The graphite plates of the semidry electroblotter were rinsed with distilled water. Six layers of filter paper which were soaked in anode solution No. 1 (0.3 M Tris., 20% v/v methanol, pH 10.4) were placed on the anodic graphite plate. Sheets of filter paper were dipped in electrolyte one at a time, letting excess liquid drip off. Each sheet was laid with a progressive rolling motion to displace air bubbles. A Trans-Unit was assembled on top of the six layers of filter paper, in sequence, as follows: a) Place three layers of filter paper, soaked in anode solution No. 2 (25 mM Tris., 20% v/v methanol, pH 10.4). b) Place nitrocellulose membrane pre-rinsed in distilled water, blotted dry and rinsed in anode solution No.2. c) Place the SDS-PAGE gel, wetted with a very small amount of cathode solution, and to eliminate any air bubbles, squeeze out the bubbles using a gloved finger. d) Place three layers of filter paper soaked in cathode solution ( 25 mM Tris., 40 mM 6 -amino-n-hexanoic acid, 20% v/v methanol, pH 9.4). The Trans-Unit was covered with six layers of filter paper soaked in cathode solution and then covered with the cathode plate which was attached to the lid of the apparatus. The apparatus was connected to a power supply at a constant current of 0.8 mA/cm2 of gel for 2 hours at room temperature. Note that the graphite plate attached to the base of the apparatus is the anode;the graphite plate attached to the lid of the apparatus is the cathode. It is essential that all filter papers and nitrocellulose paper be exactly the same size as the SDS-PAGE slab gel. To avoid transfer of protein from the skin, wear gloves when handling these materials. xi. Immunodetection Before immunodetection was carried out, remaining protein-binding sites on the nitrocellulose were blocked by immersing the nitrocellulose membrane in 1 0 % nonfat dry milk in Tris-buffered-saline(TBS; 100 mM Tris-HCl, pH 7.5 containing 0.9% w/v NaCl). The nitrocellulose membrane was covered and allowed to stand overnight in the cold room. The nitrocellulose membrane was washed with 0.1% (v/v) Tween 20 in Tris-buffered saline (TTBS). The moist nitrocellulose membrane was cut into corresponding channels. It was helpful in locating the channels to lay the Teflon gel comb on the membrane. The unused strips were sorted and saved for repeated immunoassays, if necessary. Each strip was placed in an S&S Accutran tray and rinsed three times with TTBS. The tray was agitated gently and the slots were aspirated with a pasteur pipet, tilting to drain the liquid. Diluted rabbit anti-human dCyd kinase serum was placed in each slot and agitated gently for more than 30 min., making sure the solution completely wetted the strip of nitrocellulose membrane. The slots were aspirated and washed with 3-4 changes of TTBS over 15 min. with gentle agitation. To each slot a 5ug/ml solution of biotinylated secondary antibody (goat anti-rabbit IgG, 1 drop of VECTASTAIN ABC-AP kit in 10 ml of TTBS), was added and incubated for 30 min. with gentle agitation. The slots were aspirated and washed 3 times with TTBS containing 0.5 M NaCl. Avidin-alkaline phosphatase complex (VECTASTAIN ABC-AP reagent) was added and allowed to stand for 30 min. with gentle agitation. This reagent was prepared by adding 2 drops of reagent A (avidin DH) to 10 ml of TTBS containing 0.5 M NaCl, followed by 2 drops of reagent B (biotinylated alkaline phosphatase); it was mixed immediately and allowed to stand 30 min. to complete complex formation before use. The slots were aspirated and washed 3 times to remove all excess enzyme complex. Nitrocellulose strips were transferred to new S&S Accutran trays and substrate solution was added. [ The substrate solution was prepared immediately before use by adding 2 drops of reagent 1 of VECTASTAIN substrate kit II to 5 ml of 100 mM Tris-HCl, pH 9.5. After mixing and, 2 drops of reagent 2 were added with mixing, followed by 2 drops of reagent 3 and further mixing. ] Development of the insoluble black product was allowed to proceed for 15-30 min, or until suitable color was seen. Membranes were washed twice with distilled water, over 10 min. and allowed to air dry. Immunostained membranes were stored in the dark. RESULTS 1. dCyd kinase purified by dCp^A-Sepharose affinity chromatography An ammonium sulfate fraction (30-63%) of human T-ALL cells was applied to dCp4 A-Sepharose column with protease inhibitors (1 ,1 0 -phenanthroline, benzamidine, and diphenylcarbamyl chloride) and dCyd kinase activity was eluted biospecifically by its inhibitory end-product, dCTP. A near-saturating mixture of both substrates, dCyd and ATP was considerably less effective than dCTP as an eluent, and salt and ATP did not displace the enzyme from the column. Therefore enzyme interaction with the column was probably not due to ion-exchange or broadly-specific nucleotide interactions with the phosphate-donor site of the enzyme. The amount of activity in the run-through fraction depended on the load of protein applied, and activities in the eluted fraction varied from 13-60% of the activity applied. The specific activity of the enzyme eluted was about 21 nmole/min/mg. Columns were 42 43 used repeatedly, over a period of a year or more, with only a gradual deterioration of performance being noted. Regardless of sample load or eluent concentration, not all of the activity could be eluted immediately on addition of dCTP, and pronounced tailing was seen. An additional peak always emerged if the column flow was resumed after standing overnight in the presence of dCTP. Extensive comparisons were made in establishing the common identity of the two peaks; the apparent Kj^s, native and SDS-gel electrophoretic patterns, isoelectric pH values and molecular weights were virtually identical (8 6 ). We conclude that enzyme dissociation is a slow step. The fact that all of the recoverable enzyme emerges in a very small volume— if the column sets overnight with dCTP— suggests that it is a slow off rate rather than an unfavorable dissociation equilibrium which causes initial tailing of the activity. Therefore, the column was allowed to stand overnight with dCTP before enzyme was collected as a single peak. 2. Enzyme purity and subunit molecular weight Judging from the single band obtained with the affinity-purified enzyme in SDS-polyacrylamide gel electrophoresis, this preparation was virtually homogeneous. Only one band of protein was observed upon electrophoresis in nondenaturing gels, as well ( 8 6 ). Assurance that the stained band was, in fact,, the enzyme was provided by activity assays in gel slices from a parallel channel. A series of such assays on gels containing several concentrations of acrylamide monomer revealed the congruent migration of activity and the protein band. Had the protein bearing the activity and the protein yielding the stained band been different molecular species, they could have been expected to diverge as the gel concentration was changed (82). Since they