A BSTRACT the Purification and Properties of Deoxyadenosine

A BSTRACT

The Purification and Properties of Deoxyadenosine Kinase

Vivien Krygier July, 1970 Department of Biochemistry Degree sought: Ph. D.

Deoxyadenosine (dAR) kinase, purified about 140 fold from calf thymus, was shown to catalyze the transfer of a phosphate group from specifie nucleoside 5'-triphosphate donors to the 5' position of dAR. A purification procedure involving fractionation by streptomycin, protamine sulfate, Sephadex G-150 and DEAE-cellulose was employed.

The enzyme required a divalent cation and a phosphate donor for activity. The presence of mercuric ions inhibited the enzyme. The molecular weight of dAR kinase was determined by gel filtration to be about

63,000. ATP, GTP, UTP and dTTP were ail capable of serving as phosphate donors. dAR, deoxyguanosine (dGR) and cytidine (CR) but not deoxycytidine were shown to be substrates for the enzyme. The enzyme was subject to inhibition by adenine, guanine, and cytosine deoxynucleoside 5'-mono-, di- and triphosphates as weil as by the phosphorylated derivatives of cytosine arabinosine and cytidine. 4 The Km values for dAR, dGRand CR were 7.4 x 10- M, 3 4 1.1 x 10- M and 6.3 x 10- M respectively and each was found to competitively inhibit the other. The inhibition produced by dA TP, dGTP, dCTP, CTP and araCTP appeared to be competitive with respect to A TP and non competitive with respect to dAR. Short running title: Purification and Properties of Deoxyadenosine

Kinase THE PURIFICATION AND PROPERTIES OF DEOXYADENOSINE KINASE

Vivhn Krygier

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Department of Biochemistry July, 1970 McGill University Montreal, Quebec

o Vivien Krygier 1971 -i-

ACKNOWLEDGEMENTS

1 wish to express my sincere appreciation and gratitude to my

research director, Dr. R. L. Momparler for his guidance and encouragement during this investigation.

The polyacrylamide disc electrophoresis was carried out by

Mrs. M. Roebber to whom 1 am deeply indebted.

The technical assistance of Miss A. Labitan during the performance of many experiments and especially in the preparation of araCTP is much appreciated.

1 wish to thank Miss K. Harvan for her patience and co-operation in typing the manuscript, and Mrs. M. Oel tzschner for drawing the diagrams.

This work was supported by the Medi cal Research Council of

Canada (Grant No. MA-2843).

A portion of this work has been published (V. Krygier and R. L.

Momparler, Biochim~ Biophys. Acta, 161, 578, 1968). -jj-

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS

TABLE OF CONTENTS jj

LIST OF ABBREVIATIONS vi LIST OF TABLES viii LIST OF FIGURES ix

INTRODUCTION 1. Regulation of Metabol ic Pathways

ab) Feedback Inhibition 1 ) Allosteric Enzymes 2

.!) AFunction T b 1 1 2 Il) spartate ranscar amy ase: an examp e of allosteric regulation 3

l (Yl pcrfltni!e- c) H~eet of Km and Substrate Concentration 4 d) Control of Enzyme Synthesis 5

Il. de ~ Biosynthesis of Deoxynucleotides 6 a) Interconversions of Deoxynucleotides 7 III. Control of Deoxynucleotide Biosynthesis 8 a} Ribonucleotide Reductase 8 b) Deoxycytidylate Deaminase 12 IV. Metabolism and Control of the Salvage Pathway of Deoxynucleosides 13

Deoxythymidine Kinase 14 Deoxycytidine Kinase 16 Nucleoside Monophosphokinase 18 Nucleoside Diphosphokinase 20 -iii-

Page

V. The Role of Deoxyadenosine in the Control of DNA Synthesis 21

VI. Cytosine Arabinoside 23

MA TERIAlS AND METHODS 28

1. Materials 28

Il. Methods 29

a) Assay 29 b) Testing of DEAE-cellulose Disc Capacity 30 c) Determination of Protein Concentration 30 d) Preparation of Sephadex G-150 Col umn 30

i) Swelling of Gel 30 ii) Packing of Gel 31

e) Preparation of DEAE-cellulose Column 32

Preparation of DEAE-cellulose Suspension 32 Packing of Column 32

f) Thin-layer Chromatography 33 g) Enzymatic Synthesis uf araCTP 34

i) Synthesis of araCMP 34 ii) Synthesis of araCTP 35

h) Purification of Deoxynucleoside 5 1-Mono-, Di and Triphosphates 36 i) Polyacrylamide Disc Electrophoresis 37

RESUlTS 39

1. Purification Procedure 39

a) Extra ct 39 b) Streptomycin Treatment 39 cl Protamine Sulfate Treatment 41 Ammonium Sulfate Treatment 41 ~ Chromatography on Sephadex G-150 42 f) Ouomatographyon DEAE-cellulose 45 e- g) Polyacrylamide Disc Electrophoresis 45 -iv-

Page

Il. Properties of Deoxyadenosine Kinase 50

Identification of Product 50 b~ Requirements for Reaction 56 c) Effect of pH on Rate of Reaction 56 d) Molecular Weight Determination 60 e) Stoi ch iometry 60 f) Test for Presence of Contaminating Enzymes 64

i) Monophosphokinase and Nucleoside Diphospho- kinase 64 ii) ATPase 64

g) Phosphate Donors and Acceptors 65

i) Specifi city for Phosphate Donors 65 ii) Specifi city for Phosphate Acceptors 65

h) Inhibition by Nucleosides of the Phosphorylation of Deoxyadenosine, Deoxycytidine and Cytidine 68 i) Stabil ity Studies During Dialysis 73 j) Inhibition Studies 73

i) Inhibition by Deoxynucleotides 73 ii) Inhibition by Cytosine Derivatives 78

III. Kinetic Studies on Deoxyadenosine Kinase 80

a) Effect of Deoxyadenosine and Deoxyguanosine on the Phosphate Acceptors 80 b) Comparison of Michaelis Constants During Purification 87 Effect of dA TP and dCTP on Deoxyadenosine 90 d~ Effect of dA TP, dGTP and CTP on the Phosphate Donor 90 e) Effect of araCTP and dCTP on the Phosphate Donor 90 f) Initial Velocity Patterns 101

DISCUSSION 112

1• Purification 112 Il. General Properties 113 -v-

Page

III. Substrate Specifi ci ty 114

a) Phosphorylation of Deoxyadenosine 114 b) Phosphorylation of Deoxyguanosine 114 c) Phosphorylation of Cytidine 115 d) Phosphorylation of Deoxycytidine 116

IV. Inhibition Studies 120

V. Kinetic Analysis 121

a) Mechanism of Reaction 121 b) Mechanism of Inhibition 125

VI. Regulation of Deoxyadenosine Kinase Activity 126

a) Control Through Km Values 126 b) Significance of Inhibition 128

SUMMARY 133

CLAIMS TO ORIGINAL RESEARCH 135

BI BU OGRAPHY 138 -vi-

LIST OF ABBREVIATIONS

clAR Deoxyadenos i ne

dGR Deoxyg uanos i ne

dCR Deoxycytidine

dTR Deoxythymidine

AR Adenosine

GR Guanosine

CR Cytidine

UR Uridine araC Cytosine arabinoside

(d)ATP (Deoxy )adenos i ne 5 1-tr iphosphate

(d)ADP (Deoxy)adenosine 5 1-diphosphate

{d)AMP (Deoxy)adenosine 5 1-monophosphate

(d)GTP (Deoxy)guanosine 5 1-triphosphate

(d)GDP (Deoxy)guanosine 5 1-diphosphate

(d)GMP (Deoxy)guanosine 5 1-monophosphate

(d)CTP (Deoxy)cytidine 5 1-triphosphate

(d)CDP (Deoxy)cytidine 5 1-diphosphate

(d)CMP (Deoxy)cytidine 5 1-monophosphate dTTP Deoxythymidine 5 1-triphosphate dTDP Deoxythymidine 5 1-di phosphate dTMP Deoxythymidine 5 1-monophosphate

UTP Uridine 5 1-triphosphate -vii-

UDP Uridine 5'-diphosphate

UMP Uridine 5'-monophosphate araCTP Cytos i ne arabi nos ide 5'-tr iphosphate araCDP Cytosine arabinoside 5'-diphosphate araCMP Cytosine arabinoside 5'-monophosphate

DNA Deoxyribonucleic acid

RNA Ribonucleic acid

E. col i Escherichia coli

Pi Inorganic phosphate

THFA Tretrahydrofol ic acid

DHFA Dihydrofol i c acid

NAD Nicotinamide adenine dinucleotide

NADP N!cotinamide adenine dinucleotide phosphate dR-l-P Deoxyribose-l-phosphate dR-5-P Deoxyr i bose-5-phosphate

DEAE-cellulose Diethylaminoethyl-cellulose

Tris Tris (hydroxymethyl)-aminomethane s sedimentation coefficient s Svedburg unit -viii-

LIST OF TABLES

Table Page

A Comparison of the Activators and Inhibitors of Enzymes Involved in DNA Metabol ism 19

Il Gradient System to Separate Various Deoxynucl eotides 38

III Purification of Deoxyadenosine Kinase 40

IV Properties of the Reaction 57

V Stoichiometry of Reaction with UTP as Phosphate Donor 63

VI Specificity for Phosphate Donors 66

VII Specificity for Phosphate Acceptors 67

VIII Effect of Deoxyguanosine, Adenosine and Guanosine on the Phosphorylation of Deoxyadenosine 69

IX Effect of Deoxyadenosine and Deoxyguanosine on Deoxycytidine Kinase 70

X Effect of Nucleosides and Deoxynucleoside Monophosphates on the Phosphorylation of Cytidine 72

XI Stability to Dialysis 74

XII Inhibition of Deoxyadenosine Kinase by Deoxynucl eotides 76

XIII Inhibition of the Phosphorylation of Cytidine by Deoxynucl eotides 77

XIV Inhibition of Deoxyadenosine Kinase by Cytosine Derivatives 79

XV A Comparison of the Properties of Nucleoside Kinases 117 -ix-

LIST OF FIGURES

Figure Page

The Metabol ism of Deoxynucleotides 9

2 A Comparison of the Structures of Deoxycytidine (dCR), Cytosine arabinoside (araC) and Cytidine (CR) 24

3 Sephadex G-150 (10-40 1-') Chromatography of Fraction IV 43

4 DEAE-cellulose Chrolnatography of Fraction VI 46

5 Polyacrylamide Disc Electrophoresis 48

6 Effect of Time on the Amount of clAMP Formed 51

7 Effect of Enzyme Concentration on the Amount of dAMP Formed 53

8 Effect of pH on the Reaction Rate 58

9 Determination of the Molecular Weight of Deoxyadenosine Kinase by Filtration on Sephadex G-150 61

10 Effect of Deoxyadenosine Concentration on the Inhibition Produced by Deoxyguanosine 81

11 Effect of Deoxyguanosine Concentration on the Inhibition Produced by Deoxyadenosine 83

12 Effect of Cytidine Concentration on the Inhibition Produced by Deoxyadenos i ne 85

13 Effect of Different Concentrations of Deoxyadenosine on the Velocity of the Reaction 88

14 Effect of Deoxyadenosine Concentration on the Inhibition Produ ced by dA TP 91

15 Effect of Deoxyadenosine Concentration on the Inhibition Produced by dCTP 93

16 Effect of ATP Concentration on the Inhibition Produced by dATP 95 -x-

Figure Page

17 Effect of ATP Concentration on the Inhibition Produced by dGTP 97

18 Effect of ATP Concentration on the Inhibition Produced by CTP 99

19 Dixon Plot of the Inhibitory Effect of dCTP on A TP 102

20 Effect of A TP Concentration on the Inhibition Produced byaraCTP 104

21 Effect of ATP Concentration on the Inhibition Produced by dCTP with Deoxyguanosine as Substrate 106

22 Effect of Varying Concentrations of ATP on the Initial Rate of Reaction with Deoxyadenosine 108

23 Effect of Varying Concentrations of Deoxyadenosine on the Initial Rate of Reaction with ATP 110 -l-

I NTRODUCTI ON

ln order to understand the mechanisms involved in the control of

DNA synthesis a thorough knowledge of the metabol ism of deoxynucleotides is essential. Many enzymes in this field have been studied to sorne extent and several of them have been shown to be finely regulated by various activators and inhibitors. The exact interrelationship between these enzymes leading to the production of the correct amount of deoxynucleotides for DNA synthesis have yet to be fully investigated. The characterization of one of these enzymes, deoxyadenosine (clAR) kinase from calf thymus, and its possiblfl role in the control of DNA synthesis is the subject of this thesis.

1. Regulation of Metabol i c Pathways

a) Feedback Inhibition

Cellular functions are largely mediated through the catalytic action of enzymes. The concentrations of products formed by the various biosyntheti c pathways must be in a dei icate balance with each other for efficient functioning of a cell. Therefore sorne control must be exercised over the enzymes in order to ensure this. One means of doing so is through feedback inhibition. By this mechanism the end product of a biosynthetic pathway inhibits the first enzyme

(or regulatory enzyme) unique to its synthesis (1,2). Thus a control is exerted such that the rate of formation of those compounds such as amino acids or nuc\eotides is dependent upon their removal to form either proteins or nucleic -2- acids. Most enzymes known to be subject to feedback control catalyze physiologically irreversible reactions which are frequently kinetically second order, or higher, with respect to substrates and regulating metabol ites

(3,4). Deoxythymidine (dTR) kinase and deoxycytidylate deaminase are two such enzymes, both being inhibited by dTTP, the end product of their respective pathways (5,6).

b) Allosteri c Enzymes

i) Function

It was reported in 1961 that several enzymes (7-9) lost sensitivity to their inhibitors under various conditions while retaining their catalytic activity. Those observations led both Monod and Jacob (10) and Gerhart and

Pardee (4) to propose the existence of separate but interacting sites for substrate and inhibitor. Monod and Jacob (10) emphasized the fact that the inhibitor need not be a steric analogue of the substrate by referring to such interaction as "allosteric inhibition". \t has further been observed that these allo.teric enzymes were also subject to activation. The activators and inhibitors or metabolic effectors thus modulate enzyme activity through their binding at specifie regulatory sites that are distinct From the catalytic (substrate) binding sites (2). It was first suggested by Gerhart and Pardee (4) that binding of effectors leads to conformational changes in the enzyme with a concomitant alteration of affinity for substrate at the catalytic site. This formed the basis for the model proposed by Monod, Wyman and Chang eux (11). In this model a -3- regulatory enzyme consists of two or more identical units, each containing one binding site for each substrate or effector. Each unit can exist in two

(or more) states that differ in affinity for the various 1igands.

ii) Aspartate Transcarbamylase: an example of allosteric regulation

One of the first enzymes whose inhibition was interpreted as a feedback control mechanism (12) and which later was shown to be an allosteric enzyme (4, 13) is aspartate transcarbamylase from ~. col i. This enzyme is involved in the first step in the de ~ synthesis of pyrimidine nuc\eotides and catalyzes the following reaction:

( 1 ) COOH

NH - Ç? - 0 - g - OH -----~:::;,. H N ~CH 2 ~ P6H / 2 1 2 O=t /CH-COOH "N/ Carbamyl Phosphate COOH-CH2-îH-COOH 4

NH2

L-Aspartic Acid N-Carbamyl-Aspartate

The kinetics of the enzyme are typically allosteric. The substrate (aspartate) saturation curve is sigmoidal, indicating that more than one molecule of substrate is bound to the enzyme and that the binding of one molecule of substrate fa cil itates the binding of the next (4). CTP, an end product of the pyrimidine biosynthetic pathway, and a specific inhibitor of the enzyme, -4-

enhances the sigmoidal nature of the aspartate saturation curve toward higher aspartate levels (4). The inhibitory effect of CTP con be overcome by ATP

(4) which 0150 has a capacity to activate the inhibited enzyme. This activation by high ATP concentrations causes an apparent increase in affinity for aspartate and a shift to Michael is-Menton kinetics. Gerhart and Schachman

(13) conclusively showed that each molecule of native enzyme is composed of six subunits, two of which are involved in catalysis and four in binding of CTP.

The mammal ion aspartate transcarbamylase is very sensitive to inhibition by uridine derivatives and thymidine as weil as cytidine compounds

(14). Surprisingly dAMP and dGMP as weil as their corresponding deoxy nucleosides were found to be the most potent competitive inhibitors of aspartate

(14). The next enzyme in the de ~ pyrimidine synthesis, dihydroorotase, is 0150 effectively inhibited by dAR (14). Thus the purine deoxynucleosides and their derivatives appear to occupy a key position in the regulation of pyrimidine biosynthesis, inhibiting not only the de-- nove pathway but 0150 the enzyme ribonucleotide reductase (15, 16).

IrnpQrf.nf-t!. c) Hfeet of Km and Substrate Concentration

The rate of a reaction is dependent upon the substrate concentration as weil as the Km of the enzyme for the substrate(s). If an enzyme has a high

Km the amount of product formed will be low unless the concentration of substrate(s} is sufficiently high in order to overcome the low affinity of the enzyme. A low Km produces a correspondingly greater rate of reaction. Thus having a limited amount of substrate will result in regulation of a metabolic -5-

pathway. In the ideal case, the concentration of substrate in the cell should be of the same order of magnitude as the Km of the enzyme. When the substrate concentration equals the Km value, the enzyme is very sensitive to its metabolic effectors.

d) Control of Enzyme Synthesis

The amount of enzyme available will determine the rate of meta bol •IC actlvlty.•• Synt h e5IS . 0 fi·enzymes cata yzmg rna..n::j GAY meta bol·IC sequencescan be turned on or off, dependi ng on the demands for that specifi c sequence. This is accomplished by the process of induction or repression (17).