did not, and since only one band was detected by either procedure, it can be concluded that the enzyme was practically homogeneous. The molecular weight of 52,000 + 2000 was determined for the silver- stained protein band in SDS-polyacrylamide gel electrophoresis (Figure 3). Silver-staining gave a sharper band, with a slightly higher relative mobility, than did larger amounts of protein stained with Coomassie Blue. 3. Molecular weight of native enzyme Sedimentation equilibrium determinations (80) were carried out with different batches of dCyd kinase prepared in the presence of protease inhibitors. An average molecular weight of 59,300 + 3700 was obtained for the 45 5.0 4.8- 4.6-- C7> O 4.4-- 4.2- 4.0 0.0 0.2 0.4 0.6 0.8 1.0 Rf Figure 3. Determination of the polypeptide molecular weight by SDS gel electrophoresis. Standard proteins are as follows: a, bovine albumin; b, egg albumin; c, glyceraldehyde-3- phosphate dehydrogenase; d, carbonic anhydrase; e, trypsinogen ; f, trypsin inhibitor; g, a-lactalbumin. E indicates the of the purified dCyd kinase. 46 native enzyme, assuming a partial specific volume of 0.725. An identical value was derived from analysis of the mobilities of the enzyme activity after electrophoresis in non-denaturing polyacrylamide gels of various concentrations, by the method of Hedrick and Smith (82), even though the latter determination is affected by the shape as well as the mass of the molecule. Although the molecular weight estimated for the native enzyme (Figure 4) is somewhat larger than the 52 Kd value determined for the denatured polypeptide, it seems clear that this enzyme consists of a single polypeptide chain. 4. Substrate and donor specificity The pure protein retained the ability to phosphorylate dAdo and dGuo, but dCyd is clearly the preferred substrate in terms of its apparent and efficiency ratio, as shown in Table 2. Potential phosphate donors were also compared, each at 10 mM (with 12 mM MgCl2). Relative rates are shown in Table 3. These patterns of specificity are similar to those seen with a variety of other mammalian dCyd kinase preparations, and it seems likely that the purine deoxvnucleoside kinase activities are inseparably associated with the dCyd kinase protein of humans, as well as of other mammals (20,21,46,59,60,67,75). These nucleotides were used I 47 0.10 0.08- 0.04 •• 0.0 2 - 0.00 20 40 60 80 - 3 M. W. x 10 Figure 4. Estimation of the molecular weight of the native dCyd kinase by the "Ferguson" relationship. The slopes of plots of mobility vs. total acrylamide concentration were determined for a series of standard proteins, and were re-plotted to reveal the affect of Mr. Standard proteins are as follows: a, bovine serum albumin; b, ovalbumin; c, carbonic anhydrase ; d, p-lactoglobulin monomer. E indicates the purified dCyd kinase. 48 Table 2 Substrate specificity of human deoxycytidine kinase Nucleoside ^(app) vvmax Efficiency Ratio (uM) (nmol*min-1 *mg-1) *1^ expressed in mM units. 49 Table 3 Phosphate donor specificity of human dCyd kinase Phosphate donor Relative rate ( % ) ATP 1 0 0 GTP 61 dGTP 59 dTTP 37 CTP 24 dATP 23 UTP 6 dCTP 0 50 straight from the bottle without any repurification, so it is possible -that the relatively low activity seen with UTP could have been due to an inhibitory level of UDP (58). 5. Conditional proteolytic modification during purification Enzyme purified in the absence of protease inhibitors showed evidence of substantial proteolysis during affinity chromatography, with the parental 52,000 dalton band giving place to bands of 30,000 and 33,000 dalton. Figure 5 shows very clearly that nearly all of the native protein in the eluate was replaced by two smaller fragments which stain with roughly equal intensity. The discrete character of the proteolyzed bands made it appear likely that the 30,000 and 33,000 dalton bands were the result of a proteolytic cleavage event. The proteolyzed enzyme retained activity despite altered molecular properties, but that activity was labile and was soon lost. The electrophoretic Rj of the proteolyzed enzyme activity in nondenaturing gels shifted to about 0.60 (Figure 6 ), compared with 0.47 for the native enzyme run under the same conditions. A less active minor component was also seen at Rj 0.35, corresponding to a barely-detectable band on the stained gel. The increased Rj after proteolysis is presumed to be 51 1 2 3 kDa 68- Figure 5. SDS-polyacrylamide gel electrophoresis of dCyd kinase purified with and without protease inhibitors. Lane 1, kinase purified with protease inhibitors; Lane 2, kinase purified without protease inhibitors; Lane 3, diluted kinase purified with protease inhibitors. Arrows denote R^s of molecular weight marker proteins. 52 proteolysed dCk proteolysed dAk native dCk native dAk 20,000 10,000 L_ 0.2 0.4 0.6 0.8 1.0 Rf Figure 6 . Non-denaturing polyacrylamide gel electrophoresis of parental and proteolysed dCyd and dAdo kinase. Assays were performed on gel slices from an unstained adjacent channel. The acrylamide monomer concentration was 1 2 %. 53 due to a reduced molecular mass, but activity losses prevented the completion of molecular weight determinations. Figure 6 also shows that, for both the parental and proteolyzed enzyme, dAdo kinase activity is exactly congruent with dCyd kinase activity, as would be expected if both substrates were phosphorylated by the same protein in each fraction. However, it should not necessarily be inferred from this figure that the ratio of dCyd/dAdo kinase is constant; since the assay of gel slices extended over a number of hours, differential loss of enzyme activities could have occurred, so the radioactivities plotted should, be viewed in qualitative rather quantitative terms. 6 . Kinetic effects of proteolysis We wondered whether a mixture of the parental and proteolyzed enzyme might account for the non-linear kinetics often observed with dCyd kinase. Although other human leukemic cell extracts studied have yielded such broken line kinetics (39,46,67), with this T-ALL cell preparation, we did not succeed in trapping the optimum blend of molecular species which would show this most clearly. Therefore, approximately equal amounts of partially proteolyzed and unproteolyzed protein were mixed and the kinetic results were shown in Figure 7. Apparent 54 2 0 0 0.2 0.4 0.6 0.8 1.0 1/[dCyd], jjM-1 60 60 40 40 v v 20 20 5 1/[dCyd],/ j M~ 1 1/[dCyd], pM-1 Figure 7. Lineweaver-Burk kinetics of mixture of parental and proteolyzed dCyd kinase. Each 80 ul reaction mixture contained 0.19 ug parental enzyme and/or 0.16 ug proteolyzed enzyme. ATP was 10 mM and MgCl2 was 12 mM. A. Mixture of parental and proteolyzed enzyme proteins; B. Parental protein fraction only; C. Proteolyzed fraction only. 55 values of 1.8 and 9.3 uM were obtained for dCyd from the slopes of this plot, 7A. Individually (7B & C), each preparation yielded a straight line, but the proteolyzed enzyme appeared to have a significantly larger apparent (13 uM, compared with 3.3 uM for the parental enzyme). 