Induction can be defined as an increase, and repression as a decrease in the amount of one or more specific proteins produced as the result of exposing the cell to sorne regulatory substance. This latter compound is usually a small molecule and is frequently identical with, or structurally related to the end product in the case of repression, or to the starting material in the case of induction, of a metabolic sequence. An example of induction was shown by Eker (18) with dTR kinase of human liver cells in tissue culture. He found that amethopterin or 5-fluorodeoxyuridine caused a marked increase in the specific activity of dTR kinase. As this increase could be prevented by puromycin he suggested that synthesis of new protein was occurring. The author, furthermore, postulated that a phosphorylated derivative of dTR was responsible the. o~ -\-hi!. derlV'«.tive for the phenomenon as -H& concentration"decreases in the presence of amethopterin.

The addition of dTR to the medium of amethopterin treated cells lowered the -6- specifie activity of the enzyme presumably because it formed the intermediate necessary to cause repression.

Il. -de nove Biosynthesis of Deoxynucleotides

There are two pathways involved in the biosynthesis of deoxynucleotides: de ~ and salvage. In the former, purines are synthesized in the cel! in the form of their nucleoside monophosphates, AMP and GMP, which are then phosphorylated by kinases to give ADP and GDP respectively. The de ~ pyrimidine deoxynucleotide biosynthetic pathway involves a series of enzymic reactions giving rise to a parent compound, UMP, which can then be phosphorylated through the di phosphate stage to UTP. It is at the nucleoside triphosphate level that amination of the base occurs producing CTP (19), as in the following equation~

UTP + NH +ATP (2) 3 ---~> CTP + ADP + Pi

An additional route for the formation of these structures is the salvage pathway whi ch util izes exogenous and endogenous sources of preformed bases or nucleosides. The various reactions involved in this pathway will be discussed in section IV.

The conversion of ribose to deoxyribose takes place at the nucleoside diphosphate level without breakage of the glycosidic linkage (20) by the enzyme ribonucleotide reductase (15,16). Three of the products of this enzyme, dCDP, dGDP and dADP, as weil as dTDP, are phosphorylated by -7- another enzyme, nucleoside diphosphokinase (21,22), to their respective triphosphates. These deoxynucl eoside triphosphates become substrates for the DNA polymerase reaction (23).

CI) Interconversions of Deoxyribonucleotides

Formation of dTTP occurs via a series of steps From dCDP.

This latter compound is dephosphorylated to dCMP (24) which is then deaminated to dUMP by deoxycytidylate deaminase (25). In~. col i, which lacks this enzyme, dUMP is formed From dUDP by the same enzyme which dephosphorylates dCDP (24,26). The next step involves the methylation of dUMP to dTMP by thymidylate synthetase. This enzyme, first described by

Friedkin and Kornberg (27) in an extract of~. coli and more recently by

Lorenson ~~. (28) in chicken embryo, catalyzes the reductive transfer of a methyl group From methylene tetrahydrofolic acid to dUMP in which tetra hydrofolate (TffFA) itself acts as a reductant, resulting in the simultaneous formation of dTMP and dihydrofol i c acid (DHFA).

dUMP + Methylene-THFA --~) dTMP + DHFA (3) ln order to become active again in th is methy lating reaction the dihydrofol i c acid has to be reduced to tetrahydrofol ic acid, thus necessitating the presence of the enzyme dihydrofolate reductase (29).

NADH NAD 2 DHFA + or -----~) THFA + or (4) NADPH NADP 2 -8-

Thymidylate kinase (20) followed bya nucleoside diphosphokinase (21,22) execute the last two steps leading to the formation of dTTP.

The above reaction sequences are summarized in Figure 1 •

III. Control of Deoxynucleotide Biosynthesis

As has been described, the formation of deoxynucleotides is the result of a long and complex chain of enzyme reactions which must be under a tight system of control in order to maintain a balanced suppl y of these precursors of DNA synthesis. A very intri cate pattern of control is effected through feedback mechanisms.

a) Ribonucleotide Reductase

One of the key enzymes which is involved in the regulation of deoxynucleotide metabolism and has been extensively studied both in bacterial and mammalian systems, is ribonucleotide reductase. Reichard and his co-workers (31) were the first to ascribe the abil ity of direct reduction of ribonucleoside diphosphates to their corresponding deoxynucleoside diphosphates to th is enzyme from É.. col i •

The requirements for the reaction to occur are: the enzyme consisting of two nonidentical subunits called B1 and B2 (32,33), a suitable hydrogen donor system made up of NADPH, thioredoxin reductase (34) and thioredoxin (35). Thioredoxin is a protein with two cysteine residues, the sulfhydryl groups of which act as active and direct hydrogen donors. It is -9-

Fig. l The metabol ism of deoxynucleotides -10-

UDP

CDP ----~) dCDP --~,> dCTP ---7 DNA

GDP dGDP dGTP

ADP dADP dATP /r\~ dUMP dCMP dGMP dAMP T T V dUR dCR dGR, dAR -11-

form oxidized to the disulfide during reduction reaction. The reduced of thioredoxin is regenerated by NADPH by the action of the flavoprotein, thioredoxin reductase. The rate of reduction of ribonucleoside diphosphates is markedly affected by the presence of low concentrations of nucleoside the reduction triphosphates (15,33,36,37). dTTP stimulates and dATP inhibits for GDP of ail four diphosphates. dG TP is si ightl y stimulatory, especially been reduction. Of the ribonucleoside triphosphates, whose effects have of CDP reported in detail, ATP was shown to be a positive allosteric effector (33). and UDP reduction, affecting GDP and ADP reduction only slightly maximal dTTP stimulated CDP reduction to the same extent as did ATP but was able stimulation was obtainec' at much lower concentrations. Only ATP did dTTP to overcome the allosteric inhibition of dATP (15). In fact not only caused a increase the inhibitory effects of dA TP but in combination with ATP have been pronounced inhibition of the enzyme reaction. Many of these results that explained in a recent paper by Brown and Reichard (38). They showed

TP, at concentrat~ons which inhibit the presence of the negative effectorl dA 4 enzyme activity (10- M) (37), resulted in an increase in the sedimentation i nterpreted coeffi ci ent of the enzyme compl ex from 9. 7s to 15. 5s • Th is was the s val ue. as dimer formation. Increas ing the A TP concentration decreased 6 enhanced The addition of dTTP at a low concentration of dA TP (3 x 10- M) 4 M) the formation of a heavy corrplex which still existed at higher (10- no effect concentrations of dA TP. Although ATP and dTTP individually had which inhibit on complex formation, upon combining the two at concentrations -12- enzyme activity a large increase in the sedimentation coefficient was observed.

Ribonucleotide reductase has also been partially purified by Moore and her colleagues (16,39,40) From a mammal ian source,

Novikoff hepatoma. This enzyme system shows a close similarity to the reductase From ~. coli in that it appears to consist of two subunits (41), and util izes ribonucleoside di phosphates as substrates and a thioredoxin system as a hydrogen transport system (42). A similar regulatory mechanis m to that observed with the~. col i enzyme is functioning in the mammal ian system (16). The reduction of CDP and UDP is activated by ATP and inhibited by dTTP, dUTP, dG TP and dA TP • Reduction of ADP is activated by dG TP or dTTP and inhibited by dA TP. These deoxynucleotide activators and inhibitors produce marked effects at concentrations between 0.1 mM and 0.001 mM.

b) Deoxycytidylate Deaminase

There is another key enzyme, dCMP deaminase, which is involved in regulation of the production of a deoxynucleoside triphosphate.

This enzyme is specific for the deamination of dCWPand its 5-methyl, 5- hydroxymethyl and 5-halogeno derivatives to dUMP and its 5-substituted derivatives. It was first discovered in sea urchin eggs by Scarano (43,44) and has since been purified from many sources (45-47). Extensive kinetic investigations have been undertaken by Maley and Maley (46,48) with chicken embryo enzyme and by Scarano, Geraci and Rossi (6) with nearly homogeneous spleen enzyme. dCMP deaminase has been shown to be an -13-

as an allosteric enzyme having the end product of the pathway, dTTP for lack inhibitor and requiring dCTP, a compound that would accumulate of the end product, as an activator. dTMP, dUMP, dAMP and dGMP have been found to be strength c\assical competitive inhibitors of dCMP (47,6). Glycerol, ionic enzyme (6). and changes in pH appear to affect the conformation of the spleen of dCTP or Aggregation of this enzyme could not be induced by the presence deaminase dTTP (47). In contrast, the sedimentation of the chicken embryo 7.5 in in sucrose gradients gave S20,w values of 2 in the presence of dCMP,

dCTP-Mg and 3.3 in dTTP-Mg (46,48).

IV. Metabol ism and Control of the Salavage Pathway of Deoxynuc\eosides

The abil ity of tissues to util ize preformed based as precursors thus possible of DNA was first indicated by Reichard and Estborn (49). It was of deoxy to predict that deoxynucleotides are synthesized by phosphorylation of DNA nuc\eosides (50) (equation 5) which are derived from the degredation of the nucleoside or deoxynucleotides or from bases and deoxyribose-1-P by reversai

phosphorylase reaction (51) (equation 6).

(5) dXR + ATP ----~) dXMP + ADP

(6) dXR + Pi ------)~ X + dR-1 P

pathway These above two reactions constitute a pathway called the salvage

for the reutilization of deoxynuc\eosides, preformed bases and deoxyribose. -14-

Although the latter reaction is reversible it is most likely involved in the degradation of deoxynucleosides (52). One of its products, dR-1-P,

is isomerized through a reversible reaction catalyzed by an enzyme called deoxyribose phosphate mutase (53,54) to dR-5-P (equation 7) which is in turn metabolized to acetaldehyde and glyceraldehyde-3-P (equation 8) by deoxyribose aldolase (55,56).

dR-1-P --7 dR-5-P (7)

dR-5-P ---1 acetaldehyde + glyceraldehyde-3-P (8)

The catabolic function of the last three reactions appears to be indirectly supported by Larsson and Nielands (57) who through double labell ing experiments with regenerating rat 1iver showed that formation of dCMP occured via the reductase enzyme and not via the deoxyribose aldolase pathway.

a) Deoxythymidine Kinase

Of the enzymes involved in the salvage pathway, dTR kinase, has been the moxt extensively studied. Among the first investigators were

Okazaki and Kornberg (5,58) whose work first suggested a regulatory role for the enzyme from ~ col i. The kinetic characteristics of this enzyme point to its being allosteric. Plots of initial velocity versus A TP concentration are sigmoidal. Upon plotting l/v against 1/ [ATP] 2 a straight 1ine is obtained indicating the reaction is bimolecular with respect to ATP. The activity of the enzyme, particularly at low ATP concentrations, is very markedly stimulated bya variety of deoxynudeoside diphosphates, especially dCDP. Activation -15- is accompanied bya shift from sigmoidal to normal Michaelis-Menten kineti cs and a decrease in the apparent Km of the enzyme for ATP and also for dTR. This can be interpreted as indicating the presence of an allosteric site at which an activator combines and suggesting that, in the absence of an activator, ATP combines at this site with low affinity as weil as the catalytic site. Two apparent Km values for dTR are observed with low ATP concentrations in the absence of dCOP. At saturating concentrations of A TP , onlyone Km, corresponding to that for low dTR concentrations is found.

When dA TP is used as a phosphate donor only a single Km, analogous to that found at high ATP levels is obtained, possibly due to strong binding of dA TP to the allosteric site. In addition to the above properties, dTR kinase, in the presence or absence of the activator, dCOP, is very strongly inhibited by dTTP, the end product of that particular biosynthetic pathway. The inhibitor appears to be non competitive with respect to ATP but competitive with respect to dTR. Although c1assical competitive inhibition seems to be evident, the fact that dTMP and dTOP show negl igible inhibition suggests that dTTP binds at an allosteric site (not the same as the activator) which overlaps or competes sterically with the catalytic or dTR site. More recent work with!,.. coli dTR kinase has shown that the presence of either inhibitor or activator causes an aggregation or dimerization of the enzyme (59).

Bresnick and Thompson (60) have purified dTR kinase from animal tumors and have found the properties to be similar to the bacterial enzyme. dTTP was also an inh ibitor of the enzyme but it was not found to be competitive with dTR. This apparent contradiction can be partially explained by the -16- aggregation of the enzyme in dilute solutions (61). The inhibition by dTTP is simple competitive with respect to dTR in the presence of the disaggregated enzyme whereas with the aggregated form dTTP is non competitive with respect to dTR. Control of enzyme activity by dTTP inhibition appears to be a widé)pread phenomenon and has been reported by many workers (62-66).

dTR kinase has often been considered to play an important role in DNA synthesis in various cells as increased activity of thisenzyme is associated with rapid growth and increased rates of DNA synthesis (50,67-69).

Thus dTR kinase is almost undetectable in adult liver, but increases sharply during liver regeneration (67,69-72). This increase can be prevented by inhibitors of either protein or RNA synthesis and is not the result of a change in the turnover rate of enzyme protein (73), but of synthesis of new enzyme. dTR kinase activity is also greatly enhanced in animal cells infected with certain viruses in tissue culture (74-77).

b) Deoxycytidine Kinase

The only other enzyme that catalyses the phosphorylation of a deoxynucleoside and has been studied to any extent is deoxycytidine (dCR) kinase (78-80). This enzyme is of importance from a chemotherapeutic point of view since it was reported by Chu and Fischer (81) in crude extracts of

L5178Y Iymphoma cells to catalyze the phosphorylation of an analogue of dCR, cytosine arabinoside (araC) , an antitumor agent and potent inhibitor of the reproduction of mammalian cells (82-84). -17-

dCR kinase, purified from Ll210 cells, does not appear to consist of subunits as it is insensitive to surface active agents (79) although

Kessel (79) and others (80) have observed complex kineti cs. Durham and

Ives (80) have reported, with very 1ittle evidence and contrary to the results of Momparler and Fischer (78) and those presented in this thesis, that the enzyme can also catalyze the phosphorylation of dAR and deoxyguanosine

(dGR). As supporting evidence these authors refer to recent studies by Schrecker

(85) whose work on a subi ine of leukemia cells resistant to araC appears to indicate that dGR is phosphorylated by dCR kinase. Not only was this work done using a crude extract but Schrecker (86) found that dAR was not a substrate for dCR kinase. Furthermore, dGR was not capable of interfering with the phosphorylation of araC (86) whereas Durham and Ives (80) reported that dGR was a competitive inhibitor of araC phosphorylation (80).

dCR kinase is subject to feedback regulation by dCTP (63,87).

This deoxynucleoside triphosphate was shown by Momparler and Fischer (78) to be competitive with respect to A TP (with either dCR or araC as phosphate acceptor) but by Kessel (79) to be competitive with respect to dCR. This apparent contradiction can perhaps be explained by the observation of Durham and Ives (80) that while dCTP at low concentrations competitively inhibits araC phosphorylation, at higher concentrations the inhibitor becomes non competitive. The inhibition caused by dCTP could be alleviated by the c;sddition of dTTP (79,87). AraC was found to be more sensitive to the inhibitory effects of dCTP than dCR (78, 80). -18-

dCR kinase can utilize most nucleoside triphosphates as phosphate donors (78,80) but increasing the concentration of CTP and dA TP results in inhibition (80). The results of Durham and Ives (80) showed biphasic curves upon plotting the reciprocal of the apparent velocity as a function of l/ATP-Mg, with araC as the phosphate acceptor. Kessel (79) observed the same phenomenon with UTP as the variable substrate. He also found that his Lineweaver-Burk plots were bimodal over a wide range of dCR concentrations, using ATP as a donor. This bimodality could be eliminated by the presence of dTTP.

A comparison of the activators and inhibitors of dCR kinase, dTR kinase, dCMP deaminase and ribonucleotide reductase are presented in Table 1 •

c) Nucleoside Monophosphokinase

Sable =!~. (88) in 1954 were the first to show that a muscle mitochondria preparation catalyzed the phosphorylation of dAMP from ATP to the corresponding di- and triphosphates. These reactions as weil as the phos phorylation of the other deoxynucleoside monophosphates were found by other workers in homogenates of normal and regenerating liver (89,90), and in aqueous extracts of red bone marrow and muscle (91,92). Mantsavinos and Canellakis

(93,94) demonstrated that dAMP, dGMP, ciCMP and dTMP were ail precursors of DNA. Several investigations have been undertaken with purifjed enzyme preparations (95-97), the most extensive having been carried out by Sugino

=.t~. (98-100). His work indicates that there appear to be at least four distinct -19-

TABLE 1

A Comparison of the Activators and Inhibitors of Enzymes Involved in DNA Metabolism

Enzyme Activator Inhibitor Reference

Reductase ATP dTTP 15,16 dGTP dATP dCMP deaminase dCTP dTTP 6,46-48

Deoxythymidine kinase dCDP dTTP 5,58-61

Deoxycytidine kinase dCTP 78-80 -20- nucleoside monophosphokinases which are specific for nucleotides containing the bases adenine, guanine, cytosine or thymine in calf thymus. Adenine and guanine specific enzymes were highly purified and were found to catalyze the phosphorylation of only AMP and dAMP, and GMP and dGMP respectively

(101). The cytosine specific enzyme from calf thymus not only serves CMP and dCMP but also appears to phosphorylate UMP but not dUMP (98). This latter deoxynucleotide is perhaps phosphorylated by dTMP kinase (30,101). However, in the ~. col i system a separate UMP kinase and dTMP kinase was shown to exist (100). The dCMP/CMP kinase from calf thymus is extremely unstable without added thiol (98,99) whereas the.É. col i enzyme is not affected by its absence (100). A common characteristic of these nucleoside monophospho kinases is their phosphate donor specificity, for it was found that only ATP or dA TP can serve as phosphate donors (98,1 Ol). dTMP kinase is the exception to this generalization in that it can utilize ail kinds of ribo- and deoxyribonucleotide triphosphates as phosphate donors (lOl).

d) Nucleoside Diphosphokinase

Nucleoside diphosphokinase is an enzyme common to both the de ~o and salvage pathways. It catalyzes a transphosphorylation reaction between a nucleoside triphosphate and a nucleoside diphosphate as shown in equation 9. (x, y = any base) XDP YTP XTP YDP (9) or + or )- or + or dXDP dYTP dXTP dTDP -21-

This enzyme was first described in 1953 (l 02,103) and was isolated about ten

years la.ter in crystall ine form from yeast (104). Several investigations with

preparations of nucleoside diphosphokinase of various states of purity from many tissues (21,22,104-106) indicate that the enzyme is remarkably non specific with regard to the di- and triphosphate nucleotide substrates. Kinetic studies on the enzyme show the reaction follows a "ping pong U mechanism

(l 07),i .e .,the first product dissociates before the second substrate combines with the enzyme (21,108). These observations led workers (108-110) to isolate a relatively stable phosphorylated intermediate. The enzyme was also subject to product (108) and substrate inhibition (21,108). Since nucleoside diphospho kinase has a very high level of activity in most cells (21,109) as compared to many enzymes in the pathway leading to DNA synthesis, and is relatively nonspecific, it seems uni ikely to occupy a key position in the regulation of nucl eotide synthesis. v. The Role of Deoxyadenosine in the Control of DNA Synthesis

Many laboratories have reported that dAR causes a pronounced inhibition of DNA synthesis in a variety of tissues (111-116). It was first found by Klenow (111,117) that dAR in contrast to either aden:;.e or adenosine

(which have slight stimulatory effects) caused a marked decrease in the incorporation of 14C-formate and 32p into DNA thymidylic acid of Ehrlich ascites tumor cells in vitro. The rate of incorporation was inhibited 50% by about 1 mM of dAR and with a concentration of 2 mM, DNA synthesis was -22-

almost completely inhibited. Upon testing other deoxynuc\eosides such as dGR and deoxyinosine, no inhibition of incorporation of the carbon of formate into DNA thymidyl i c acid was found.

Further studies revealed that the addition of dAR to an Ehrlich ascites tumor cell suspension resulted in an accumulation of dA TP in the cells (118), whereas no signifi cant increase in dCTP, dGTP or dTTP could be detected when the cells were incubated in the presence of dCR, dGR or dTR respectively (119). When equimolar concentrations of adenosine and dAR were added to suspensions of cells in vitro, the accumulation of dA TP was prevented and the inhibition of the DNA synthesis was alleviated (119).

This appeared to establ ish a relationship between dA TP concentrations and

DNA synthesis. It has since been shown by Reichard (15,37) and Moore (16) 6 that dATP (10- M) is a potent inhibitor of the enzyme ribonuc\eotide reductase. Klenow (117) first reported that the simultaneous addition of dGR and dAR in equimolar amounts overcame the inhibiting effects of the latter compound. In a later publ i cation (119) he revised th is statement by an nouncing that the inhibition of DNA synthesis by dAR could be completely reversed only by the addition of dCR together with dGR.

Among the other workers to observe the effect of dAR on DNA synthesis were Maley and Maley (113) and Prusoff (112). The former found that dAR exerted a marked inhibitory effect on the incorporation of labelled cytidine and uridine into chick embryo DNA cytosine and thymine. Using

Ehrli ch ascites tumor cells, Prussoff (112) discovered that the addition of dAR -23-

and (0.4 mM) to the incubation medium inhibited the utilization of formate to a lesser extent that of dTR for the biosynthesis of DNA-thymine. Inhibition of RNA (111,113,114,116,120) and protein (114, observed. 120) synthesis by high concentrations of dAR (2-7 mM) has also been dAR The work of Kim et~. (114) showed that Hela cells exposed to was (2-4 mM) lost their viabil ity. The content of DNA, RNA and protein are in considerably reduced after the addition of dAR. These latter results in the contrast to those found by the same authors upon incubating the cells a normal presence of dTR. In this case, RNA and protein was synthesized at

rate while DNA synthesis was inhibited. An explanation for this observation activity in was not offered. dAR has also been shown to stimulate dTR kinase the Chang liver cells (120). Except for the inhibition of DNA synthesis but it is mechanism by which dAR exerts its effects on these systems is unknown

most likely through a phosphorylated derivative.

VI. Cytosine Arabinoside

Cytosine arabinoside (araC; 1-B-D-arabinofuranosyl cytosine) is the pentose a synthetic member of a c1ass of nucleosides having D-arabinose as associated moiety. A comparison of the three sugar components that could be with cytosine is presented in Figure 2. AraC has been shown to inhibit the growth of a wide range of of neoplasms in both animais (84,121) and humans (122-124). The growth and embryos (126) other proliferating tissues such as regenerating bone marrow (125) -24-

Fig. 2 A comparison of the structures of deoxycytidine (dCR),

cytosine arabinoside (araC) and cytidine (CR) -25-

HO H

dCR

HO· H HO OH araC CR -26-

by this as weil as the repli cation of DNA viruses (127) are also affected decrease nucleoside. In studies with mammal ian cells araC produced a marked during in the synthesis of DNA (123,128-131), exerting its effect primarily be due the S phase of the cell cycle (132). Its lethal effects could possibly

to its incorporation in very small amounts into DNA (123,128,133,134). be phosphorylated Thus in order to function as an lnhibitor of cell growth araC must of the (81). The harmful effects of araC was first thought to be a result derivative inhibition of ribonuc\eotide reductase (15,16) by its phosphorylated (131) nor (83). This finding, however, was not confirmed in cell cultures AraCTP !!' vitro where araCTP was shown to be a very weak inhibitor (135). mammal ian has since been found to be a potent inhibitor of partially purified competitive DNA polymerase (134,136); the inhibition produced by araCTP was by araCTP of with respect to dCTP (136). Just as dCTP reversed the inhibition the inhibitory DNA polymerase (134,136), the addition of dCR could counteract (83,123, effect of araC on tumor and viral systems and in vivo DNA synthesis lie in the 128,131,132,137). A possible mechanism for this reversai could (78-80). fact that deR inhibits the phosphorylation of araC by dCR kinase inhibitor of Furthermore,the end product of dCR phosphorylation, dCTP, is an AraC dCR kinase (63,87) thus blocking, also, araC phosphorylation (78,80). The may then be deaminated to the noncytotoxic uracil arabinoside (138). found in enzymes capable of deaminating araC are widely distributed being Thus the toxity bacteria (139), and in mammals (140,141) including man (142). deamination of araC would be dependent on the ratio of the phosphorylation to

reactions. -27-

The enzyme responsible for catalyzing the phosphorylation aroused of dAR has not until now been studied. Interest in this enzyme was that the by the work of Klenow (111,117) and others (113-115) who showed synthesis, addition of dAR to mammalian cells resulted in an inhibition of DNA c inhibitor possibly due to an accumulation of dATP (118), a potent allosteri thus of ribonuc\eotide reductase (16,37). The present investigation was control of undertaken with the view of studying the mechanism of action and purified the phosphorylation of dAR by dAR kinase. This enzyme was partially nuc\eotides from calf thymus and kinetic analysis performed. The effect of various on enzyme as weil as the phosphorylated derivates of an analogue of dCR, araC, activity was also studied. -28-

MATE RIALS AND METHODS

1. Materials

The carbon-14 or tritium labelled purine and pyrimidine

nucleosides were obtained from Schwarz Bio Research, Inc. Nonradioactive P-l nucleosides and nucleotides were purchased from Calbiochem and (type 81) , laboratories. Serva DEAE-celtulose powder, DEAE-cellulose paper

ammonium sulfate (enzyme grade), protamine sulfate (type l), Sephadex obtained G-150 (10-40 fJ), and cellulose-coated (avicel) glass plates were Company from Gallard-Schlesinger (Carl Place, New York), Reeve Angel

(Clifton, New Jersey), Mann Research laboratories, Sigma, Pharmacia

Fine Chemicals, and Analtech (Wilmington, Delaware), respectively. The labelled nucleosides were purified by descending chromatography

for 24 hours on Whatman No. 3M paper in 86% n-butanol-concentrated were ammonium hydroxide (94.5 : 5.5, by volume). The chromatograms to the air dried at room temperature, and the radioactive spots corresponding 0 at -20 • material to be purified were eluted with 50% ethanol and stored nucleoside Before use the ethanol was evaporated and the concentration of the

adjusted. -29-

Il. Methods

a) Assay

dAR kinase activity was assayed by measuring the incorporation of 14C_dAR or 3H- dAR into deoxyadenosine 5 1-monophosphate (clAMP) by the binding of this latter compound to DEAE-cellulose dises (143). The incubation mixture (0.1 ml) contained 10.0 jJmoles of Tris-HCI pH 8.0,

1.0 jJmole of MgCI , 0.1 jJmole of ATP, 0.1 ~ole of dithiothreitol (or 2 4 1.0 jJmole of 2-mercaptoethanol), 100 mjJmoles of 14C_dAR (9.0 x 10 cpm) 5 or 3H- dAR (1.9 x 10 cpm) and 0.1-2.0 units of enzyme. Following the

0 addition of the enzyme to the preheated (37 ) reaction mixture, the mixture was incubated for 5 minutes at 3~ and then immediately diluted to 5 ml with water. The solution was permitted to flow by gravity through 2.5 cm diameter

DEAE-cellulose dises, on a Mil 1ipore filter apparatus, that had been previously washed with 1.0 ml of 0.01 N HCI and 20 ml of water. The dises were then washed with 20 ml of water, dried and placed in scintillation vials containing

10 ml of scintillation solution (4 9 of 2,5-diphenyloxazole and 100 mg of p bis (2-(5-phenyloxazolyl»-benzene in one liter of toluene). The vials were counted in a Packard Tri-Carb 1iquid scintillation counter with a counting effi ciency for carbon-14 of 30% and for tritium of 2% under these conditions.

One unit of enzyme was defined as the amount catalyzing the conversion of

1.0 mjJmole of dAR to dAMP in one minute und~r the described essay conditions. -29-

Il. Methods

a) Assay dAR kinase activity was assayed by measuring the incorporation- of 14C_dAR or 3H- dAR into deoxyadenosine 5 1-monophosphate (dAMP) by the binding of th is latter compound to DEAE-cell ulose dises (143). The

incubation mixture (0.1 ml) contained 10.0 J.lmoles of Tris-HCI pH 8.0,

1.0 iJmole of MgCI , 0.1 iJmole of ATP, 0.1 iJmole of dithiothreitol (or 2 4 1.0 iJmole of 2-mercaptoethanol), 100 miJmoles of 14C_dAR (9.0 x 10 cpm) 5 or 3H- dAR (1.9 x 10 cpm) and 0.1-2.0 units of enzyme. Following the

DEAE-cellulose dises, on a Millipore filter apparatus, that had been previously washed with 1 .0 ml of 0.01 N HCI and 20 ml of water. The dises were then washed with 20 ml of water, dried and placed in scintillation vials containing

10 ml of scintillation solution (4 9 of 2,5-diphenyloxazole and 100 mg of p

bis (2-(5-phenyloxazolyl»-benzene in one liter of toluene). The vials were

counted in a Packard Tri-Carb liquid scintillation counter with a counting efficiency for carbon-14 of 30% and for tritium of 2% under these conditions.

One unit of enzyme was defined as the amount catalyzing the conversion of

1.0 miJmole of dAR to dAMP in one minute und~r the described assay conditions. -30-

b) Testing of DEAE-cellulose Disc Capacity

It WOlS necessary to perform several experiments in order to determine the optimum conditions for maximum binding of nucleotides to

DEAE-cellulose dises. (i) Upon allowing 3.2 I-'moles of AMP in 3 ml of water to gradually flow through the dises, it was found, by measuring the absorbance of the eluate at 260 ml-', that 97% of the nucleotide was bound to the dises. Increasing the flow rate decreased the amount bound to 70%. (ii)

Doubl ing the amount of AMP permitted 23% to pass through the filter but upon first washing the disc with 1 ml of 0.01 N HCI followed by 20 ml of water, 97% of the AMP was bound. (iii) Provided this acid wash procedure was followed, the presence of 10 I-'moles of Tris-HCI pH 8.0 in 5 ml of a solution containing 4 I-'moles of AMP did not affect the amount of nucleotide bound (95%). Throughout the experiments described in the Results section, the total nucleotide concentration placed on the dises did not exceed 0.25 I-'mole.

c) Determination of Protein Concentration

Throughout the assay procedure, protein was determined by the method of Lowry ~~. (144) using crystalline albumin as a standard. The amount of protein eluted from the Sephadex and DEAE-cellulose columns was monitored by measuring the absorbance of the various fractions at 280 ml-'.

d) Preparation of Sephadex G-150 Col umn

i) Swell ing of Gel

Sephadex G-150 (10-40 1-') powder (100 g) was suspended in

9 1iters of glass distilled water, heated for 5 hours at 900 and then placed in -31-

2 liter graduated cylinders. The resulting slurry was put in the cold room

and after permitting it to settle for about 4 hours, the fines were decanted.

The cyl inders were refilled with distilled water, mixed, and the process

repeated until 50% of the Sephadex was lost.

ii} Packing of Column

Ali of the following procedure was performed in the cold

room. After 200-300 ml of buffer was placed in a Sephadex column 2 (4.9 cm x 50 cm), any resulting air bubbles were removed by use of a syringe. Then the swollen gel containing 1 part Sephadex and 3 parts buffer

(by volume) was degassed by vacuum for 15 minutes, poured into a reservoir whi ch had been attached to the top of the column and permitted to settle.

When a 20 cm layer of Sephadex had formed on the bottom of the column the outlet was opened. A hydrostatic pressure no greater than that resulting from

a 15 cm difference in height between the inlet and outlet to the column was

maintained. After the column had packed a flow adaptor was filled with

buffer and hooked to a syringe. It was gently lowered onto the top of the

column. At the same time the syringe was aspirated in order to remove the

layer of buffer covering the Sephadex. When the flow adaptor reached the

top of the Sephadex, manual pressure was placed upon it 50 that the Sephadex

wa5 compressed about 5 cm. Before use, the column was equil ibrated with

buffer by downward flow. Just prior to application of the protein sample it was shifted to upward flow. When the column was not in use a solution of -32-

0.02% sodium azide in 20 mM Tris-Hel pH 8.0 was passed through it.

e) Preparation of DEAE-cellulose Column

i) Preparation of DEAE-cellulose Suspension

DEAE-cellulose powder (100 g) was suspended in 4 liters of After glass distilled water and then poured into 2 1 iter graduated cyl inders. cylinders permitting the material to settle the fines were removed and the Then refilled with distilled water. This process was repeated 7-8 times. concentration concentrated NaOH was added slowly to the suspension until a on of 0.2 N was reached. It was mixed for 30 minutes and then filtered the cellulose Whatman 3 MM paper placed in a buchner funnel. Following this, care was taken so was washed with water until neutrality. During this procedure in 4 liters of that it never became dry. The cellulose powder was resuspended above. 0.2 N HCI, mixed for 30 minutes and once more filtered as described was re- After it had been washed with water until neutrality, the cellulose cold room. suspended in 2 liters of 20 mM Tris-HC/ pH 8.0 and stored in the

ii) Packing of Col umn

The following procedure was carried out in the cold room. cm2 x 30 cm) About 5 ml of buffer was placed in a small Sephadex column (0.63 a 30 ml portion and care was taken that no air bubbles formed. At the same time it was de of the prepared DEAE-cellulose was diluted 2:1 with buffer. After into the gassed for 20 minutes, about 10 ml of the slurry was gently poured -33-

to column. The air bubbles were removed and the mixture was permitted of the settle. As soon as a 2-3 cm layer of cellulose covered the bottom column until column, the outlet was opened. More slurry was poured into the column was the packed cellulose reached a height of 18 cm. Before use the compressed equil ibrated with buffer under whose pressure the cellulose was the si ightly. After the protein sample was appl ied and allowed to enter to a cellulose, the column was hooked up to a pump which was attached flow rate reservoir. The pump was adjusted so that a constant and accurate

was maintained.

f) Th in-Layer Chromatography

This method may be used for the separation of bases, nucleosides

and nucleotides (145). The DEAE-cellulose for th in-layer chromatography (20 g) was was prepared in the following manner. DEAE-cellulose powder for 30 suspended in 500 ml of 0.5 N NaOH and stirred at room temperature the cellulose minutes. The mixture was filtered through a buchner funnel and in 500 ml was washed with water until neutral ity. The resin was resuspended filtered of 0.5 N HCI and mechanically stirred for 30 minutes. It was again was then on a buchner funnel and washed until neutral ity. The cellulose shaken suspended in 200 ml of water, degassed under vacuum and the suspension 13 for a few minutes until a homogeneous slurry was obtained. (Approximately cellulose.) plates (12.7 cm x 16.7 cm) could be made from this quantity of plate, evenly This homogeneous slurry was then poured onto a very c1ean glass oyen spread, dried overnight at room temperature and then put in a ventilated -34-

0 for one hour at 40 • The plates, thus made, can be stored in a dry place until use.