7. Kinetic mechanism of dCyd kinase from T-ALL cells i. Time-course of dCMP production dCyd kinase was incubated in the assay mixture and activity was assayed every 5 minutes to determine the optimal conditions for the kinetics. Figure 8 shows that up to 25 minutes, the production of dCMP was linear and that 12% of the dCyd was converted to dCMP when the dCyd concentration was 0.5 uM. But after 25 minutes, the production of dCMP leveled off. Therefore, whenever the kinetic studies were performed, the incubation time was 25 minutes. ii. Initial velocity measurements in the absence of products All the figures in the kinetic studies were drawn by a computer-driven plotter, using the non-weighted linear regression analysis feature of Sigma Plot. Lineweaver-Burk plots of reciprocal velocity versus reciprocal concentration of dCyd at various fixed rdc, dCMP. product, Figure Figure % Conversion to dCMP 20 10 15 5 0 0 8 Tm oreo h ovrin f Cd to dCyd of %conversion the of course Time . 10 20 Time (minutes) 30 050 40 070 60 56 57 concentrations of MgATP give a family of lines which intersect to the left of the vertical axis. A sequential mechanism leading to a ternary complex is suggested by the converging reciprocal plots shown in Figure 9. Since neither family of lines converges on the abscissa, it is apparent that the limiting values are not identical with the respective dissociation constants of the individual substrates. The apparent values for dCyd increase as the fixed concentration of MgATP decreases. Replotting the slope against reciprocal concentrations of MgATP provides the value for the of dCyd, 0.94 + 0.15 uM. Using the same set of data points, the Lineweaver-Burk plots of variable MgATP concentration at various fixed concentration of dCyd give a similar plot which intersects at a common point (Figure 10). Slope and intercept replots, which were all linear, yielded the kinetic constants, listed in Table 4. Thus, a process which requires the dissociation of one product before the addition of the second substrate is ruled out, and the mechanism is a sequential one in which all the substrates bind to the enzyme before the first product is formed. Figure 9. The effect of varying dCyd concentration on the on concentration dCyd varying of effect The 9. Figure reaction rate at fixed concentrations of MgATP. of concentrations fixed at rate reaction Slope V 10 20 15 0 5 0 20 30 40 50 5 /MAp-,m- /MAp-, mM"1 1/[MgATp2-], mM-1 1/[MgATp2-J, 10 0.4 20 0.8 /dy] u ^ uM 1/[dCyd], 25 . 1.6 1.2 10 15 0 5 0 5 [MgATP]: 10 0.15 mM0.15 0.042 mM0.042 .90 mM.90 2.0 520 15 58 25 iue 0 Teefc fvrigMAPcnetain on concentration dCyd. ofMgATP varying of effect concentrations The fixed at rate reaction the 10. Figure Slope 0.5 0.0 1.0 0.0 0.4 /MgT2-] m 1 - mM ], - gATP2 1/[M 50 30 40 2.0 0 30.0 20.0 5 0.0 0.0 10 0.4 [dCyd]: /dCyd] uM ], d y C l/[d 0.8 15 . uM 2.5 • .3 uM 0.833 1.2 rB25 20 -1 uM 59 25 2.0 60 Table 4 Summary of kinetic constants for the two substrates Kinetic A Value Varied Pattern constant (uM) substrate KmA(ATP) 30 + 1.5 dCyd, MgATP Converging Kia(ATP) 73 + 20 II K ^ (dCyd) 0.94 + 0.15 11 •A? Kinetic constants were obtained from slope and intercept replots of primary kinetic plots. 61 iii. Initial velocity measurements in the presence of products Initial velocity measurements in the presence of products were conducted to determine whether substrate binding is an ordered process, or not. Products may act as inhibitors by occupying the same site as the substrate from which it is derived. Inhibition experiments with ADP were difficult since it was necessary to use high concentrations of ADP to produce variations in slope due to relatively weak inhibition by this product. Under these conditions, high concentrations of ADP may form a complex with magnesium and deplete the magnesium in the assay mixture although ADP is a far weaker chelator than ATP. Therefore, the total magnesium concentration should be changed according to the ATP and ADP concentrations to keep the concentration of free magnesium (Mg+2) constant (Appendix). If ADP used in kinetics was contaminated with ATP, the kinetic results would be subject to error. Therefore, the purity of ADP was checked by chromatography on Pharmacia FPLC unit equipped with a Mono Q anion-exchange column., For comparison, ATP was also chromatographed. As seen in Figure 11, the ADP used in kinetic experiments did not show any contamination of ATP. 62 0.09 M A __f 0.18 M Figure 11. Purity analysis of reagents by FPLC. 0.05 umole of ADP or ATP was loaded onto a Mono-Q column and eluted with linear gradient of 0-0.5 M NaCl in 0.01 N HCl. The Absorption Unit was 0.5. A. ADP elution profile; B. ATP elution profile. 63 The results of product inhibition studies with ADP against variable MgATP or dCyd are shown in Figures 12 and 13, respectively. The inhibition patterns generated by ADP were noncompetitive types with both varied dCyd and MgATP when the fixed substrate was present at a level below saturation. On the other hand, the inhibition of dCMP against varied MgATP was competitive when the fixed concentration of dCyd was present below saturation (Figure 14). But when dCyd was the variable substrate, dCMP inhibited the enzyme in a mixed noncompetitive fashion, as shown in Figure 15. Replotting the slopes against inhibitor concentrations, Figures 14 and 15, provided the values for the K^'s of the products. Table 5 summarizes the kinetic constants and inhibition patterns of dCMP and ADP. With only one competitive relationship, one mixed- type and two noncompetitive inhibition patterns at subsaturating fixed substrate concentrations, it is apparent that the substrates bind in an obligatory order in the steady state, with ATP being the first substrate to bind, and dCMP being the last product to dissociate (87,88). The proposed kinetic mechanism is illustrated in Figure 16. 64 300 [ADP]: 25 mM 200 16 mM 1 0 0 CONTROL 3 6 9 12 1/[MgATP2 - ], mM"1 2 0 d) CL .2 10 cr) 20 25 [ADP], mM Figure 12. Product inhibition by ADP ; the effect of varying MgATP concentration on the reaction rate, with a 2.2 uM dCyd concentration. A minimum of 97% of the total ATP is in the form of MgATP and unbound Mg^ is fixed at 0.5 mM. 65 [ADP]: 40 10 m mM 1 mM CONTROL 1 20 V 0.4 0.8 1.2 1.6 1 /[dCyd], uM“ 1 2 0 10 0 0 5 10 [ADP], rriM Figure 13. Product inhibition by ADP ; the effect of varying dCyd concentration on the reaction rate, with a 2.1 mM MgATP concentration. A minimum of 92% of the total ATP is in the form of MgATP and unbound Mg2 is fixed at 0.5 mM. 66 2 5 0 [dCMP]: 200 90 uM 150 60 uM 77100 30 uM CONTROL 50 9 1 2 15 1/[MgATP2 - ], mM-1 20 15 10 5 0 30 60 90 [dCMP], UM Figure 14. Product inhibition by dCMP ; the effect of varying MgATP concentration on the reaction rate, with a 2.2 uM dCyd concentration. 67 [dCMP]: 1 0 0 ^"^90 uM 80 60 uM 1 60 V 40 CONTROL 2 0 0.4 0.8 1.2 1.6 1 /[dCyd], uM“ ^ 50 40 tn 20 0 30 60 90 [dCMP], uM Figure 15. Product inhibition by dCMP ; the effect of varying dCyd concentration on the reaction rate, with a 1.0 mM MgATP concentration. 