As little as 10-15 mJJmoles ef seltltief16 of substances to be separated were applied at a line 2.5 cm from one end of the plaie and dried.

The plate was placed in a small chromatographic tank filled with solvent to a height of 0.5-1.0 cm. The resulting spots were detected by ultraviolet light.

If the material were labelled, the radioactivity may be determined by scraping off the cellulose from the glass plate and placing it in a scintillation vial filled with the same scintillant as described in the assay section.

g) Enzymatic Synthesis of AraCTP

i) Synthesis of araCMP

0 for one hour at 40 • The plo;.,;:s, thus made,can be stored in a dry place until use.

As 1ittl e as 10-15 mjJmol es ef seltltieFl6 of substances to be separated wer e appl ied at a 1ine 2.5 cm from one end of the plate and dried.

The plate was placed in a small chromatographic tank filled with solvent to a height of 0.5-1.0 cm. The resulting spots were detected by ultraviolet light.

g) Enzymatic Synthesis of AraCTP

i) Synthesis of araCMP

AraCTP was synthesized enzymatically from araC by a two step procedure. The first step involved the phosphorylation of araC in the presence of ATP and dCR kinase to araCMP. The incubation mixture contained in a total volume of 10 ml, 500 jJmoles of Tris acetate pH 8.0, 100 jJmoles of Mg acetate, 50 jJmoles of ATP, 5 jJmoles of araC, 200 jJmoles of 2-mercaptoethanol Frllm CClI~ ~~tr\u.!o and 50 units of dCR kinase.. ,.(78). The mixture was incubated at 370 for 30 minutes. The reaction was stopped by heating in aboi 1ing water bath for 2 minutes and the resulting denatured protein removed by centrifugation in a cl ini cal centrifuge. The supernatant was carefully removed, diluted to 30 ml 2 with water, and placed on a Dowex 1 HC03 column (0.63 cm x 52 cm). After ail of it had entered the col umn, the walls were washed with 5 ml of water. The mixture was eluted with a linear NH HC0 gradient (146). The mixing chamber 4 3 -35- contained 300 ml of water as weil as 0.1 ml of 1 N NH 0H and the 4 reservoir contained 300 ml of 0.8 M NH HC0 (19 g/300 ml of water). 4 3 During elution, the column was connected to a recorder that measured optical density at 260 m~. Fractions of 15 ml were collected at 30 minute intervals. The contents of the tubes from the second peak were pool ed as they contained araCMP and their optical density checked at 280 m~ and

260 m~ using water as a blank. The solution of araCMP was then Iypholized and the crystals that were formed were dissolved in a small volume of water which was evaporated to dryness with a stream of air. This latter step was repeated twice. The material was finally dissolved in about 1.0 ml of water and the concentration determined by absorbance measurements at 280 m~.

The column was prepared for the next step by washing it with 200 ml of 1 N

NH HC0 (15.8 g/200 ml of water) fol\owed by 200 ml of water. 4 3

ii) Synthesis of araCTP

The second part of the procedure involves two reactions.

AraCMP phosphorylation to araCDP is catalyzed by dCMP kinase (98) and then another enzyme, nuc\eoside diphosphokinase (22) catalyzes the phosphorylation of araCDP to araCTP. The reaction mixture contained in

5 ml, 250 ~moles of Tris acetate pH 8.0, 50 ~moles of Mg acetate, 20

~moles of ATP, 2.5 ~moles of araCMP, 100 ~moles of 2-mercaptoethanol and about 20 mg of an enzyme fraction containing both dCMP and dCDP ~"D~ calf +1-.';\"' .... $ kinases.1\. (These enzymes were purified according to the method of Sugino et__ al.

(98) with modifications as follows: Sugino's Fraction CIII, the ammonium -36- sulfate precipitate, was suspended in 35 ml of 50 ml of Tris-HCI pH 8.0,

20 mM 2-mercaptoethanol and 20% glycerol and dialyzed overnight in

1800 ml of this sa me buffer. It was dialyzed overnight again with a fresh change of buffer whose glycerol concentration was inaeased to 50%. The

0 enzyme fraction was then stored at -20 .) The mixture was incubated at

370 for 30 minutes. The reaction was stopped as desaibed for the first step.

The supernatant was di! uted to 12 ml with water and placed on the Dowex 1

HC0 column. The same elution procedure was followed. The contents of 3 the tubes containing araCTP were Iyphol ized and twice dissolved in water and evaporated to dryness as described before. The araCTP was redissolved 2 in 2 ml of water and placed on a DEAE-cellulose column (1.75 cm x 27 cm) as it was contaminated by AMP. The material was eluted with 300 ml of a linear gradient of 0-0.3 M NH HC0 (3.5 g/150 ml of water). Fractions of 4 3 10 ml were collected every 10 minutes. The contents of the tubes containing the purified araCTP were pooled, concentrated and the NH HC0 removed 4 3 as described above. The araCTP was then suspended in 1 ml of water and its concentration detcrmined. The Dowex 1 HC0 column was washed as 3 previously described whereas the DEAE-cellulose column was washed with

100 ml of 1 N NH HC0 (7.9 g/100 ml of water). 4 3

h) Purification of Deoxynucleoside 5 1-mono-, di- and triphosphates

These deoxynucleotides were purified in the same manner as described in the previous section for the separation of araCTP from AMP.

0.5 ml of a 10 mM solution of deoxynucleotides was placed on the same size -37- column of DEAE-cellulose and eluted with a Iinear gradient of 300 ml of varying molarity of NH HC0 , depending on the material to be fractionated. 4 3 The purHied fractions were Iypholized; twice dissolved in water and evaporated to dryness; and finally redissolved in a small quantity of water and the concentration determined.

Table 2 describes the eerreet gradient system to use in order to achieve a good separation of the various deoxynucleotides.

i) Polyacryiamide Disc Electrophoresis

The disc electrophoresis apparatus used in this experiment was

Model 1200 from Canal co Research.

The method was essentiall y that of Davis (147) and Ornstein

(148) with some modification as specified in the technical literature issued by Canal Industrial Corporation (Md., USA) dated September 1968 and titled Chemical Formulations for Disc Electrophoresis.

Electrophoresis was performed at pH 9.5. A 15% running gel,

5 cm in length, was used in conjunction with a 2.8% stacking gel, 0.5 cm in length. About 0.020 mg of protein from Fraction VIII in 0.09 ml of 15 mM

Tris-HCI, 7 mM 2-mercaptoethanol and 20% glycerol was appl ied to the top of the gel and el ectrophores is was carried out for 2 hours at 3 ma/tube. At the end of this time the gels were removed from the tubes and stained for 30 minutes with 0.5% amido black in 7% acetic acid. The gels were destained by washing in 7% acetic acid for 24 hours. -38-

TABLE Il

Gradient System to Separate Various Oeoxynucleotides

Separation Gradient System

Mixing Cham ber Reservoir dCR, dCMP, dCOP and dCTP 150 ml of water 150 ml of 0.23 M NH4HC03 dTR, dTMP, dTOP and dTTP Il Il

UR, UMP, UOP and UTP Il Il

Il dAR, dAMP, dAOP and dA TP 150 ml of 0.30 M NH4HC03 dGR, dGMP, dGDP and dGTP Il Il -39-

RESULTS

1. Purification Procedure

Cal f thymus was chosen as the source of the enzyme to be purified as the rate of DNA synthesis is very high in this tissue. Many attempts were made at determining the optimum conditions necessary to achieve a good purification. The procedure finally arrived at is described below and summarized in Table III.

a) Extract

Throughout the purifi cation procedure the temperature was

0 0 maintained around 4 • Frozen (-60 ) calf thymus (300 g) was homogenized in a Phill ips blender in 600 ml of 20 mM cold Tris-HCI buffer pH 8.0, for

3 minutes. The homogenate was centrifuged at 23,000 x 9 for 30 minutes and the supernatant (565 ml) was passed through 3 layers of gauze. To the sup ernatant, 0.81 ml of 14 M 2-mercaptoethanol was then added yielding

Fraction 1.

b) Streptomycin Treatment

As streptomycin is highly positively charged, it will bind to nucleic acids, effectively removing them from solution and thus preventing them from interfering with further purification. -40-

TABlE III

Purification of Deoxyadenosine Kinase

Fraction Volume Protein Total Specifie Units Activity

ml mg units/mg

1. Extract 565 9,600 5,000 0.5

II. Streptomyci n 565 6,800 4,100 0.6 III. Protamine 102 1,530 7,800 5. 1

IV. Ammonium Sulphate 14.9 450 4,100 9. 1

V. Sephadex G-150 39 42 1,605 38.2

VI. 20% g Iycerol 9 44 1,650 38.2

VII. DEAE-cellulose 13.5 9.0 652 72.5

VIII. 60% glycerol 2.4 8.9 650 73.0 -41-

To 565 ml of Fraction l, 28.2 ml of 10% streptomycin

sul fate (neutral ized with 1 N KOH) were added slowly with stirring,

by a magnetic stirrer. After mixing for 30 minutes, the suspension was

centrifuged at 23,000 x 9 for 30 minutes and the supernatant collected.

As the pH was found to have fallen to 6.5, 1 N KOH was added dropwise

until the pH was raised to 8.0 (Fraction Il).

c) Protamine Sul fate Treatment

Protamine sulfate was used to separate the acidic (dAR kinase)

and basic proteins as it selectively binds the former.

To 565 ml of Fraction 1\, 114 ml of 2% protamine sul fa te

(neutralized with 1 N KOH) were added with stirring over a 10 minute period.

Increasing its concentrations in the mixture did not bind any more dAR kinase.

After the mixture was stirred for 30 minutes, it was centrifuged at 23,000 x 9

for 30 minutes and the precipitate retained. To the protamine sulfate

precipitate 100 ml of 2% ammonium ~ulfate in 50 mM Tris-HO pH 8.0 and

20 mM 2-mercaptoethanol were added. An ultrasonic probe was then used with continuous stirring for 17 minutes to disperse the precipitate. After the suspension was centrifuged at 23,000 x 9 for 30 minutes, the supernatant was

col\ected giving Fraction III.

d) Ammonium Sulfate Treatment

To 102 ml of Fraction III, 21.3 9 of ammonium sulfate (0-35% saturation) were added with stirring over a 15 minute period. After 20 minutes -42- of additional stirring, the suspension was centrifuged for 30 minutes at

23,000 x g. The precipitate was discarded white the supernatant was retained. Following this, 17.2 9 of ammonium sulfate (35-60% saturation) were again added with stirring, over a 10 minute period, to 105 ml of the supernatant from the previous step. After an additional stirring of

20 minutes, the precipitate was obtained by centrifugation for 30 minutes at 23,000 x 9 and dissolved in 15 ml of 50 mM Tris~HCI pH 8.0 and

20 mM 2-mercaptoethanol (Fraction IV).

e} Chromatography on Sephadex G-150 2 . A col umn of Sephadex G-150 (10-40 1-1, 4.9 cm x 50 cm) was prepared and equilibrated with 1200 ml of 50 mM Tris-HCI pH 8.0 and 20 mM 2-mercaptoethanol (buffer A). Through the column were passed, by upward flow, 14.5 ml of Fraction IV. The protein was eluted from the column with buffer A, at a flow rate of 12.5 ml per hour. Fractions of 19 ml were collected at 90 minute intervals and protein concentration and enzyme activity determined. The elution profile can be seen in Fig. 3. The two fractions (#15 and #16) of maximal specific activity and containing 45% of the activity appl ied to the column were pooled and concentrated 4.1 fold by dialyzing them (in 1/4 inch diameter tubing) against 500 ml of 70% glycerol in buffer A for 4 hours. Then in preparation for the next column, they were dialyzed against 500 ml of 20% glycerol in 20 mM Tris-HCI pH 8.0 and

20 mM 2-mercaptoethanol (buffer B) yielding Fraction V. -43-

Fig. 3 Sephadex G-150 (10-40 !J) chromatography of Fraction IV.

Fraction IV (14.8 ml) containing 445 mg of protein and 4100

units of enzyme activity was eluted from a column of 2 Sephadex G-150, 10-40 !J, (4.9 cm x 50 cm) with 50 mM

Tris-Hel pH 8.0 and 20 mM 2-mercaptoethanol. Fractions

containing 19 ml were collected at 90 minute intervals, and

the deoxyadenosine kinase assay WIlS performed as described

under Methods. -44-

Sephadex G - 150

3.0 70

o • 1 1 2.5 60 : o • ::l.-. - E 02.0 a:> (\J -o Cl) 1.5 o c > o .0 30 u L- -o o (/) .0 1.0 Cl) « E 20 ~ c • W 0.5 ) 10 .~.

o 5 10 15 20 25 Fraction number -45-

f) Chromatographyon DEAE-Cellulose 2 A column of DEAE-cellulose (0.63 cm x 16 cm) wos prepared and equilibrated with buffer B. Fraction V (9 ml) was placed on the column and eluted with a 1inear KCI gradient. The mixing chamber contained

43 ml of buffer B and the reservoir contained 43 ml of 0.3 M KCI in buffer B.

Fractions of 3 ml were collected every 8 minutes and assayed for protein and enzyme activity (Fig. 4). The fractions containing the highest specific activities (#16-#20) were combined and concentrated by dialysis against 250 ml of 3 different concentrations of glycerol in buffer A: an 18 ho ur dialysis against 70% glycerol, followed by a 5% glycerol dialysis for 4 hours and a final dialysis against 60% glycerol for another 4 hours.

A 140 fold purification was achieved with 13% recovery. The o a.Pfr()-;'1f'1~tel'f Il> '7<:1 enzyme can be stored in 60% glycerol at -20 for at least 3 months with",~ loss of activity.

g) Polyacrylamide Disc Electrophoresis

Two equally staining major components, one of which is presumed to be dAR kinase, migrated toward the anode (Fig. 5). The 51 ight separation between the two bands was observed by eye inspection but was not detected by photography due to the swell ing of the gel during the washing procedure. Traces of three visible minor contaminants are apparent. s:" Electrophoresis was also tried at pH 4-;a but the protein precipitated out upon contact with the pH 5.0 buffer and thus the run could not be performed. -46-

Fig. 4 DEAE-cellulose chromatography of Fraction VI.

Fraction VI (9 ml) containing 44 mg of protein and 1650

units of enzyme activity was eluted from a column of 2 DEAE-cellulose (0.63 cm x 16 cm) with a linear gradient

of 0.3 M KCI in 20% glycerol, 20 mM Tris-HCI pH 8.0

and 20 mM 2-mercaptoethanol. Fractions containing 3 ml

were collected at 8 minute intervals, and the deoxyadenosine

kinase assay was performed as described under Methods. e e

DEAE - Cellulose • 1.2 j60! 1• 1.0 50e- ::J.- 1'-' fO,\ E fi) "+- 0 c CX) 1- \ °:" 0,\ C\J 0.8 . \ 40 :l . 1 ~ - \ y-, ~ +- ~ "-1 0 ..li ."., \, +- 1 ., > Cl) 0.6 '...' . , 30 += 0 • • 0 c .· \l' 0 0 : '.'. ..c'- • Cl) o o '~ 0.4 \ 20 E Cf) •' 0 • ~ . .c ,• '<\, N « / . c ,. , '0'. W ,• \ ,. 0.21- , '0, ...... 0 .... ., 10 \ . ,• \ •,- '0_0/ 0 0

0 5 10 15 20 25 30 Fraction number -48-

Fig. 5 Polyacrylamide disc electrophoresis.

About 0.020 mg of protein from Fraction VIII in 0.09 ml of

15 mM Tris-Hel, 7 mM 2-mercaptoethanol and 20% glycerol

was applied to the top of the gel and electrophoresis, at

pH 9.5, was carried out for 2 hours. The gel was stained with

0.5% amido black in 7% acetic acid. -49-

pH 9·5 -49- e

" .. : . i ,i

1, 1 l ' 1 l' ',- 1

pH 9-5 -50-

Il. Properties of Deoxyadenosine Kinase

Several essential properties of the enzyme need to be

known before any meaningful kinetic studies can be undertaken. First

it was necessary to determine the range within which the formation of

product was linear with respect to time and enzyme concentration.

From the results in Fig. 6 it can be seen that possibly due to enzyme

instabil ity the rate of formation of clAMP from clAR with A TP as the phosphate

donor is 1inear with respect to time only up to 5 minutes. Beyond 7 fJl of

enzyme, product formation was no longer linear (fig. 7). This could be the

result of the inàbility to saturate the enzyme with clAR due to the insolubility of this deoxynucleoside, thus limiting the substrate available to the enzyme.

a) Identification of Product

Identification of the product and proof that the 51 position is

being phosphorylated were obtained by the following experiments. Tritiated clAR (3,500 fJc/fJITlole) was incubated at 3~ for 1 hour together with 0.02 ml of enzyme Fraction IV, 1.0 fJmole of UTP, 1.0 fJmole of MgCI , 5.0 jJmoles of 2 Tris-HCI pH 8.0 and 0.2 fJmole of 2-mercaptoethanol in a total volume of 0.1 ml.

At the end of 1 hour cold clAR (0.04 fJmole) plus more enzyme was added to the mixture. The incubation was continued for another ho ur at which time more cold clAR (0.12 fJmole) was added. At the end of a total incubation time of 3 hours, the reaction mixture was heated in a boiling water bath for 1 minute, -51-

Fig. 6 Effect of time on the amount of dAMP formed.