68 Table 5 Product inhibition constants and patterns Kinetic Value Varied Cone. of Pattern constant (uM) substrate fixed s. Kiq(dCMP) 31+ 3.1 MgATP 2.2 uM C Kiq(dCMP) 49+ 4.8 dCyd 1.0 mM Mixed Kip(ADP) 7870+ 1900 MgATP 2.2 uM NC Kip(ADP) 19800+ 6700 dCyd 1.0 mM NC ATP dCyd ADP dCMP 1 E ATP ADP E—ATP E-dCMP dCyd dCMP Figure 16. The proposed kinetic mechanism for human dCyd kinase purified from T-ALL. cn VO 70 iv. End-product inhibition dCyd kinase is known to be subject to strong feedback inhibition by its distal product, dCTP, in several systems (91-93). The end product inhibition of (deoxy)nucleoside kinases by dNTP has now been found to be virtually universal for all (deoxy)nucleoside kinases, except for adenosine kinase (1 ). Ikeda, et al., in studies of the kinases from L. acidophilus, (51) showed that the end products, dNTP exhibited kinetic effects quite similar to those of the corresponding multisubstrate analogs, dNp4 A. Several cases have been documented in this laboratory supporting the view that the end-product nucleoside triphosphates may function as bisubstrate inhibitors of the deoxynucleoside kinases, bridging the subsites for both ATP and deoxynucleoside (51). For an ordered kinetic pathway, a multisubstrate analog should compete only with the leading substrate. Experiments were carried out to determine the type of inhibition patterns., produced by the end product, dCTP. Lineweaver-Burk plots for the inhibition kinetics are shown in Figures 17 and 18. dCTP is competitive versus MgATP, but exhibits purely noncompetitive inhibition with dCyd as the varied substrate, fully in accord with the view that it behaves as a multisubstrate analog in its 71 [dCTP]: 200 8 uM 150 5 uM 1 0 0 CONTROL 50 4 8 12 16 1/[MgATp2-], mM-1 1 0 5 0 0 2 4 6 8 [dCTP], u M Figure 17. End product inhibition by dCTP ; the effect of varying MgATP concentration on the reaction rate, with a 2.2 uM dCyd concentration. 72 [dCTP]: 150 5 uM 1 2 0 90 2 uM V 60 uM 30 CONTROL 0.4 0.8 1.2 1.6 2.0 1 /[dCyd], uM ^ 60 a) 40 a. _o in 2 0 0 1 2 3 4 5 [dCTP], uM Figure 18. End product inhibition by dCTP ; the effect of varying dCyd concentration on the reaction rate, with a 0.12 mM MgATP concentration. 73 interaction with the enzyme. Table 6 shows the inhibition constants of dCTP. In terms of the multisubstrate model of control, the specificity of dCTP derives from the binding of its nucleoside moiety to the dCyd site , while its phosphate groups overlap the ATP site, preventing that substrate from binding. There are absolutely no kinetic indications of an allosteric control site for this enzyme, so this structurally simpler mechanism serves, apparently, to bring about feedback inhibition. 8 . Looking for possible isozymes with new affinity media i. dCp^-Sepharose affinity chromatography Hoping to find a means of fractionating a putative more-weakly-binding dCyd kinase isozyme, and also to increase the affinity column capacity, a new affinity ligand has been synthesized. While the dCp^A-Sepharose medium used for purification of the T-ALL dCyd kinase has been highly specific, its capacity has been limited by apparent steric interference with binding, causing a substantial portion of the activity to run through the column. The run-through fraction might conceivably consist of a different isozyme. Therefore, a medium which retains all of the dCyd kinase activity, and which yields nearly all the enzyme activity as pure protein, is the ideal. 74 Table 6 End-product inhibition constants Kinetic Value Varied Cone, of Pattern constant (uM) substrate fixed s. Apparent inhibition constant Kjl(dCTP) 2.1+ 0.22 dCyd 120 uM NC K^dCTP) 4.6+ 0.67 MgATP 2.2 uM C True inhibition constant Kis (dCTP) = 0.7 uM ( extrapolated to zero MgATP)* •fg The true dissociation constant of dCTP was estimated by extrapolation on a plot of apparent vs. MgATP concentration (51). 75 Observing that dCTP inhibits the dCyd kinase more potently than dCp4 A, a new affinity medium has been synthesized in our laboratory (52), based on its structure, by joining the phosphate group of 6 -amino hexyl phosphate to the terminal phosphate of dCTP. This ligand has an extra phosphate group to compensate for the 4th negative charge lost upon derivatization. The new affinity dCp4-Sepharose column was tested first with human T-ALL cell ammonium sulfate fraction to compare with dCp4 A-Sepharose column. The result is shown in Figure 19. Although dCp4-Sepharose column contained only 1 . 8 ml of gel, and the sample was the same size as was normally applied to a 4 ml column of dCp4 A-Sepharose, the run-through activity was cut nearly in half and relatively good recovery of activity was obtained. Very little activity was eluted with 0.3 mM dCTP immediately after applying the sample and most of the activity emerged after the column stood overnight ( with protease inhibitors such as 1 ,1 0 -phenanthroline, benzamidine, and diphenylcarbamyl chloride, in the sample). Thus the slow release of kinase is even more pronounced than in experiments with dCp4 A-Sepharose. dCyd kinase from a variety of other leukemic cell lines has been purified with this new affinity column. As shown in Figure 20, dCp4-Sepharose column retained much Enzyme Activity, units/ml 2 0.0 1 otiig m T n 0 lcrl 3-3 ammonium 30-63% 20%glycerol. and DTE mM 2 containing dCp4-Sepharose by kinase dCyd of Purification 19. Figure qiirtd ih 0 M rsHl p 75 a 4°C) at 7.5,process. elution (pH Tris-HCl mM 20 with equilibrated h clm. ro dntsa vrih pue n the in pause overnight an denotes Arrow was column. column the to applied was protein) 339 mg (37 dCp*-Sepharose Units, fraction sulfate ml 1.8 A column. affinity . . 0 0 - - ape Buffer Sample rcin No Fraction + 0.5 MNaCI 0.5 + 0.3 mM dCTP0.3 mM 0.3 mMATP 0 2 25 77 AML £- 0.9 mM ATP 0.19 mU 0.9 mU co Sampl* Buff«r + 0.9 M NaCI dCTP dCTP "E 3 1 .0 - 49* |> O < (I) E 0.5 S' 1 2 * c LlI \ 0.0 A,. — 9— 10 15 20 25 30 35 40 Fraction No. I CML 0.9 mU ATP 0.9 mU + 0.9 U NoCt dCTP 0 .2 - 0.0 0 10 30 40 50 Fraction No. Figure 20. Purification profile of dCyd kinase from different kinds of human leukemic cell lines and cultured human T-lymphoblasts by dCp^-Sepharose affinity column. A 1.4 ml column was equilibrated with 20 mM Tris-HCl (pH 7.5, at 4°C), containing 2 mM DTE and 20% glycerol. Similar dCyd kinase activities (1.6 Units) of crude extract from a variety of human leukemic cell lines were applied to the column. 