The incubation mixture contained, in 0.1 ml, 10 ~moles of

Tris-HCI pH 8.0, 1.0 ~mole of MgCI , 0.1 ~mole of 2 dithiothreitol, 0.1 ~mole of ATP, 100 m~oles of 14C_ 4 deoxyadenos i ne (9. 1 x 10 cpm), and 1 .7 uni ts of Fraction

VIII. The mixture was incubated for the time indicated and

assayed as described under Methods. -52-

15 le

"t:J CI) E le L- 0 10 't- • a. :;;E « "t:J

't- 0 • fi) CI) -0 5 E :::L- / E / / •

0 2 4 6 8 10 Time (minutes) -53-

Fig. 7 Effect of enzyme concentration on the amount of clAMP formed.

The incubation mixture contained, in 0.1 ml, 10 I-Imoles of Tris-

HCI pH 8.0, 1.0 I-Imole of MgCI 2, 0.1 I-Imole of dithiothreitol, 0.1 1Jffi0ie of ATP, 100 ml-lmoles of 14C-deoxyadenosine 4 (9.1 x 10 cpm) and the indicated volume of Fraction VIII

(270 units per ml). The mixture was incubated for 3 minutes 0 at 37 and assayed as described under Methods. e. -54-

c::" "ë rf) • ...... -g ro E '- 0 't- a. «~ -0

't- 0 5 V) Cl) 0 E ::J.- E

o 2 468 ro Enzyme (pl) -55-

centrifuged (in a clinical centrifuge), spotted on a DEAE-cellulose thin- layer plate and developed in 0.02 N HCI. The radioactive spot correspondin9 to clAMP, the product of the reaction, was scraped off the plate and eluted with

2 ml of 0.5 N KCI. It was then diluted to double its volume with water and 2 placed on a charcoal column (0.2 cm x 1.0 cm) to desalt. After washin9 the column with water, 3H- clAMP was eluted with 2 ml of ethanol-water-ammonia

(2:2:1, by volume), ~vaporated to dryness with a stream of air and dissolved in 0.5 ml of water.

ln order to positively identify this purified product, a portion of it was chromat09raphed on an avicel plate in n-propanol-NH 0H-water 4 (60:30:10, by volume). It produced an RF identical to that of 5'-dAMP and different from the RF's of 51-AMP, 5'-dGMP and 51-IMP.

Proof that the phosphate 9rouP is in the 51 position was shown by incubatin9 the purified 3H-dAMP with Crotalus adamanteus 5 1-nucleotidase.

The basic reaction mixture contained 2.5 !Jmoles of Tris-HCI pH 8.0, 0.5 !Jmole of M9C12' 0.1 !Jmole of 2-mercaptoethanol and 0.09 !Jmole of cold dAMP

(added after 30 minutes of incubation)in a total volume of 0.05 ml. Six tubes held this mixture with the followin9 additions; #1 - 20 !JI of purified 3H- clAMP ,

#2 - same as #1 but with 0.02 m9 of 51-nucleotidase, #3 - 0.18 !Jmole of cold clAMP, #4 - same as #3 but with 0.02 m9 of 51-nucleotidase, #5 - 0.20 !Jmole of cold UMP - 21 - 3', #6 - same as #5 but with 0.02 m9 of 51-nucleotidase.

The tubes were incubated for 1 hour at 37', the reaction stopped as mentioned above and the contents spotted on a DEAE-cellulose plate which was developed -56-

in 0.005 N Hel. The spots corresponding to dAMP and dAR were scraped

off the plate and counted. In the presence of the enzyme, dAMP lost its

phosphate group resulting in the formation of dAR. UMP-21 - 31 was

unaffected by the enzyme.

b) Requirements for Reaction

The presence of a divalent cation and ATP in the reaction

mixture was essential for activity (Table IV). Of the three cations tried,ata COIJceht,.qtlll? ++ prcdI.J.ced the nis"esf ++ ++ resuJtin! "1/ ~f 1t),.,1tII. Mg (10 MM) r86l:1l~ea iR efJtiMal activity, with Mn and Ca prQgW&liRS

a lower rate of reaction. ADP was not able to replace ATP as the phosphate

donor. The sensitivity of the enzyme to mercuric ions in the absence of the

sulfhydryl compound, dithiothreitol, was demonstrated by the presence of

0.01 mM and 0.1 mM of the heavy metal causing 25% and almost complete

i nh i bi tion respectivel y •

c) Effect of pH on Rate of Reaction

Under conditions of the routine assay, the partial! y purified

enzyme (Fraction VIII) had a broad pH optimum range with the Tris-Maleate

Bicine buffer (Fig. 8). A pH range of 7.7-9.1 could be covered by the

Tris-Maleate buffer while the Bicine buffer was useful between pH 5.5 and

8.3. There was hardly any difference (10%) in activity within the pH range

of 6.5-8.5. Below pH 6.5 a sharp drop was evident. -57-

TABLE IV

Properties of the Reaction

The complete system contained, in 0.1 ml, 10 fJmoles of Tris-HCI pH 8.0, 0.1 fJmole of ATP, 1.0 fJmole of MgCI:M 0.1 fJmole dithiothreitol, 100 mfJmoles of 14C-deoxyadenoxine (1.35 x 10'-' cpm), and 0.4 unit of Fraction VIII. The mixture was incubated at 370 for 10 minutes and assayed as described under Methods.

Conditions dAMP Formed

mfJmoles

Complete 3.5 Omit Mg++ 0.5 Omit Mg ++ and add Ca++ (10 mm) 1.0 Omit Mg ++ and add Mn ++ (10 mm) 2.6 Omit ATP o Omit A TP and add ADP (1 mm) 0.5 Omit Oithiothreitol 3.4 Omit Oithiothreitol and add Hg * (0.1 mm) 0.5 Omit Oithiothreitol and add Hg* (0.01 mm) 2.7 -58-

Fig. 8 Effect of pH on the reaction rate.

The reaction mixture contained, in 0.1 ml, 10 J.UI1oles of Tris Maleate-8icine buffer at the indicatecl pH, 1.0 ",mole of MgCI , 2 1.0 ",mole of 2-mercaptoethanal, 0.4 unit of Fraction VIII, 110 ml-'moles of 3H-deoxyadenosine (1.85 x 105 cpm),

and 0.2 ",mole of A TP. The mixture was incubated at 370 for

10 minutes and assayed as described under Methods. At pH 7.45, 2.1 mJ.Ul1oles of dAMP were formed. -59-

l'-:C c.

~----~----~~----~------~------~~ o o ~ o o ~ (\J -60-

d) Molecular Weight Determination

The molecular weight of dAR kinase was determined by gel fil tration according to the method of Andrews (149). Various protein lseE.. lege.nd "h fijo q) markers of known molecular weights",were used to cal ibrate the column.

Fig. 9 shows that a 1inear relationship between the logarithm of the molecular weight of a protein and the ratio of its elution volume to the void volume from a Sephadex G-150 (10-40 fJ) column, exists up to a molecular weight of at least 70,000. Thus applying this relationship to dAR kinase, a molecular weight of about 63,000 can be obtained. An attempt was also made to see if the potent inhibitor of the enzyme, dCTP, could cause aggregation. The same molecular weight, however, was chrorol1"tE)~ro..fhj of observed after u-adioRCiltÏl:ag the enzyme in the presence of 0.3 fJrTIole of dCTP.

e) Stoi chiometry

The stoichiometry of the reaction was determined as shown in Table V. Upon incubating UTP (the phosphate donor in this experiment) and dAR with enzyme Fraction VIII, the amounts of UTP and dAR that disappeared were approximately equal and matched the formation of UDP

(and UMP) and dAMP. In the absence of dAR from the reaction medium there was an increase in the amount of UMP produced and a decrease in UTP concentration. During the incubation, with both substrates and enzyme present, a somewhat larger amount of UMP than UDP was formed. These results -61-

Fig. 9 Determination of the molecular weight of deoxyadenosine kinase

by fil tration on Sephadex G-150.

A mixture of 1 mg each of myoglobin, ovalbumin, bovine serum

albumin (BSA) and y-globulin in a total volume of 0.25 ml

together with 0.05 ml (13.5 units) of Fraction VIII was passed through 2 a col umn of Sephadex G-150, (10-40 fJ) (0.63 cm x 50 cm)

equilibrated with 100 mM Tris-Hel pH 8.0, and 20 mM 2-mercapto

ethanol. The positions of the markers were determined by following

the absorbancy of the fractions (1 ml) at 280 mfJ. Samples (30 fJl)

were taken from each fraction and assayed for deoxyadenosine kinase

activity as descri bed under Methods. e e

Molecular Weight Determination Sephadex G - 150

3 Myoglobin

E o+- Ovalbumin' 1 0- o i'.) ~2 BSA 1 Q) ------'(t::'+- > ." .~.- "'( Globulin

dAR Kinase

o 1 1 l ,JI' 1 4.0 4.4 4.8 5.2 5.6 log mol. wt. -63-

TABLE V

Stoi ch iometry of Reaction with UTP as Phosphate Donor

The basic reaction mixture contained, in 0.1 ml, 10 ..,moles of Tris-Hel ~~ 8.0, 1.0 ..,mole of MgCI2' 0.1 ..,mole of dithiothreitol, 370 m..,moles of C-UTP (62 cpm per m..,mole) and 0.6 unit of enzyme incubated at 3~C for 30 minutes. 360 m..,moles of 14C-deoxyadenosine (95 cpm per m..,mole) were added to the B series only. The reaction was stopped by placing the tubes in ice. A 40 ..,1 portion of each tube was spotted together with 0.1 1JI1l0ie of UTP, UDP, UMP,clAR and dAMP on two DEAE-cellulose thin-layer plates. The plates were chromatographed in 0.10 N and 0.015 N HCI respectively and dried at 400 • The separated compounds were scraped off the plates and counted. No enzyme was added to the zero time sample.

Series Time UTP UDP UMP clAR dAMP

min. m..,moles A o 370 25 5 o o 30 366 25 10 D. -4 o +5 o o

B o 370 25 5 360 o 30 204 78 103 174 177

-166 +53 +98 -186 +177 -64- indicate the presence of UTP and UDP phosphatases. Thus based on the above observations, the following stoichiometric equation can be written.

dAR + UTP ---~) dAMP + UDP (10)

f) Test for Presence of Contaminating Enzymes

i) Monophosphokinase and Nucleoside Diphosphokinase

These two enzymes were shown not to be present in the final enzyme fraction as there was virtually no formation of dADP or dA TP from dAMP.

The experimental conditions were as follows. A mixture containing 10 jJRloles of Tris-HCI pH S.O, 1.0 IJmole of MgCI , 0.1 IJmole of dithiothreitol, 2 0.ljJRloleofATP,100mlJmolesof14C-dAR(1.S5x 105 cpm) and 0.4 unit of enzyme (Fraction VIII) in a total volume of 0.1 ml, was incubated at 3-,0 for 20 minutes. The reaction was stopped by placing the tubes in ice. The material was spotted on a DEAE-cellulose plate together with cold dAR, dAMP, dADP and dA TP and developed in 0.02 N HCI. After the plate was dried, the spots corresponding to these 4 markers were scraped off the plate and counted. Radioactivity was detected only in the dAR and dAMP positions and not in the dADP nor dA TP ones.

ii) ATPase

An experiment similar to the previous one was performed to 14 discount the presence of an ATPase. 1.0 mlJmole of C-ATP replaced the labelled dAR in the above reaction mixture. After a 20 minute incubation -65-

0.05 N the material was spotted on a DEAE-cellulose plate, developed in

HCI, dried and the spots corresponding to A TP, ADP, and AMP were breakdown scraped off the plate and counted. The ATP itself contained sorne AMP was products and upon taking this into account no formation of ADP or detected. Thus the enzyme fraction was not contaminated by ATPase.

g) Phosphate Donors and Acceptors

i) Specificity for Phosphate Donors

Various nucleotides and deoxynucleotides were tested for their were the ability to phosphorylate dAR and dGR (Table VI). ATP and GTP were 90% most effective phosphate donors, followed by UTP and dTTP which the order and 75% respectively as active. With either dAR or dGR as substrate deoxy of activity of the donors was the same. Deoxyguanosine, cytidine,

1 of adenosine and deoxycytidine 5 -triphosphates were much less capable

serving as phosphate donors.

il) Specificity for Phosphate Acceptors The abi! ity of enzyme Fraction VIII to catalyze the phosphorylation and of a variety of nucleosides is shown in Table VII. dAR, dGR, dCR monophosphates. cytidine (CR) were phosphorylated to their corresponding nucleotide own Whether they ail serve as substrates for dAR kinase or whether their following kinases are present in the enzyme fractions was determined by the uridine (UR) series of experiments. Neither adenosine (AR), guanosine (GR), -66-

TABLE VI

Specificity for Phosphate Donors

The incubation mixture contained,in 0.1 ml, 10 fJmoles of Tris-HCI pH 8.0, 1.0 fJmole of MgCI2' 0.1 !-Imole of dithiothreitol, 100 mfJmoles of 3H-deoxy adenosine (1.85 x 1()5 cpm) or 100 mfJmoles of 14C-deoxyguanosine (2.6 x 104 cpm) as required, 0.5 unit of Fraction VIII, and 0.1 fJmole of the nucleotide tested as a phosphate donor. The mixture was incubated at 370 for 10 minutes and assayed as described under Methods.

Phosphate Donor Phosphate Acce~tor Oeoxyadenos i ne eoxyg uanos i ne

mfJmoles nucleotide formed

Adenosine 5 1-triphosphate 4.2 5.8

Guanosine 5 1-triphosphate 4.1 5.3

Cytidine 5 1-triphosphate 0.8 1.1

Uridine 5 1-triphosphate 3.8 4.4

Deoxyadenosine 5 1-triphosphate 0.4 0.9

Deoxyguanosine 5 1-triphosphate 1.2 1.3

Deoxycytidine 5 1-triphosphate 0.2 0.2

Deoxythymidine 5 1-triphosphate 3.2 4.3 -67-

TABLE VII

Specificity for Phosphate Acceptors

pH 8.0, The incubation mixture contained, in 0.1 ml, 10 I-Imoles of Tris-Hel 0.1 unit: 1.0 tJIIIOle of MgCI2, 0.1 tJI1lole of ATP,. 0.1 I-Imole of dithiothreitol, VIII and 20 mtJl1loles and 20 I-Ic/ml of the tritiated nucleoside (except y~ Fraction for 10 minutes C-uridine) tested as acceptor. The mixture was incubated at 3]0 and assayed as desaibed under Methods.

Phosphate Acceptor Nucleotide Formed ml-lmole

Adenosine o

Guanosine o

Cytidine 0.14

Uridine o

Deoxyadenosine 0.78

Deoxyguanosine 0.75

Deoxycytidine 0.10

Deoxythymidine o -68-

nor dTR were active as substrates nor were their corresponding kinases detected.

h) Inhibition by Nucleosides of the Phosphorylation of Deoxyadenosine,

Deoxycytidine and Cytidine

One means of testing to see if any of the above mentioned capacity to nucleosides is an actual substrate of the enzyme is to measure its The inhibit the conversion of a known substrate to its nucleotide derivative. not ability of these nucleosides to interfere with phosphorylation does enzyme necessarily imply that their own conversion is catalyzed by the same byanother that they inhibit. One of their derivates (its formation catalyzed question. enzyme) could possibly be responsible for inhibiting the enzyme in the addition An example of this situation was found when it was observed that (Table of cold dCR to labelled dAR prevented any dAMP from being formed extremely VIII). Upon investigating this further, dCMP was shown to be an dAR (Table potent inhibitor of the enzyme catalyzing the phosphorylation of next section, XII). (dCDP and dCTP are also inhibitors, as will be shown in the but as no nucleoside diphosphokinase was detected the latter deoxynucleoside necessary triphosphate cannot be considered in this case.) Therefore, it was to labelled to perform the reverse experiment, i.e., the addition of cold dAR of 3 _ dCR. It can be seen (Table IX) that there was no effect on the amount H or dGR in dCMP formed from 3H- dCR upon the addition of either cold dAR

about 200 fold excess with respect to dCR. -69-

TABLE VIII

Effect of Deoxyguanosine, Adenosine and Guanosine on the Phosphorylation of Deoxyadenosine

The complete reaction mixture contained, in 0.1 ml, 10 ",moles of Tris-HCI, pH 8.0, 1.0 ~101e of MgCI2, 0.1 p-mole of ATP, 0.7 ",mole of 2-mercaptaethanol, 0.1 ",mole of C-deoxyadenosine (2.7 x 104 cpm) and 1.0 unit of enzyme. The mixture was incubated at 3~ for 10 minutes and assayed as described under Methods.

Addition Concentration clAMP Formed

mM m",moles

None 8.7

Deoxyguanosine 2.6 3.6

Deoxycytidine 3.0 0

Cytidine 1.0 5.5

Adenosine 1.2 9.0

Guanosine 0.5 9.3 -70-

TABLE IX

Effect of Deoxyadenosine and Deoxyguanosine On Deoxycytidine Kinase

The complete reaction mixture contained, in 0.1 ml, la IJmoles of Tris-HCI pH 8.0, 1.0 IJmole of Mgg 2, 0.2 IJmole of ATP, 1.0 ~mole of 2-mercaptoethanol, 1.0 mlJmole of H-deoxycytidine (3.6 x la cpm) and 2 units of enzyme. The mixture was incubated at 370 for la minutes and assayed as described under Methods.