00 Enzyme Activity, un^ts/ml I > CD E ‘ 3 c to >3 ? + o EE O CM O N> —o o Enzyme Activity, units/ml MOLT4T o a. EE + O + Ot O u3 u Enzyme Activity, units/m l cn to cn o Ol “H Figure 20. (cont. 79 more dCyd kinase from all different kinds of human leukemic cell lines and MOLT 4 T cells ( cultured human T- lymphoblasts ). The recovery of activity was higher with myeloblastic leukemia cells ( AML and CML ) than with lymphoblastic leukemia cells (B-CLL, B-ALL, T-ALL and MOLT 4 ). When MOLT 4 cell extract was applied to dCp4 A- Sepharose column, all the activity ran through the column. This indicates that new dCp^-Sepharose column which retained MOLT 4 kinase activity, is much more powerful for purifying dCyd kinase and for finding isozymic forms of the deoxynucleoside kinases if more than one type of enzyme is present. ii. dAp4-Sepharose column An analogous medium directed towards dAdo kinase, dAp4 -Sepharose, was synthesized in our laboratory and tested with one of the human myeloblastic leukemic cell lines, CML cells, in attempts to isolate a putative dAdo kinase which is distinct from the dCyd kinase-related activity. As seen in Figure 21, only 8 % of the total dAdo kinase activity applied was eluted with dATP after the column stood overnight. This purified protein also had multiple specificities for the dAdo, dCyd and dGuo substrates, but it didn't have Ado kinase activity. 0.075 Enzyme Activity, units/m! 0.5- 0.0 f M cls A m dp-ehrs clm was column dAp^-Sepharose ml 1 A cells. CML of qiirtd ih 0 MTi-C (H ., t 4°C) at 7.5, (pH mM Tris-HCl 20 glycerol.20% and DTE 2 mM containing with equilibrated iue 1 A^Spaoecrmtgah fcue extract 21. dAp^-Sepharose Figure crude of chromatography ape ufr 05 M 0>5 NaCi + Buffer Sample A A , dGuo kinase kinase dGuo ; , A A O ---- / Aokns D Q, Cdkns ; Q ,kinase dCyd D /kinase dAdo o ; . mMATP 0.5 rcin o ' No. Fraction 0 5 0 5 0 45 40 4 35 30 25 20 ____ 0 ,kinase. Ado . mM dATP 0.3 81 mM dATP inhibited the dAdo kinase of crude extract of CML cell by 15% and of crude extract of B-ALL cell only by 7%. When crude extract of B-ALL was applied to a different batch of dAp4-Sepharose in a column, a similar amount of activity ran through the column ( 79% ) but no activity was eluted, even with overnight contact with dCTP (results not shown). iii. Purity analysis Proteins purified by dCp4-Sepharose column from the various human leukemic cell lines and MOLT 4 cell were analyzed for purity. The peak fraction of each dCp4- Sepharose chromatography was concentrated by centricon 10 micro-concentrator, and applied to 12% SDS-polyacrylamide electrophoresis gel. For comparison, the enzyme purified from T-ALL on the dCp4 A-Sepharose column was also applied. Protein bands were stained with 0.1% Coomassie Brilliant Blue R-250. As seen in Figure 22, enzymes purified from B- CLL, AML and CML cell lines showed one major band that has the same Rf as the enzyme purified from T-ALL cells (52 Kd). On the other hand, enzyme proteins from B-ALL and MOLT 4 cell showed one lower molecular weight band (30 Kd). Protein purified on the dAp4-Sepharose column from CML cell extract was also analyzed for purity (Figure 22). 82 1 2 3 4 5 6 7 8 PO f I Figure 22. SDS-polyacrylamide gel electrophoresis of dCyd kinase purified from different human leukemic cell lines. Lanes 1-7 contain dCp^-Sepharose-purified enzymes from different cell lines: Lane l r T—ALL; Lane 2 r B—ALL; Lane 3/ B—CLL; Lane 4, MOLT-4; Lane 5, AML; Lane 6 , CML; Lane 7, dAp^-Sepharose purified enzyme from CML; Lane 8 , standard proteins ( Sigma dalton-markers). 83 The . purified protein from CML cells exhibited only one stained band, at the same position as the T-ALL protein (52 kd). iv. Electrophoretic mobility To compare the electrophoretic mobilities of the native enzymes purified from different cell lines on the dCp^-Sepharose column, 12% non-denaturing polyacrylamide gel electrophoresis was performed. dCyd kinase activity was assayed as described in Experimental Procedures. The enzymes purified from CML and AML cells were applied to 1 2 % non-denaturing electrophoresis gels, and for comparison, with T-ALL enzyme (Figure 23). Figure 24 shows the results obtained when MOLT-4, B-CLL and B-ALL enzymes were applied to one gel, along with T-ALL enzyme. dCyd kinase purified from either lymphoid cells (T-ALL, B- ALL, B-CLL and MOLT 4 T) or myeloid cells (AML and CML) showed the same electrophoretic mobility in non denaturing polyacrylamide gels. However, B-ALL , B-CLL, and MOLT 4 T cell, had an extra minor peak (all at the same position) in addition to the major peak. dAdo kinase activity was also assayed in the lane containing B-ALL cell protein. Both dAdo and dCyd phosphorylating activities co-migrated, indicating that these activities are associated with the same protein 100 84 o TJ3 CML - O w 80 - Q. O c o 60 - 'e > 0 5 10 15 20 100 o -o3 T-ALL uo . 80- CL -*-1o c. o 60- ’55 Slice No Figure 23. Electrophoretic mobility profiles of dCyd kinase purified from CML, AML cell lines and comparison with Llva enzyme from T-ALL cells (in one gel). • — • / dCyd kinase ; o — 0 / dAdo kinase. 85 100 MOLT—4 a. 60- o c o e 40- 4) C> ao K 20 100 B-CLL 3a 00 - 7 3 a.s o 60- c o '« 40- a>u >e o a 20- 20 100 B-ALL 80- a . o 60- c o e 40 4) C OO 2 0 - 0 5 10 15 20 100 T-ALL o 80- -o3 2 CL 5 60- oc 4 0 - 0)e > c oa 2 0 - M 20 Slice No. Figure 24. Electrophoretic mobility profiles of dCyd kinase purified from MOLT-4, B-CLL, and B-ALL cell lines and comparison with the enzyme purified from T-ALL cells (in one gel). • -- • , dCyd kinase ; o o , dAdo kinase. 86 (also in Figure 24). 9. Immunoassay i. ELISA The relatively small amounts of enzyme protein make conventional antibody production impractical. Instead, a new technique, disk implantation method which has proven successful in the laboratories of Sarah Elgin and Mark Muller ( unpublished procedure) was used. A total of 2 ug of purified enzyme was applied to 1 0 small disks of nitrocellulose. After 15 ul of Freund’s complete adjuvant was applied to the disks, pairs of disks were implanted in five incisions in the back muscle of an anesthetized rabbit. A booster dose was administered after 3 weeks, using 1 ug of enzyme and incomplete adjuvant. Polyclonal antibodies against dCyd kinase was developed with purified enzyme protein as an antigen. To determine the titer of the antiserum, ELISA was performed on an Immulon II microplate. As shown in Figure 25, a titer of 80 was determined with crude antiserum. The nitrocellulose powder method of immunization was attempted as a variation designed to increase the titer. However, the titer was not increased as shown in Figure 26. O.D. at 490 nm 0.5 - 0.5 0.0 1.5-- 1 Figure 25. Determination of the titer, by ELISA, of crude of ELISA, by titer,the of Determination 25. Figure antiserum raised by the disk implantation method. implantation disk the by raised antiserum . 0 0.7 -- . 1.9 1.3 o dlto factor dilution Log o — • --- 2.5 12 days afterdays2ndboost12 boost 1st afterdays 20 preimmuneserum 12 days after 1st boost 1st afterdays 12 3.1 00 O.D. at 490 nm 0.5-- 0.0 1.5-- 1 . 0 . 1.3 0.7 -* Figure 26. Determination of the titer, by ELISA, of crude of ELISA, by titer,the of Determination 26. Figure antiserum raised by the nitrocellulose powder method. powder nitrocellulose the by raised antiserum o dlto factor dilution Log . 