Addition Concentration dCMP Formed

mM mlJmoles

None 595

Deoxyadenosine 2.0 588

Deoxyguanosine 2.5 580 -71-

Table VIII 0150 reveals that the presence of either dGR or

CR has an adverse effect on the phosphorylation of clAR. The addition of AR or GR does not prevent the formation of product, dAMP. Although not indicated here, another experiment yielded results which showed that

0.38 mM of clAR inhibited the phosphorylation of dGR by 30% but the addition of either cold AR or GR to the reaction mixture had no effect upon the enzyme with dGR as substrate. Due to these results the phosphorylation of dGR was further explored and the data will be presented in Results,

Section III.

The results from Table VIII suggesting that CR could possibly be a substrate for dAR kinase were rather surprising as it appears that the enzyme is 50 unspecific as te catalyze the phosphorylation of a pyrimidine nucleoside as weil as two purine deoxynucleosides. To substantiate these findings the effects of various nucleosides and deoxynucleoside monophosphates on the phosphorylation of CR were observed (Table X). In contrast to the experiment with dCR as substrate, the addition of either cold dAR, dGR or dCR but not UR decreased the amount of product formed from 3H- CR • The monophosphorylated derivatives of these deoxynucleosides, at a concentration of 0.3 mM, were also tested for their effect on 3H- CMP formation. The addition of clAMP resulted in 051 ight inhibition of the phosphorylation of 3H- CR; dGMP had no effect; and dCMP was completely inhibitory. The possibility that CR 4 could be contaminated by dCR was explored. 20 ml-'moles of 3H- CR (7.8 x 10 cpm) together with cold CR and dCR were spotted on an avi cel plate and developed in -72-

TABLE X

Effect of Nucleosides and Deoxynucleoside Monophosphates On the Phosphorylation of Cytidine

The complete reaction mixture contained,in 0.1 ml, 10 ",moles of Tris-HCI pH 8.0, 1.0 "'rlJ0le of MgCI2! 0.2 ",m.fle of ATP, 1.0rmole of 2-mercaptoethanol, 1.0 m",mole of H-cytidine (1.8 x 10 cpm), 2 units 0 Fraction VIII and the indicated additions. The mixture was incubated at 370 for 10 minutes and assayed as described under Methods.

Addition Concentration CMP Formed

mM m",mole

None 0.19

Deoxyadenosine 0.8 0.06

Deoxyg uanos i ne 1.0 0.12

Deoxycytidine 1.0 0

Uridine 1.0 0.19

Deoxyadenos ine 5'-monophosphate 0.3 0.18

Deoxyguanosine 5'-monophosphate 0.3 0.19

Deoxycytidine 5'-monophosphate 0.3 0 -73-

86% n-butanol-concentrated ammonium hydroxide (94.5:5.5, by volume).

After drying the plate the spots corresponding to CR and dCR were scraped off tne plate and counted. As no radioactivity was detected in the dCR position, labelled CR was free from 3H- dCR •

i) Stabil ity Studies During Dialysis

As enzyme Fraction VIII was found to be able to catalyze the phosphorylation of clAR, dGR, dCR and CR, it raised the possibility that one enzyme could have four substrates. Further experiments indicated that dCR kinase and dAR kinase are separate enzymes, or at least that these two deoxynucleosides are phosphorylated at different catalytic sites.

Additional evidence to support this observation was sought by the following study. A portion of Fraction IV was subjected to dialysis in buffers at two different ionic strengths with and without 2-mercaptoethanol. (For details see Table XI.) When dCR was tested as a substrate the amount of product formed was not affected by dialysis, whereas with either dAR, dGR or CR, dialysis in the absence of 2-mercaptoethanol resulted in about a 50% decrease in product formation.

j) Inhibition Studies

i) Inhibition by Deoxynucleotides

The nucleotides which were not capable of serving as phosphate donors were tested as to their effect on the activity of dAR kinase, -74-

TABLE XI

Stabil ity to Dialysis

Fraction IV was divided into 4 parts and each part diluted 3-fold (to a concentration of 10 mg/ml) by the buffer in which it was then dialysed for 48 hours. Buffer A: - 200 mM Tris-HCI pH 8.0, 20 mM 2-mercaptoethanol. Buffer B: - 200 mM Tris-HCI pH 8.0; Buffer C: - 20 mM Tris-HCI pH 8.0, 20 mM 2-mercaptoethanolj Buffer D: - 20 mM Tris-HCI pH 8.0. The incubation mixture contained, in 0.1 ml, 10 J.lmoles of Tris-HCI pH 8.0, 1.0 J.lmole of MgCI2, 0.1 J.lmole of ATP (0.2 J.lmole with deoxycytidine and cytidine), 1.0 p-mole of 2 mercaptoethanol, 30 J.l1 of enzyme, 100 mJ.lmoles 4 of deoxyadenosine ll.85 x 10S cpm), 100 mlJm~les of deoxr~anosine (2.6 x 10 cpm). ~ mJ.lmole of deoxycytidine (3.6 x 10 cpm) and ~ mJ.lmole of cytidine (1.05 x loS cpm). The mixture was incubated at 370 for 10 minutes and assayed as described under Methods.

High Salt Low Salt

Substrate Buffer Buffer B/ Buffer Buffer D/C Tested A B A C D

mJ.lmoles nuc\eotide formed mJ.lmoles nuc\eotide formed clAR 142 75 0.53 140 82 0.59 dGR 298 164 0.55 300 193 0.64 dCR 13.1 13,0 1.00 13.0 13ft 1.03

CR 1.63 0.69 0.42 -75- as shown in Table XII. Adenine, guanine and cytosine deoxynucleoside

5 1-mono-, di- and triphosphates were ail active as inhibitors of the enzyme. The most rotent inhibitors were the deoxynucleotides of cytosine, followed by adenine and then guanine. The relative inhibitory activity of these deoxynucleotides with respect to the number of phosphate groups was trit di., mono-. The end product, dA TP, was 100 times less potent an inhibitor than dCTP which at a concentration of 0.5 .,.M produced an inhibition of 60% of the dAR kinase activity.

If CR is a substrate of dAR kinase and is phosphorylated at the same catalytic site as dAR itself, the same pattern of inhibition as shown in

Table XII would be expected to emerge. Indeed, this was found to be the case (Table XIII). dA TP, dGTP and dCTP but not dTTP were shown to interfere with the phosphorylation of CR. As was observed with dAR as substrate, dCTP was again the most potent inhibitor followed by dATP and dGTP. l~e $c:.""e Although not presented here, siFRiigr concentrations of dATP, dGTP and dCTP have been found to inhibit enzyme activity to about the same extent, with dGR as the substrate.

The addition of dTTP alleviated somewhat, the inhibition produced by dA TP. This was most 1ikely due to the phosphate donor capacity of dTTP, as it was found that increasing the ATP concentration decreased the inhibition caused by dA TP and dG TP • -76-

TABLE XII

Inhibition of Deoxyadenosine Kinase by Deoxynucleotides

The incubation mixture contained, in 0.1 ml, 10 tJmoles of Tris-HCI pH 8.0, 1.0 tJmole of ~CI2' 0.1 ",mole of ATP, 0.7 ",mole of 2-mercaptoethanol, 0.1 tJmole of C-àeoxyadenosine (2.7 x 104 cpm), 1.0 unit of Fraction VIII and the deoxynucleotide tested as inhibitor. The mixture was incubated at 370 for 10 minutes and assayed as described under Methods.

Addition Concentration clAMP Formed Inhibition

",M m",moles %

None 9.7 0 dATP 50 3.1 68 dADP 50 2.9 70 clAMP 200 6.1 36 dGTP 200 3.5 64 dGDP 200 5.2 46 dGMP 500 6.6 32 dCTP 0.5 3.9 59 dCDP 1.0 3.9 60 dCMP 10 1.1 88 -77-

TABLE XIII

Inhibition of the Phosphorylation of Cytidine by Deoxynucleotides

The incubation mixture contained, in 0.1 ml, 10 IJmoles of Tris-HCI pH 8.0, 1.0 IJmole of MgCI 2 , O.~ IJmole of ATP, 1.01J~le of 2- mercaptoethanol, 1.0 mlJmore of H-cytidine (1.18 x 10 cpm), 2 units of Fraction VIII and the indicated additions. The mixture was incubated at 370 for 10 minutes and assayed as described under Methods.

Addition Concentration CMP Formed Inhibition

IJm mlJmoles %

None 0.20 0

dATP 50 0.13 35

dGTP 200 0.14 30

dCTP 0.5 0.12 38

dTTP 300 0.19 4 -78-

ii) Inhibition by Cytosine Derivatives

A comparison of the inhibitory effects of the various

phosphorylated products of CR, araC and dCR on clAR kinase was attempted,

in Table XIV, in order to reveal any pattern of specificity in the enzyme

towards the sugar component of these nucleotides. The relative inhibitory activity of these nucleotides with respecte the number of phosphate groups

is the same as above. The derivatives of dCR were the most potent inhibitors

followed closely by those of araC. AraCTP was about 10 times weaker than

dCTP but nonetheless a strong inhibitor as it is about 10 fold more effective

than the end product, dATP. The CR derivatives are very weak inhibitors of clAR kinase. -79-

TABLE XIV

Inhibition of Deoxyadenosine Kinase By Cytosine Derivatives

The incubation mixture contained, in 0.1 ml, 10 tJmoles of Tris-HCI pH 8.0, 1.0 tJmole offr9Cl2' 0.1 tJmole of ATP, 0.1 ",mole of dithiothreitol, 100 mtJmoles of 1 C-deoxyadenosine (9 x 104 cpm), and 1.8 units of deoxyadenosine kinase. The mixture was incubated at 3]0 for 10 minutes and assayed as described under Methods.

Addition Concentration dAMP Formed Inhibition

tJM mtJmoles %

None 17.9 0

araCMP 100 7.3 59

araCDP 20 11.9 33

araCTP 5 10.7 40

dCMP 5 5.5 69

dCDP 9.5 47

dCTP 0.5 9.5 47

CMP 1,000 12.7 29

CDP 1,000 9.9 45

CTP 500 13.3 26 -80-

III. Kinetic Studies on Deoxyadenosine Kinase

a) Effect of Deoxyadenosine and Deoxyguanosine on the Phosphate

Acceptors

ln the previous chapter it was observed that the addition of

dGR to a reaction mixture containing 14C-dAR, and vice versa, resulted

in a decreased formation of 14C_dAMP and 14C-dGMP respectively. To

determine the mechanism of this inhibition a study of the effect, in the first

case (Fig. 10) of dGR and in the second (Fig. 11) of dAR on the reaction

rate in the presence of different concentrations of dAR and dGR respectively.

The data have been plotted according to the method of Lineweaver and 4 3 Burk (150). The Km and Ki values for dAR were 7.4 x 10- M and 1.1 x 10- M. 3 4 The corresponding values for dGR were 1.1 x 10- M and 7.8 x 10- M. As

can be seen the Km value for each is very close to its Ki. The inhibition

in both plots was competitive with respect to the deoxynucleoside substrates.

The V values for dAR and dGR were 10.5 and 12.5 respectively. max Since it was discovered that the presence of dAR inhibited the

phosphorylation of CR, it became necessary to determine the effect of dAR

on this reaction in order to substantiate the evidence indicating that CR is

. also a substrate of dAR kinase. As observed with dGR as the substrate, dAR

was found to be a c-ompetitive inhibitor of CR phosphorylation (Fig. 12). The 4 Km and V values for CR are 6.3 x 10- M and 8.4 respectively, while max 4 the Ki for dAR is 3.5 x 10- M, a value fairly similar to its Km. -81-

Fig. 10 Effect of deoxyadenosine concentration on the inhibition produced

by deoxyguanosine.

The reaction mixture contained, in 0.1 ml, 10 ... moles of Tris-

HCI pH 8.0, 1.0 I-Imole of MgCI 2, 0.1 ... mole of dithiothreitol, 0.1 ... mole of ATP, 1.2 units of Fraction VIII, and the indicated 5 concentrations of 14C-deoxyadenosine (1.5 x 10 cpm) and

deoxyguanosine. The mixture was incubated at 370 for 5 minutes

and assayed as described under Methods. -82-

0 a::: \ (,!) • "'0

0 C a::: (,!) "'0 :E E v 0 -

. Il (Il ~~\ C\J- ~ "'\o •

~------~------~------~----~~--~o ~ o

C\J ______1 -83-

Fig. 11 Effect of deoxyguanosine concentration on the inh ibition

produced by deoxyadenosine.

The reaction mixture contained, in 0.1 ml, 10 jJmoles of

Tris-HCI pH 8.0, 1.0 jJmole of MgCI , 0.1 jJmole of 2 dithiothreitol, 0.1 jJmole of ATP, 1.2 units of Fraction VIII,

and the indicated concentrations of 14C-deoxyguanosine 4 (1.7 x 10 cpm) and deoxyadenosine. The mixture was

incubated at 3i> for 5 minutes and assayed as described under

Methods. -84-

1.0 0.4mM dAR

-c 0.8 E l!) "- no v dAR/ 0 • c -~ 0 "!- 0.6 CL 2 C) v

U) Ç,) 0 E 0.4 ::J.... E Il -> -~ 0.2

-1 o 2 4 6 I/s (s = mM d GR) -85-

Fig. 12 Effect of cytidine concentration on the inhibition produced

by deoxyadenosine.

The reaction mixture contained, in 0.1 ml, 10 ",moles of

Tris-HCI pH 8.0, 1.0 ",mole of MgCI , 1.0 ",mole of 2- 2 mercaptoethanol, 0.2 ",mole of ATP, 2 units of Fraction VIII, 5 and the indicated concentrations of 3H-cytidine (1.5 x 10 cpm)

and deoxyadenosine. The mixture was incubated at 370 for 5

minutes and assllyed as described under Methods. -86-

0.4 mM dAR

_ 1.5 c E ll') ...... "'0 Q) E o'- '+- a.. 1.0 ~ u

Cf) Q) -o E ::s- E ~ 0.5 _-..?-

-2 o 12 4 6 8 10 15 (5 = mM CR) -87-

For the determination of Ki values either of the two following

equations were util ized.

(11 ) Siope t ~ ) Km V

(12) Ki = Q] ~ - 1 Km

(Kp = the point on the graph where the 1ine representing the inhibited reaction

intersects the abscissa.)

b} Comparison of Michaelis Constants During Purification

When clAR kinase was only about 75 fold pure, that is before

column chromatography on DEAE-cellulose, the velocity of the reaction was measured using different concentrations of dAR and the data plotted in a

lineweaver-Burk plot. The apparent Km of the enzyme for clAR was determined from this plot and found to be about 5 mM (Fig. 13). Substituting dGR as the substrate, a similar Km value was obtained. As was observed in

the previous section, further purification of the enzyme resulted in about a

5-7 fold decrease in the apparent Km with either clAR or dGR as substrate. -88-

Fig. 13 Effect of different concentrations of deoxyadenosine on the

velocity of the reaction.

The incubation mixture contained, in 0.1 ml, 8 ..."oles of

Tris-HCI pH 7.8, 0.8 iJmole of MgCI 2, 0.8 iJmole of ATP, 0.6 iJmole of 2-mercaptoethanol, and the indicated concen trations of 14C-dl3oxyadenosine (2.7 x 104 cpm), and 1.0

unit of Fraction VI. The mixture was incubated at 370 for 5

minutes and assayed as described under Methods. -89-

1'"'2 E ,lO -0 Q) E ~ 0 "t- 0.2 a.. «~ -0

fn Q) -o E ~O.I E Il ->

2.0 o l, ( 1.0 's S = mM d AR ) -90-

c) Effect of dA TP and dCTP on Deoxyadenosine

The effects of dATP and dCTP on the reaction rate in the presence of different concentrations of dAR are shown in Figures 14 and 15. The Km values determined from the Lineweaver-Burk plots were similar to the one in

Figure 10. The inhibition produced by dATP and dCTP appeared to be non 5 competitive with dAR, the respective Ki constants being 5.9 x 10- M and

3.3xl0-7 M.

d) Effect of dA TP, dG TP and CTP on the Phosphate Donor

The presence of either of these deoxynucleoside triphosphates

in the reaction mixture has been observed to cause an inhibition of dAR kinase.

Thus the effect of dA TP, dG TP and CTP on the rate of formation of 3H-dAMP with different concentrations of A TP has been studied. Lineweaver-Burk plots of the results are presented in Figures 16,17 and 18. The plot of the reciprocal of the apparent velocity as a function of l/[ATP] yielded a straight

line with ATP concentrations over a ten fold range (0.087 mM to 0.83 mM). 4 The Km constants for ATP was 2.2 (± 0.4) x 10- M. The inhibition produced by dA TP, dGTP and CTP appeared to be competitive with respect to ATP. The

Ki values, under the conditions described in the legends to the figures, were -5 -5 -5 found to be 2.7 x 10 M, 4.5 x 10 M and 8.0 x 10 M respectively.

e) Effect of araCTP and dCTP on the Phosphate Donor

The mechanism of inhibition of dCTP and its analogue, araCTP, on -91-

Fig. 14 Effect of deoxyadenosine concentration on the inhibition

produ ced by dA TP •

The reaction mixture contained, in 0.1 ml, 10 fJmoles of

Tris-HCI pH 8.0, 1.0 fJmole of MgCI , 0.1 fJmole of dithiothreitol, 2 0.1 fJmole of ATP, 1.6 units of Fraction VIII, and the indicated 4 concentrations of 14C-deoxyadenosine (3.4 x 10 cpm) and dA TP.

The mixture was incubated at 370 for 5 minutes and assayed as

described under Methods. -92-

\ o

a.. ~ "0

~ E oLO o

-0:::

°"0 . -...::

~------~------~------~~--~~--~o.Ç" C\J o~ o 0

C\J1 ______-93-

Fig. 15 Effect of deoxyadenosine concentration on the inhibition

produced by dCTP.