2.5 1.9 — □ •— 7 days after 1afterdays boost7 st preimmuneserum 20 daysboostafter2nd 20 10 days afterdaysboost2nd 10 3.1 00 00 89 ii. Purification of the serum Antibodies are immunoglobulins that occur in the beta and gamma globulin fractions of serum. Of the total globulin, the fraction of antibody protein is relatively small, usually less than 1 0 % of the total protein, even in a hyperimmunized animal. The purification of immunoglobulins, especially the gamma globulin (IgG) was attempted. Immunoglobulins were precipitated in 40% saturated ammonium sulfate (pH 7.4) as a preliminary purification and concentration step. The ammonium sulfate fraction containing the IgG was dialyzed and centrifuged for 15 minutes at 1500 x g, and the sediment was discarded. Purification of IgG free of contamination, with other serum proteins or immunoglobulin classes can be accomplished with DEAE-Sephadex A-50 and Blue Sepharose chromatography. A 5 ml DEAE-Sephadex A-50 column was equilibrated with 0.015 M sodium phosphate buffer , pH 7.2 (PB) and about 34 mg of protein from the 40% ammonium sulfate fraction was applied. The optical density of each fraction was checked at 280nm by UV monitor. After chromatography, the fractions with highest protein content in the run-through peak were pooled and applied to a Blue- Sepharose column equilibrated with 0.015 M PB, pH 7.2. Very little protein ran through the column, and the 90 retained protein was eluted by 1.0 M NaCl in PB. The purity of each preparation was checked by SDS- polyacrylamide gel electrophoresis. As seen in Figure 27, the run-through fraction from the DEAE-Sephadex A-50 column was still contaminated with serum albumin ( 6 6 Kd). But the Blue-Sepharose column removed the serum albumin successfully, since the eluate of the Blue-Sepharose column showed only IgG ( 55 Kd heavy chains and 22.5 kd light chains). iii. Western Immunodetection Western immunoassays of T-ALL crude extract, and of purified enzyme, were performed with antiserum raised by the disk implantation method, diluted to 80 times (Figure 28). Immunodetection revealed a single sharp band of immunoreactive material at 50 Kd in the affinity- purified enzyme fraction, but several bands in the crude extract, including a thin one at 30 kD, and a heavy band at 60 Kd leading the dCyd kinase band. Immunostaining the lane containing the T-ALL ammonium sulfate fraction exhibited the same pattern as crude extract. It was not clear whether the 30 Kd band might represent a cross reactive dCyd kinase isoenzyme, or a proteolyzed product. 91 K D a 66 45 5 29 20 Figure 27. SDS-polyacrylamide gel electrophoresis of purified IgG preparations. Lane 1, Standard proteins (Sigma dalton-markers); Lane 2, Fraction retained and eluted from the Blue-Sepharose column; Lane 3, Fraction retained and eluted from the DEAE-Sephadex A-50 column; Lane 4, Run-through fraction of the DEAE-Sephadex A-50 column; Lane 5, Ammonium sulfate fraction (0-40%) of crude antiserum. 92 1 2 3 4 5 Figure 28. Western immunodetection of dCyd kinase. A duplicate of the stained 12% SDS-polyacrylamide gel on the left was electroblotted onto nitrocellulose and immunostained with rabbit antikinase and alkaline phosphatase-coupled goat anti-rabbit IgG. Lane 1, Standard proteins (Sigma dalton-markers); Lane 2, Crude extract of T-ALL; Lane 3, Ammonium sulfate fraction (30-63%) of T-ALL; Lane 4, Immunostained crude extract; Lane 5, Immunostained pure dCyd kinase fraction. 93 10. Attempt to determine the N-terminal amino acid sequence It appeared likely that the N-terminus of human dCyd kinase was blocked. An 80 pmole sample, submitted to gas- phase sequence analysis after extensive filtration dialysis on a centricon unit, yielded a maximum of only about 8 pmoles Edman-reactive material (results not shown). DISCUSSION dCyd kinase was purified to apparent homogeneity from a variety of human leukemic cell lines ( T-ALL, B-ALL, CLL, AML, and CML), and from cultured T-lymphoblasts (MOLT 4), using two newly developed affinity media, dCp4 A- Sepharose and dCp4 -Sepharose. These affinity systems, developed and synthesized in our laboratory, provided a simple and efficient method for isolating apparently homogeneous dCyd kinase. The recent purifications of human dCyd kinase from leukemic spleen (67), leukemic T-lymphoblasts and myeloblasts (46), and cultured T-lymphoblasts (75) involved several purification steps, such as ion-exchange chromatography, hydrophobic chromatography, and general specificity affinity chromatography. This study showed that highly biospecific affinity ligands could be used for the purification of dCyd kinase from mammalian cytosol. Ligands based on structural analogs of nucleoside triphosphates, including Blue Sepharose, have been useful but not completely selective (46,66). On the other hand, modifications of the substrate, the deoxynucleoside toward which the enzyme is 94 95 most specific , drastically interfere with binding (42). Affinity media based on the multisubstrate-type inhibitor dNp4A were very useful in distinguishing different kinase species in bacterial extract (49) and in purifying human dCyd kinase from T-ALL (50). However, their capacity as practical affinity media was found to be limited and comparative kinetic studies indicated that dNp4A did not fit optimally within the active site of a kinase (52). The natural end-product inhibitor, dNTP, has exhibited the tighter binding to the bacterial kinase than the corresponding dNp4 A, while functioning also as a multisubstrate analog (51). An extra phosphate was inserted between the gamma-phosphate and the hexyl linker group to compensate for the fourth negative charge lost in the covalent attachment of dNTP (52). Datta et al. (75) used a dCTP-Sepharose 4B column in the last purification step for human dCyd kinase from MOLT-4 cells. The enzyme activity was eluted even with 2 mM ATP and 2.4 mM MgCl2 and final enzyme elution was made with 0.1 mM dCyd, 2 mM ATP, 2.4 mM MgCl2 and 2 mM dTTP. It seems that the triphosphate group alone was not sufficient for efficient binding of dCyd kinase. Comparing the binding efficiency of the enzyme to dCp4- Sepharose with that of dCTP-Sepharose, the extra phosphate might strengthen considerably the interaction of a 96 derivatrzed dCTP with the enzyme. With the dCp^-Sepharose column, the binding was so tight that enzyme activity was not eluted continuously in a day even with dCTP. It seems that enzyme dissociation is a slow step. The purified dCyd kinase has broad substrate specificity and has a small apparent for dCyd, but much larger K^s for dAdo and dGuo. dCyd kinase purified from human leukemic spleen (67), MOLT-4 (75) and human leukemic T- Lymphoblasts and myeloblasts (46) contained also the major phosphorylating activity for dAdo and dGuo. The discovery that proteolysis can occur on the column highlights the very important concept that, unless suitable precautions are taken, an enzyme isolated from human leukocytes may be a modified version of the native enzyme. Fortunately, a mixture of simple chemical inhibitors completely eliminated this effect in the study. A similar phenomenon was reported for calf thymus terminal deoxynucleotidyl transferase (89, 90). Crude thymus extracts were found to contain 58 and 44 kd active enzyme peptides, in addition to the 32 kd peptide species originally isolated as terminal transferase, and it was suggested that the alpha and beta subunits reported in the 32 kd preparation may have arisen by proteolysis of the 58 kd monomeric peptide (90). By analogy, it seems quite possible that the T-cell specific kinase reported as 97 having a molecular weight of 26.5 kd (45), may have been a proteolyzed fraction, with corresponding changes in its kinetic properties , as well. To identify any possible variations in the structure of deoxynucleoside kinase, crude extracts of different kinds of human leukemic cells were subjected to dCp4- Sepharose affinity chromatography. This new affinity column retained dCyd kinase from all different kinds of human leukemic cell lines tested, and the recovery was relatively good ( 27 - 49%). SDS-gel electrophoresis revealed that the protein purified from AML, B-CLL, CML cells exhibited the same molecular weight band ( 52 kd ) as T-ALL cells, but the B-ALL and MOLT-4 dCyd kinase showed a low molecular weight band ( 30 kd) instead. This result coincided with other observations. Datta et al. (75) found a 30.5 kd protein band derived from dCyd kinase purified from MOLT 4 cell and suggested that the enzyme consisted of two subunits. A similar subunit molecular weight was reported for the enzyme purified from human B-cell lymphomas (67). However, no subunit molecular weight was determined on the enzyme purified from T-ALL, CML, and cultured CCRF-CEM (human T- lymphoblasts cells) (46). In terms of electrophoretic mobility in non denaturing gels, all kinase preparations purified on 98 dCp^-Sepharose showed the same Rf except that MOLT-4 and B-ALL enzymes had an extra minor peak. The enzymes purified from MOLT-4 cells and B-ALL had the same broad substrate specificity spectrum as the other preparations, also phosphorylating dAdo and dGuo (67, 75). The phosphate donor specificity of the dCyd kinase from MOLT-4 cells was also similar to that purified from T-ALL; ATP, GTP and dGTP were efficient phosphate donors. The kinetic behavior of human dCyd kinase purified from B-cell lymphomas (67) showed bimodal character in the double-reciprocal plots for dCyd as the phosphate acceptor. Similar results were reported with human dCyd kinase by Sarup and Fridland (46). The kinetic pattern we obtained with a synthetic mixture of native and proteolyzed proteins bears a striking resemblance to their results ( 39, 46, 67), and also to the results obtained with calf thymus dCyd kinase ( 58. 60). There seems to be a distinct possibility, therefore, that partial proteolysis of the kinase may account for the non-linear kinetics obtained by several laboratories. Or, if the modified protein were the species isolated, the K^s and other physical properties reported would be subject to error. While we can minimize this possibility by the judicious use of protease inhibitors, we can not be certain that even the 52 kd polypeptide is unmodified 99 until its terminal sequences are compared with corresponding genetic sequences. Initial velocity studies suggest that human dCyd kinase from T-ALL cells follows an ordered kinetic pathway involving a ternary complex of the enzyme and the two substrates. The leading substrate is MgATP and the last product to leave is dCMP. The for dCyd, using the T-ALL enzyme is lower than for dCyd kinase found in any other source : human leukemic spleen (3.5 uM), L 1210 cells (24 uM), Lactobacillus acidophilus R-26 (5 uM), p815 murine neoplasm (9.3 uM), MOLT 4F T cells (1.9 uM). Ikeda et al.(51) proposed a general mechanism for feedback inhibition of (deoxy)nucleoside kinases from Lactobacillus acidophilus. in which the dNTPs also behave as multisubstrate analogs. Multisubstrate type analogs (such as dNp4A and dNTP) have proven to be simple and useful tools for distinguishing between rapid equilibrium Random Bi Bi and steady state Ordered mechanisms. In the present study it has been demonstrated that this model can be also applied to a mammalian deoxynucleoside kinase. dCTP competes with ATP, but is non-competitive toward dCyd, consistent with this inhibitor functioning as a multisubstrate analog. An identical pattern of inhibition by dCTP was reported for the dCyd kinase from human leukemic spleen by Bohman and Eriksson (67) and for two kinase activities from MOLT-4 cells by Yamada, et al. (45). Bohman and Eriksson (67) reported that a noncompetitive mixed type of inhibition was observed, with an apparent of 3 uM and a Kis of 6 uM vs. dCyd, and competitive inhibition with a low apparent of 0.03 uM for varying MgATP. However, while the apparent value of 1.9 uM for the dCyd kinase from MOLT- 4 cells reported by Yamada, et al. (45) is comparable to ours, dCTP appeared to inhibit a "T- lymphoblast-specific kinase" (TSK) friuch more weakly, with an apparent of 0.38 mM. It should be noted that the molecular properties (molecular weight, pi and electrophoretic Rf ) of our human dCyd kinase from T-ALL resemble those of Yamada's dCK preparation much more closely than they do the TSK fraction. Only the dCK type of activity was retained by our affinity column. This TSK fraction of MOLT-4 cell extracts (45) was not found by Datta et al. (75) nor by others using CCRF-CEM cell extracts (46). While it is possible, of course, that dCTP binds only to the ATP site to produce this pattern of inhibition, such a mechanism would not explain the inability of dCTP to serve as a phosphate donor, nor the strength of its interaction, as compared with ATP. The true dissociation constant of dCTP was estimated to be 101 about 0.7 uM by interpolation on a plot of apparent Kj_ vs. MgATP concentration (51). A straight line drawn between two known points ( the apparent at 0.12 mM ATP, and the value of Kj_a(ATP) ) yields the value of at zero MgATP concentration lying at the intersection with the ordinate. The multisubstrate binding model seems to provide a simpler and more plausible explanation of the inhibition effect of dCTP. Interaction of the deoxycytidine moiety of dCTP with the deoxycytidine enzymatic site accounts both for the specificity of the inhibition and the inability of dCTP to serve as a phosphate donor in this orientation (51). APPENDIX Calculation of concentrations of MgATP— complex For all nucleoside kinase reactions, ATP must be present as the MgATP2- complex. Without magnesium, human dCyd kinase showed no activity. Consequently it is of importance to determine the concentration of the MgATP2- complex for each experimental situation. In particular, careful consideration must be given to the choice of experimental conditions when studying the kinetics. Unless this is done, the resulting data may lead to erroneous conclusions about the kinetic behavior and/or to incorrect values for kinetic parameters. Sufficiently high magnesium ion concentration must be maintained to convert nearly all the ATP to MgATP2-, but, at the same time, large excesses of inhibitory free Mg2+ must be avoided. The ionization of ATP and the reaction of Mg2+ with the ATP species are shown in Figure 29 (94). Klf K2 , KH and K^jjj represent dissociation constants for the appropriate reactions. Dissociation constants, that is, the reciprocals of the stability constants, will be used to express the binding of the metal ion by the ligand. 