The reaction mixture contained, in 0.1 ml, 10 jJmoles of Tris-HCI

pH 8.0, 1.0 jJmole of MgCI , 0.1 jJmole of dithiothreitol, 2 0.1 jJmole of ATP, 0.7 unit of Fraction VIII, and the indicated 5 concentrations of 14C-deoxyadenosine (1.96 x 10 cpm) and

dCTP. The mixture was incubated at 370 for 5 minutes and assayed

as described under Methods. -94-

Cl.. 1- • U

o c:

Cl.. 1- U "0

~ ::l.... lO -0:: 0 0 ~

~----~------~------~------~----~~--~~o ll1 q lO C\I C\I

______~ C\I1 -95-

Fig. 16 Effect of A TP concentration on the inhibition produced by dA TP •

The reaction mixture contained, in 0.1 ml, 10 fJmoles of

Tris-HCI pH 8.0, 1.0 fJmole of MgCI , 1.0 fJmole of 2-mercapto 2 ethanol, 1.3 units of Fraction VIII, 95 mfJmoles of 14C-deoxyadenosine 4 (9.1 x 10 cpm), and the indi cated concentrations of A TP and dA TP • The mixture was incubated at 3.,0 for 5 minutes and assayed as

described under Methods. -96-

0.5 mM dATP

0.8 -.-c E ,L{) "C Q) E '- ....0 0.6 no dATP a.. ~

en 0 Q) 0.4 -0 E 01 ::J.... E Il -> J. 0_'> ,G/ /0 0

o 5 10 15 Ils (s=mM ATP) -97-

Fig. 17 Effect of ATP concentration on the inhibition produced by

dGTP.

The reaction mixture contained, in 0.1 ml, 10 IJmoles of

Tris-HCI pH 8.0, 1.0 IJmole of MgCI 2, 1.0 1Jm0ie of 2- mercaptoethanol, 0.8 unit of Fraction VIII, 120 mlJmoles 5 of 3H-deoxyadenosine (1.9 x 10 cpm), and the indicated

concentrations of ATP and dGTP. The mixture was incubated

at 3~ for 5 minutes and assayed as described under Methods. -98-

2.0 0.21 mM d GTP o

-.-c E 1.5 LO ...... '"0 Cl) E '- 0 ~ 0 Cl.. «~ 1.0 '"0

(/) 0 Cl) 1 0 -0 E ::J..... no d GTP E • Il 0 / -> 0.5 -~

-5 o 5 10 115 (5 =m MAT P) -99-

Fig. 18 Effect of ATP concentration on the inhibition produced

by CTP.

The reaction mixture contained, in 0.1 ml, 10 jJmoles

of Tris-HCI pH 8.0, 1.0 jJmole of MgCI , 1.0 jJmole of 2 2-mercaptoethanol, 0.9 unit of Fraction VIII, 120 mjJmoles 5 of 3H-deoxyadenosine (1.9 x 10 cpm) and the indicated

concentrations of A TP and CTP. The mixture was incubated

at 3~ for 5 minutes and assayed as described under Methods. -100-

0.8 0.1 mM CTP -.-c E LO 0 "- "'0 Q) E no CTP Cs 0.6 '+- Cl. «~ "'0

CI) ~ 0.4- 0 E 0 ::l.... E Il 01 /. > /0 - •

-5 o 5 10 1/5 (5 =mM ATP) -101- clAR kinase was studied by observing the effect of these deoxynucleoside triphosphates on the rate of formation of 3H- clAMP (Figs. 19 and 20) or

3H-dGMP (Fig. 21) with different concentrations of ATP. dCTP was found to be a competitive inhibitor of ATP with either clAR or dGR as the phosphate acceptor. The Ki values from the Dixon plot in figure 19 and from the

Lineweaver-Burk plot in figure 21 were similar; 1.6 x 10-7 M and 1.3 x 10-7 M respectivel y. With clAR as one of the substrates, araCTP was also found to 6 competitively inhibit ATP, the Ki value being 4.0 x 10- M.

f) Initial Velocity Patterns

When an enzyme-catalyzed reaction involves two (or more) substrates whose concentrations can be varied independently, the reaction may follow three possible pathways: random, ordered or ping-pong. These will be alluded to in more detail in the Discussion. In order to determine which of the three mechanislT60ccur with clAR kinase, the experiments presented in figures 22 and 23 were performed. The reciprocal plots of data obtained in initial velocity studies with clAR as a variable substrate and ATP as the changing-fixed substrate yielded a series of parallel lines characteristic of a ping-pong mechanism where one product is released before the second substrate combines with the enzyme (Fig. 22). Varying ATP and using dAR as the changing fixed substrate resulted in an identical pattern of lines (Fig. 23). -102-

Fig. 19 Dixon plot of the inhibitory effect of dCTP on ATP.

The reaction mixture contained, in 0.1 ml, 10 IJmoles of

Tris-HCI pH 8.0, 1.0 IJmole of MgCI 2, 0.1 IJmole of dithiothreitol, 100 mlJmoles of 14C-deoxyadenosine 4 (9.1 x 10 cpm), 1. 0 unit of Fraction VIII, and the

indi cated concentrations of ATP and dCTP. The mixture

was incubated at 370 for 5 minutes and assayed as described

under Methods. -103-

0.5mM ATP c 0.8 -c E LO

-0""" (J) E "- 0 0.6 't- a.. «~ -0

en (J) 0.4 -0 E ::::1- E 2 mM ATP Il >

- ~--.

o 0.5 1.0 pM d CTP -104-

Fig. 20 Effect of ATP concentration on the inhibition produced

by araCTP.

The reaction mixture contained, in 0.1 ml, 10 IJmoles of

Tris-HCI pH 8.0, 1.0 IJmole of MgCI , 1.0 IJmole of 2- 2 mercaptoethanol, 0.3 unit of Fraction Viii, 167 mlJmol es of 4 3H-deoxyadenosine (3.3 x 10 cpm) and the indicated

concentrations of ATP and araCTP. The mixture was incubated

at 370 for 5 minutes and assayed as described under Methods. -105-

....0- \ u • 0 ~ 0 0- .... 0 u c 0 ~ 0 :E ::l... &() 0 -.-Cl..

~------~------~------~----~----~orr) (\J d d

(\J 1 -106-

Fig. 21 Effect of ATP concentration on the inhibition produced by

dCTP with deoxyguanosine as substrate.

The reaction mixture contained in 0.1 ml, 10 fJmoles of

Tris-HCI pH 8.0, 1.0 fJmole of MgCI , 1.0 fJmole of 2- 2 mercaptoethanol, 1.0 unit of Fraction VIII, 100 mfJmoles of 4 14C-deoxyguanosine (2.7 x 10 'cpm), and the indicated

concentrations of ATP and dCTP. The mixture was incubated

at 3-;0 for 5 minutes and assayed as described under Methods. -107-.

a.. a.. \ t- t- 0 U U "0 "0

::2: 0 ::l.- e: lO d 0

-a.. «Jo-

lO ~ E \•, -"Cf) ct ...... Cf) \

~------~------~~oo lO . d

ln 1 -108-

Fig. 22 Effect of varying concentrations of A TP on the initial

rate of reaction with deoxyadenosine.

The reaction mixture contained, in O. 1 ml, 10 ,",moles of

Tris-HCI pH 8.0, 1.0 ,",mole of MgCI , 1.0 ,",mole. of 2- 2 mercaptoethanol, 3 units of Fraction VIII, and the indicated

concentrations of 3H-deoxyadenosine (8.0 x 104 cpm) and

ATP. The mixture was incubated at 370 for 4 minutes and

assayed as described under Methods. -109-

0.5 ,./ 0.13mM ATP

0/ 0.4 c: 0.22 mM - ATP E ~

'""C CI) E "- 0 Ii. '+- 0.3 • a. «~ / /0 "C

fi) CI) /0 -0 0.2 E 0 ::1... E -"> ~ 0.1

o 2 4 6 1/s (s = mM d AR) -110-

the Fig. 23 Effect of varying concentrations of deoxyadenosine on initial rate of reaction with ATP.

The reaction mixture contained, in 0.1 ml, 10 jJmoles of 1.0 jJmole of 2- Tris-HCI pH 8.0, 1.0 jJmole of MgCI 2, mercaptoethanol, 3 units of Fraction VIII, and the indicated 4 concentrations of 3H-deoxyadenosine (8.0 x 10 cpm) and 0 ATP. The mixture was incubated at 37 for 4 minutes and

assayed as described under Methods. -111-

0.8

-.-c E ~ 0.6 -0 (l,) E 0/ o'- 0.42mM 't- dAR Cl.. ~ « 0.4 ""0 o 1.2 mM

Cf) dAR (l,) -o E

-> -~

o 2 4 6 8 10 12 I/S(s=mM ATP) -112-

DISCUSSION

1. Purifi cation

Deoxyadenosine kinase, which was shown to catalyze the transfer of a phosphate group from specific nucleoside 5 1-triphosphate donors to the 51 position of dAR, was purified about 140 fold from calf thymus. A purification procedure involving fractionation by streptomycin, protamine sulfate, Sephadex G-1S0 and DEAE-cellulose was employed

(Table III). Throughout these steps the enzyme was fairly unstable, necessitating the presence of a reducing agent, 2-mercaptoethanol, and glycerol. Possible contaminating enzymes such as ATPose, dAMP nucleoside monophosphokinase or nucleoside diphosphokinase were not de tected in the final enzyme fraction·. Upon subjecting a portion of Fraction

VIII to polyacrylamide disc electrophoresis (Fig. 5) two major equally staining bands plus a few very minor components were observed. These bands are similar in charge (negative at pH 9.5) and size since only after

2 hours was separation achieved. Adequate separation is usually obtained after one hour. One of them might be the enzyme, dCR kinase, as it has been shown to be a contaminant in the final enzyme fraction and its purification procedure (78) is very similar to that of dAR kinase. As polyacrylamide disc electrophoresis is such a sensitive procedure it might be possible to obtain the homogeneous enzyme using the preparative apparatus provided the enzyme is -113-

stabl e under these conditions.

Il. General Properties

The requirements of the enzyme for a divalent cation and a phosphate donor are similar to the other deoxynucleoside kinases (58,60,

78-80). Mg ++ was found to be the most active cation followed by Mn ++ and then Ca++ (Table IV). These cations combine with ATP (151), for example, to give the complex, ATP-Mg, which could possibly be the active form of the phosphate donor. The presence of the heavy metal, Hg, effectively inhibited the enzyme indicating the importance of sulfhydryl groups at the catalytic site.

The molecular weight of dAR kinase, estimated to be about

63,000 by gel filtration (Fig. 9), is very close to that for dCR kinase (79) but differs considerably from mammalian dTR kinase (60) which is greater than

700,000. Certain enzymes such as dTR kinase (59,61) and ribonucleotide reductase (38) have been shown to form aggregates under various conditions.

There was no evidence of aggregation of dAR kinase in the presence of the inhibitor, dCTP.

As found with the other deoxynucleoside kinases (58,60,78-~0) there was no absolute specificity for phosphate donors (Table VI). ATP and

GTP served almost equally weil as phosphate donors with UTP and dTTP being somewhat less effective; CTP, dATP and dGTP were very weak and dCTP was -114-

nearly inactive.

III. Substrate Speci fi city

The enzyme Fraction VIII appeared to catal yze the phos

phorylation of dGR, CR and dCR as weil as clAR (Table VII). There are

three possible explanations for this observation. These nucleosides could

be phosphorylated by: (i) the same enzyme, (ii) different enzymes, or

(m) the same enzyme but at different catalytic sites. The latter

possibility cannot be excluded at the present time.

a) Phosphorylation of Deoxyadenosine

The final enzyme fraction was shown to catalyze the phosphorylation of clAR. The product, clAMP, was isolated by thin-layer chromatography and shown to be identical with the known compound. Treatment of dAMP with

Crotalus adamanteus 5 1-nucleotidase resulted in the removal of the phosphate group indicating that clAR is phosphorylated at the 51 position.

b) Phosphorylation of Deoxyguanosine

The same enzyme, dAR kinase, appears to catalyze the phosphorylation of dGR as weil as CR. In the case of dGR th is hypothesis is substantiated by the following observations: (i) the addition of cold dGR resulted in a decrease in the rate of conversion of 14C- dAR to 14C-dAMP (Table VII!); -115-

(ii) a similar decrease in stability was found during the dialysis study

(Table XI); (iii) a constant ratio of specific activity towards dAR and dGR was demonstrated during purifi cation on DEAE-cell ulose (not presented in the Results section); (iv) the same pattern of activity with inhibitors and phosphate donors was shown for dAR and dGR (Table Vl)i (v) kinetic data such as, (a) Km values decreased during purification in a similar fashion

(Figs. 10,11 and 13), (b) Km values for dAR and dGR were close to their respective Ki values with the other as inhibitor (Figs. 10 and 11), (c) the

Ki value for dCTP was the same with either dAR or dGR as the substrate

(Figs. 15,19 and 21).

c) Phosphorylation of Cytidine

The finding that dAR kinase could catalyze the phosphorylation of CR was unexpected as Skôld (152) and Orengo (153) have reported that a partially purified uridine kinase from Ehrl i ch and Novikoff ascites tumor cells respectively, can catalyze the phosphorylation of UR and CR, but not dAR or dGR. That CR could be a substrate for dAR kinase was supported by the following experiments: (i) It was observed that the addition of either cold dAR or dGR decreased the conversion of 14C_CR to 14C_CMP (Table X).

It could be argued that the phosphorylated products of these deoxynucleosides, dAMP and dGMP, inhibit the formation of CMP. However, it was shown that the addition of either dAMP or dGMP at sufficiently high concentration

(0.3 mM) had no effect on the phosphorylation of CR (Table X). The complete -116- inhibition of CMP formation in the presence of dCR is most likely caused by dCMP as dCR kinase has been shown to be present in enzyme Fraction

VIII. If dAR kinase catalyzes the phosphorylation of CR, as it appears to do, it is understandable that dCMP would interfere with the reaction as it is a potent inhibitor of the enzyme (Table XII). (ii) Since the addition of an excess of UR had no effect on the conversion of CR to CMP, it is most

Iikely that the enzyme which catalyzes the phosphorylation of CR is not the same as the one described by Skëld (152) and Orengo (153). (iii) Furthermore, the same pattern of inhibition with deoxynucleoside triphosphates as observed with dAR and dGR emerged when CR was the substrate for the enzyme (Table XII!). (iv) dAR was also shown to be a competitive inhibitor of the phosphorylation of CR, the Ki of dAR being close to its Km value (Fig. 12). Whether dAR kinase plays an active part in the phosphorylation of CR ~ vivo is uncertain, since the affinity of this nucleoside for UR kinase

is somewhat greater (Table XV). However, as UR kinase activity has been found to be very low in calf thymus (154), dAR kinase could perhaps be the main enzyme involved in its phosphorylation in this tissue.

d) Phosphorylation of Deoxycytidine

The phosphorylation of dCR was shown to be catalyzed by dCR

kinase (78,79), an enzyme that is most likely different from dAR kinase.

This was demonstrated experimentally here by the inabil ity of either dAR or dGR, added in 200 fold excess, to interfere with the phosphorylation of dCR e e

TABLEXV

A Comparison of the Properties of Nucleoside Kinases

Kinases Substrates Km Activators Phosphate Inhibitors Ki References donors dTR dTR, UR 83 ~ (dTR) dCDP ATP, dGTP, dTTP 400r 5, 58 (~.coli) dATP, dCTP {dTR dTR dTR, dUR 3 ~M (dTR) ATP dTTP 60 (mammalion)

1 --0 dCR dCR, araC 14 ~ (dCR) GTP, ATP, dCTP, dCDP, 1.8 ~M 78-80 --0 40 ~ (araC) UTP, dTTP dCMP (dCTP) 1 dGTP, CTP, " dATP

UR UR, CR 48 ~ (UR) ATP, GTP, CTP, UTP 560 fJM 152, 153 23 ~ (CR) dATP, dGTP, (CTP) dUTP, dCTP, dTTP

AR AR 1.8 ~ ATP, ITP 155,156 GTP, dATP dAR dAR, dGR, 740 ~ (dAR) ATP, GTP, dATP, dGTP, 0.2 ~M (dCTP) CR 1100 ~M (dGR) UTP, dTTP dCTP, CTP 30 ~M (dATP) 630 ~ (CR) 45 ~ (dGTP) 80 ~ (CTP) -118-

(Table IX). Further proof was shown by the distinct difference in the ability of the enzyme fraction to catalyze the phosphorylation of dAR and dCR after dialysis (Table XI). dCR kinase remained stable during dialysis in the absence of 2-mercaptoethanol whereas dAR kinase lost about half its activity under the same conditions.

Cellular studies of Bernard and Brent (157) offer additional evidence to support these findings. Kinase activity towards four deoxynucleosides was measured during the cell cycle of 3T3 cells derived from mouse fibroblasts.

80th dCR and dTR kinases increased about 4 and 10 fold respectively during

S phase, i.e., during the period of DNA synthesis, whereas the kinase activity towards dAR and dGR remained constant at a relatively high level throughout ail phases of the cell cycle.