102 103 H HATP3- — ------AT] I % " / K- Mg2+ K 1 Mg2+ k m h MgHATP 1- MqATP ------H Figure 29. Equations relating the various forms of ATP and magnesium complexes. 104 From the equilibrium reactions shown in Figure 29, it follows that Kx = [Mg2+] [ATP4"] [MgATP2"] K 2 = [Mg2+] [HATP3"] [MgHATP1"] Kh = [H+ ] [ATP4"] [HATP3"] (1) The concentration of each nucleotide species may be written in terms of the concentration of MgATP2" as [ATP4"] = K-l . [MgATP2"] [Mg2+] [HATP3-] = Kx . [MgATP2-] . [H+ ] [Mg2+] Kh [MgHATP1-] = Kx . [H+ ] . [MgATP2-] K 2 Kh (2) The total concentration of metal ion (Mgt ) and the total concentration of ATP (ATPt ) are given by Mgt = Mg2+ + MgATP2" + MgHATP1" = Mg2+ + MgATP2" ( 1 + Kx . [H+ ] ) K 2 % (3) 105 ATPt = MgATP2- + ATP4" + HATP3" + MgHATP3" = MgATP2" ( 1 + + Kx . [H+ ] + Kx . [H+ ] ) [Mg2+] [Mg2+] K„ Ko K„ (4) With a knowledge of the values for K-^, K 2 , and KH that apply under the chosen experimental conditions, equations (3) and (4) can be used to calculate the MgATP2" that is present in reaction mixture containing particular concentrations of magnesium and ATP. The data of Table 7 show that if the total magnesium and total ATP concentrations are varied in a constant ratio of 1.2:1 at pH 7.5 in the kinetic experiments, the concentration of MgATP2" may be very different from the total concentration of ATP. The difference depends on concentrations of total magnesium and ATP added, and on the pH. For example, at an ATP concentration of 0.083 mM, only 50% of total ATP forms MgATP2" complex in the presence of a 20% molar excess of magnesium. Therefore in kinetic experiments the total magnesium concentration should be adjusted to ensure that the ATP is fully complexed with Mg2+, while holding the concentration of free magnesium (Mg2+) constant. 106 Table 7 The effect of the variation of total concentrations of magnesium and ATP on the concentrations of MgATP2” and other species at pH 7.5. Total Total Magnesium ATP MgATP2- Mg2+ MgHATP1" (mM) (mM) (mM) (mM) (mM) 1 . 2 0 1 . 0 0 0.904 0.288 0.0084 0.600 0.500 0.426 0.170 0.0040 0.300 0.250 0.195 0.103 0.0018 0.240 0 . 2 0 0 0.151 0.088 0.0014 0.172 0.143 0 . 1 0 2 0.070 0.0009 0.133 0 . 1 1 1 0.069 0.063 0.0006 0 . 1 0 0 0.083 0.042 0.058 0.0004 Values used in the calculations: dissociation constants for MgATP2-, K1=0.0143 mM ; MgHATP1-, K 2 =1.44 mM. When ADP is present with ATP, the calculations become more complicated. Even though ADP is a far weaker chelator of metal ion than ATP, it should be taken into consideration that a large excess of ADP will compete with ATP to chelate magnesium. Thus, for the product inhibition studies conducted with ADP, the calculation of total magnesium concentration must be made in connection with the concentrations of both ADP and ATP. The total concentration of magnesium should be sufficiently high enough to complex at least 92% of the total ATP as MgATP2-, while holding the concentration of free Mg2+ constant (since free Mg2+ ion is slightly inhibitory). It is not known which ADP species would act as an inhibitor with respect to the enzyme. When 50% of the total concentration of ADP forms the MgADP1- complex, the free Mg^ concentration should be maintained at a fixed concentration. The relationships that would apply are : ADPt = MgADP1- ( 1 + Kx + Kx . [H+ ] + . [H+ ] ) [Mg2+] [Mg2+] KH K 2 % ( 1 + Kx + Kx . [H+ ] + K-l . [H+] ) [Mg2+] [Mg2+] Kh K2 Kh = ADPt = constant MgADP- 1 Mgt = Mg2+ + MgADP1- ( 1 + ^ . [H+ ] ) 108 Under these circumstances the total concentrations of magnesium depend on the concentrations of MgADP1- holding the free Mg2+ constant. When both ADP and ATP are present in the reaction mixture, total concentration of magnesium required is calculated on the assumption that a minimum of 92% of the total concentration of ATP preferentially forms a MgATP2- complex, and approximately 50% of total concentration of ADP form a MgADP1- by chelating the free Mg2+, maintaining the free Mg2+ constant. Thus total concentrations of magnesium required in the presence of both ATP and ADP can be calculated backward. First, total concentrations of magnesium in the presence of ADP only are calculated according to the total concentrations of ADP and MgADP1-. Then, the total concentrations of magnesium calculated (Table 8 ) may be considered as unbound magnesium (Mg2+) when both ATP and ADP are present in the reaction mixture to determine the required concentrations of magnesium and MgATP2- in the system (Table 9). Quantitative illustrations of these procedures are given by the data of Table 8 and Table 9. In the study of end product inhibition by dCTP, with varying MgATP concentrations, the total concentrations of magnesium required should vary according to the need to maintain a minimum of 95% of the total ATP in the form of MgATP2-, and the unbound Mg2+ at a fixed concentration (Table 10). 109 Table 8 The calculation of total concentrations of magnesium required when the ratio of total ADP and MgADP*"" is 1:1 O L and unbound Mg^ is at a fixed concentration . Total MgADP1" Mg2+ Total ADP Magnesium (mM) (mM) (mM) (mM) 8 4 0.496 4.59 16 8 0.496 8 . 6 8 2 0 1 0 0.496 10.7 25 12.5 0.496 13.3 *Values used in the calculationsj : dissociation constants for MgADP1-, K-^0.25 mM; MgHADPL K2=10 mM. 110 Table 9 The calculation of total concentrations of magnesium required for a minimum of 98% of the total ATP to form complex with magnesium and for unbound Mg2+ to be at fixed concentration in the presence of ADP. Total Mg2+ MgATP2" Total ATP Magnesium (mM) (mM) (mM) (mM) 0.083 4.59 0.0817 4.67 8 . 6 8 0.0820 8.77 10.7 0.0820 1 0 . 8 13.3 0.0820 13.4 0 . 1 1 1 4.59 0.109 4.67 8 . 6 8 0 . 1 1 0 8.79 10.7 0 . 1 1 0 1 0 . 8 13.3 0 . 1 1 0 13.4 0.167 4.59 0.164 4.76 8 . 6 8 0.165 8.85 10.7 0.165 10.9 13.3 0.165 13.5 0.250 4.59 0.246 4.84 8 . 6 8 0.247 8.93 10.7 0.247 1 1 . 0 13.3 0.247 13.5 0.500 4.59 0.493 5.09 8 . 6 8 0.494 9.18 10.7 0.494 1 1 . 2 13.3 0.494 13.8 Ill Table 10 The calculation of total concentrations of magnesium required according to the increasing concentrations of total ATP Total MgATP2- Mg2+ Total ATP Magnesium (mM) (mM) (mM) (mM) 0.067 0.064 0.639 0.704 0.083 0.079 0.639 0.719 0 . 1 1 1 0.106 0.647 0.753 0.167 0.159 0.644 0.804 0.250 0.238 0.639 0.879 0.500 0.475 0.640 1.119 BIBLIOGRAPHY 1. Anderson, E. P. (1973) in The Enzymes (Boyer, P. D., ed) vol.9, pp.49-96, Academic Press, New York. 2 . Henderson, J. F., Scott, F. W., and Lowe, J. K. (1980) Pharmacol. Ther. 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