The above results ar.e in contrast to the reports of Schrecker

(85,86) and Durham and Ives (80). Although the former (86) reported similar findings, with Ll210 cells, to the ones presented here, i.e., the interference of the phosphorylation of dAR by dGR and dCR, and of dGR by clAR and dCR, he concluded that the phosphorylation of dGR and possibly clAR might be catalyzed by dCR kinase. This conclusion was based solely on the observation that in a subi ine of leukemia Ll210 cells made resistant to araC, the phos phorylation of araC, of dCR and of dGR was decreased by 95%, 98% and 87% respectively, in comparison with the parent 1ine, but that of dAR decreased by about 30% (85). Furthermore, the author found that dGR did not interfere with the phosphorylation of araC. As araC is a substrate fo~ dCR kinase and a -119- competitive inhibitor with respect to dCR (78-80), the fact that dGR was not an inhibitor of araC phosphorylation casts grave doubts on the suggestion that dGR is indeed a substrate for dCR kinase.

Durham and Ives (80) also suggest that dCR kinase from calf thymus can catalyze the phosphorylation of dGR and dAR as weil as araC and dCR. They do not present much evidence to support their statement. In fact they say that the activity of the enzyme toward dGR and dAR (but not dCR), as phosphate acceptors, is susceptible to inactivation during enzyme isolation. This could not occur if dCR kinase were the enzyme responsible for catalyzing the phosphorylation of these purine deoxynucleosides. These authors also found that dGR, at high levels (2-4 mM), as weil as dAR, competitively inhibited araC phosphorylation. This phenomenon could be due to the low solubility of dGR or due to the formation of dGTP which at a concentration of 0.1 mM was shown by Schrecker (86) to inhibit the phosphorylation of araC.

Another possibil ity, which could also explain the mixed kinetics observed by Durham and Ives (80), is that the phosphorylation of araC might be catalyzed, to a limited extent, by dAR kinase as weil as dCR kinase. This suggestion should be considered as araC is an analogue of CR which serves as a substrate for dAR kinase. The reason that Durham and Ives (80) and not

Schrecker (86) observed inhibition of araC phosphorylation by dGR could be that the former's enzyme preparation had a greater ratio of dAR kinase to dCR kinase than the latter1s. Inhibition of dAR kinase in the former case would thus noticably decrease the amount of araC being phosphorylated, but in the latter -120- case any decrease would be negligable. U)i-t~ 'r\-.e.. (.all:- t~'j"'I..\S e.n~j""e. The work of Momparler and Fischer (78),I\support the findings that dCR kinase is not the same enzyme which catalyzes the phosphorylation of clAR and dGR. Upon adding excess clAR and dGR to the incubation medium they found that these deoxynucleosides did not interfere with the phosphorylation of dCR to dCMP.

IV. Inhibition Studies

clAR kinase can be inhibited to varying extents by ail the deoxynucleoside triphosphates, except for the phosphorylated derivatives of dTR regardless of the nucleoside substrate used (Table XII and XIV). Thus the enzyme does not appear to have too great a specificity for either substrate or inhibitor. It can catalyze the phosphorylation of a purine deoxyriboside, clAR or dGR, as weil as a pyrimidine riboside, CR, with about equal efficiency.

Ali of the natural inhibitors of clAR kinase except for CTP, the weakest, have the deoxyribose sugar moiety but differ in their bases. The most potent inhibitor, dCTP, contains a pyrimidine base whereas those deoxynucleoside triphosphates with a purine base, clATP and dGTP, are 100 and 400 fold respectively weaker as inhibitors. AraCTP, the next strongest inhibitor differs from dCTP only in that the former contains an hydroxy group in the 2 1 position that is trans with respect ta the 3 1 hydroxy group. Consequently it appears that optimal inhibition is obtained when a deoxyribose sugar is used in combination with a cytosine base. -121-

V. Kinetic Analysis

a) Mechanism of Reaction

The majority of the enzymatic reactions that have been studied in detai! appear to fall into just a few categories as designated by Cleland (107). The mechanism describing the situation where ail substrates must add to the enzyme before any products are rel eased is termed "Sequential". This can be further subdivided into "Ordered" reactions in which the substrates add in obligatory order and the products leave similarly,or "Random" in which there is no obligatory order of addition of substrates or dissociation of products. A third or "Ping-Pong" mechanism is said to occur when one or more products are released before ail the substrates have added to the enzyme.

ln order to further clarify a given enzyme reaction Cleland (107) has proposed that the syllables Uni, Bi, Ter, Quad etc. be designated to represent the number of kineti cally important substrates or products. Thus a reaction with two substrates and two products is termed Bi Bi and a reaction involving one substrate and two products a Uni Bi reaction.

The various reaction mechanisms can be graphically illustrated as follows, The enzyme is represented by a horizontal 1ine and substrate additions and product dissociations by verti cal arrows. Substrates are indicated by the letters A, B, C, D in the order in which they add to the enzyme and products are designated P, Q, R, S in the order in which they leave the enzyme. -122-

The letter E represents the "free" enzyme whereas E in combination with another letter indicates a transitory complex of enzyme and substrate or product. Examples of the three reaction sequences are presented below.

Ordered Bi Bi

A B P Q

E 1 EA l l EQ l E (EAB) EPQ

Random Bi Bi

A B

E EAB) E ( EPQ EP

B A Q P -123-

Ping-Pong Bi Bi

A B Q Ir---

E El E

Initial velocity studies of reactions involving two substrates, where the concentration of one substrate is varied at various fixed levels of the other, results in three linear patterns in Lineweaver-Burk plots

(158). For reactions proceding via an ordered mechanism the double reciprocal plots intersect above the abscissa and to the left of the ordinate, but for a random sequence they meet on the abscissa to the left of the ordinate.

To be compatible with a ping-pong mechanism, the enzyme kinetics have to satisfy the following equation.

v = 1 + Ka + Kb (13) v a b

This means that ail plots of l/v versus either l/a or l/b as variable substrates will be parallel 1ines of identical slope, regardless of the concentration of the fixed substrate. -124-

From the results presented in Figures 22 and 23 the mechanism of action of 'clAR kinase appears to be ping-pong Bi Bi. The kinase is thus postulated to first bind ATP with release of ADP and then dAR with the subsequent release of dAMP as illustrated below.

ATP ADP dAR dAMP

E l EATP l El l E'dAR l E E'ADP E'dAMP

A number of enzymes have been studied as to their reaction mechanisms. Many reactions catalyted bya variety of NAD- and NADP- requiring dehydrogenases are ordered whereas many kinases such as creatine kinase follow a random mechanism (158). Other}inases, e.g., UR kinase

(153) and nucleoside diphosphokinase (21,108) obey a ping-pong type mechanism.

According to the ping-pong mechanism a stable phosphoenzyme should be formed during the enzymatic reaction. The formation of such an intermediate can be shown by incubating the enzyme with A TP-r 32p and then chromatographing the reaction mixture on a gel filtration column. The elution pattern should show a peak of enzyme activity containing 32p and the ratio of

32p to enzyme activity should remain constant throughout the peak. To el iminate the possibility that ATP-r32p binds, as a whole, to the enzyme instead of transferring its r 32p, the enzyme should also be incubated with 14C_A TP. In -125-

t'his case the peak of enzyme activity should be free of radioactivity.

Using a similar technique Garces and Cleland (108) and Norman !!' ~. (110) isolated a phosphorylated intermediate of nucleoside diphospho kinase. Upon enzymatic hydrolysis of the phosphorylated enzyme, the latter group (110) found that the 32p was associated with a histidine residue. Other phosphorylated enzymes such as phosphogl ucomutase (160) have been isolated and the phosphate group shown to be bound to serine.

b) Mechanism of Inhibition

dAR kinase is subject to end product or feedback inhibition as are many of the other known enzymes in the salvage pathway (5,60, 78-

80) (Table XV). dATP was shown to be a competitive inhibitor with respect to A TP (Fig. 16) but not with respect to dAR (Fig. 14). Th is is in agreement with the observation on the mechanism of feedback inhibition of mammalian dTR kinase (60), but is in contrast to the results of Okazaki and Kornberg (5) with the É.. coli enzyme in that dTTP appeared to compete with the phosphate acceptor and not the phosphate donor, A TP • The authors suggested that dTTP probably does not bind to the catalytic site for dTR but to an adjacent region.

This paradoxical phenomenon was partially eXplained by the observation that the enzyme aggregated in dilute solutions (61). The inhibition by dTTP is simple competitive with respect to dTR in the presence orthe disaggregated enzyme whereas with the aggregated form dTTP is non competitive with respect to dTR. Durham and Ives (80) have found that at low concentration of dCTP -126-

6 (1.0-2.5 x 10- M) the inhibition of araC phosphorylation is simple competitive while at higher values the inhibition pattern changes to mixed inhibition, with an inaeasing non competitive element.

The other inhibitors of dAR kinase, dGTP, dCTP, araCTP

and CTP were ail shown to be simple competitive with respect to the phosphate donor, A TP, and probably bind at the ATP site. At low concentrations of A TP, in the presence of dATP, dGTP or CTP (figs. 16,17 and 18), the points did not fall on a straight line. This finding is most likely due to the fact that these inhibitors can also act as phosphate donors to a si ight extent (Table VI), thus inaeasing significantly the velocity of the reaction in the presence of low levels of ATP.

VI. Regulation of Deoxyadenosine Kinase Activity

a) Control Through Km Val ues

During the early steps of the purification measurement of the Km for 3 dAR was found to be 5.0 x 10- M (fig. 13). Upon further puri fi cation of the enzyme, 4 it was shown to have a value of 7.4 x 10- M (fig. 10). The Km for dGR 3 similarly deaeased to a val ue of 1 .1 x 10- M (fig. 11). As the Km 4 for CR, 6.3 x 10- M (fig. 12), is close to that for the other two substrates and the V values do max not differ greatly from each other, dAR kinase cannot be said to have a preferred substrate. CR, as mentioned previousl y, could possibly be phosphorylated ~ vivo by UR kinase (152,153) as the Km of this enzyme for CR is 2.3 x 10-5 M. -127-

The Km values of clAR kinase are 10-100 fold greater than those of the other deoxynucleoside kinases in the salvage pathway (Table XV). These apparent Km values would favor any inhibition produced by the deoxynucleotides of adenine, guanine and cytosine and would also explain why high concentrations of dAR (2-4 mM) were required to inhibit DNA . synthesis in mammalian cells (111,112,114). A possible expia n.ation for the high Km values and inhibition by deoxynucleoside triphosphates is that. these factors are some form of a control mechanism to prevent the accumulation of cIA TP. If the enzyme had a high affinity for clAR the concentration of the end product, dATP, would increase resulting in an inhibition of ribonucleotide reductase (15) and therefore producing a secondary inhibition of DNA synthesis. Ribonucleotide reductase (15,16), one of the key enzymes in the de ~ synthesis of deoxynucleotides, is controlled bya complex pattern of inhibition as weil as activation by nucleoside triphosphates. Ali of the deoxynucleoside triphosphates except dCTP are inhibitory for the reduction of one or more nucleoside diphosphate substrates. Only dATP is inhibitory for the reduction of ail substrates and dGTP is inhibitory for three of them. 80th of these nucleotides are 6 effective inhibitors at extremely low concentrations (10- M). The potent inhibition of ribonucleotide reductase by dATP or dGTP may require regulation of dAR kinase activity. One of the regulatory mechanisms for control of the activity of clAR kinase may be its high Km value as discussed here; the other is the inhibition of the enzyme by deoxynucleotides which will be discussed in the following section. -128-

b) Significance of inhibition

It is essential that the concentrations of deoxynucleoside triphosphates are in balance with each other in order to maintain fidelity of replication of DNA. If this balance is upset by too high or Iowa level of a particular deoxynucleotide the chances of mutation occuring due to incorrect base pairing are probably considerably greater. The cell has developed quite an intricate mechanism to maintain a certain concentration of the different deoxynucleotides. If, for instance, the level of dCTP is too high, the enzyme dCMP deaminase is activated and dCMP is transformed eventually into dTTP (6). The concentration of this latter deoxynucleoside triphosphate is also weil regulated. As the level of dTTP increases, it not only decreases dCMP deaminase activity (6), but also inhibits ribonuc\eotide reductase (16). However, what mechanism functions for the control of the purine deoxynucleotides? As dA TP is a very potent inhibitor of the reductase

(15,16) it is necessary to decrease its concentration to a level that will not completely inhibit thi~ enzymesince DNA synthesis will cease due to depletion of the other deoxynucleoside triphosphates. Thus control of the biosyntheti c and catabol ic pathways of dA TP is important. A possible mechanism for the regulation of dA TP levels with respect to dAR kinase could be the following.

Due to the equilibrium constantiof their respective enzymes, the concentrations of dA TP, dADP and dAMP exist in a constant ratio with each other. For example, the equilibrium constant (K) for the reaction dAMP

+ ATP, ' dADP + ADP can be represented mathematically by the formula -129-

K =[dAD~ IP.D~ / @AMfi ft. T-g • Since the concentrations of ATP and AOP are considerably greater than those of dAMP and dAOP and the ratio

I}.oij / ~ T~ is most likely constant for most cells, this ratio can be

incorporated into the equil ibrium constant to give K'a = @ADfI / @AM.f} •

The same type of analysis can be done for the reaction between dADP and

dA TP to give K'b = @A T~ / @AoIJ. To illustrate the importance of these equil ibrium constants let us assume that the relative concentrations of

dATP, dADP, and dAMP are 100: 10: 2 as illustrated below (159).

Relative Concentrations

100 50

10 5

The K'a = 2/10 = 0.2 whereas the K'b = 10/100 = 0.1. When dAR kinase is

inhibited completely by dCTP, the concentration of dAMP will decrease due

to the presence of non-specifie phosphatases which will dephosphorylate dAMP

to dAR. If we assume that the relative concentration of dAMP is reduced to l,

then the concentration of dADP must be reduced by one half to 5 to maintain the equilibrium constant (K'a = 1/5 =0.2).Also to maintain equilibrium the concentration of dATP must be reduced by one half to 50 (K'b = 5/50 = 0.1). -130-

As can be seen, a relatively small change in dAMP concentration will drastically alter the equilibrium, decreasing dATP levels. A similar mechanism could possibly function with the phosphorylated derivatives of dGR. The inhibition of dAR kinase by dCTP may possibly be a control mechanism for the regulation of the intracellular pool size of dA TP and dGTP. However, such a mechanism described here must remain hypothetical until further evidence is found to support it since the cell may have other mechanisms, unknown at this present lime, to control the equilibrium and intracellular concentrations of purine deoxynucleotides.

Since CR can act as a substrate for dAR kinase, inhibition of this enzyme could possibly also affect the pool size of CTP, dCTP and dTTP in the following manner.

dTTP 1, dTDP --~"'dl

reductase ------~.~>dCDP------~

= inhibitor

= activator -131-

The concentration of CTP would decrease, in order to maintain the equil ibrium, in a similar fashion to dATP if a phosphatase acted upon

CMP while dAR kinase was in an inhibited state. At the same time in order to maintain equilibrium the level of CDP would also fall, decreasing the amount of substrate available for the enzyme, ribonucleotide reductase, and therefore lowering the concentration of dCTP. As dCTP is required to activate dCMP deaminase, the formation of dTTP will thus be affected.

Since dCR kinase is also strongly inhibited by dCTP, this would serve to further lower the amount of this deoxynucleotide. The above description of a possible control mechanism is simpl ified in that it do es not take into account the contribution by UR kinase (152,153) wlich can also catalyze the phosphorylation of CR. 'Thus from the foregoing, it can be seen that not only must the biosynthesis of nucleotides be closely regulated but their catabolism must also be weil controlled to achieve, at each moment, the correct levels to maintain a viable properly functioning cell.

The inhibition of dAR kinase byaraCTP (Table XIV) is of interest from the point of view of cancer chemotherapy since araC is used in the treatment of acute leukemia (122-124). In mammalian cells araC is rapidly phosphorylated to araCTP (81), which is a potent inhibitor of DNA synthesis (123, 128-131). The mechanism by which araCTP inhibits DNA synthesis in mammalien cells has not been completely elucidated. This nucleotide analogue may act by inhibiting DNA polymerase directly (134,

136), by being incorporated into DNA (128), and/or by interfering with dAR metabol ism by inhibition of dAR kinase. The exact mode of action of -132-

araCTP must await further clarification of the mechanism of DNA replication in mammalian cells and of the role of dAR kinase in deoxynucl eotide metabol ism. -133-

SUMMARY

Deoxyadenosine kinase, which was shown to catalyze the transfer of a phosphate group From specific nucleoside 51-triphosphate donors to the 51 position of dAR was purified about 140 fold from calf thymus. A purification procedure involving fractionation by streptomycin, protamine sulfate, Sephadex G-150 and DEAE-cellulose was employed.

Upon subjecting a portion of the final enzyme fraction to polyacrylamide disc electrophoresis, at pH 9.5, two major equally staining bands plus three minor components were detected. The enzyme was found to require the presence of a divalent cation, Mg ++, and a phosphate donor, such as ATP, for activity. The presence of mercuric ions effectively inhibited the enzyme indicating the importance of sulfhydryl groups at the catalytic site. The rate of reaction was not affected bya change in pH

From 6.5-8.5. The molecular weight of dAR kinase was determined by gel filtration and found to be about 63,000. The final enzyme fraction was shown to be free From contamination by ATPase, nucleoside monophosphokinase and nucleoside diphosphokinase. Among the nucleotides tested for their ability to serve as phosphate donors ATP and GTP were the most effective, followed by UTP and dTTP. Enzyme Fraction VIII was found to be able to catalyze the phosphorylation of the nucleosides dAR, dGR, CR and dCR. Further experiments involving inhibition and stability studies as weil as kinetic data indicated that dAR, dGR and CR but not dCR could serve as substrates for dAR kinase. The enzyme was subject to inhibition by adenine, guanine and cytosine deoxynucleoside -134-

5 1-mono, di- and triphosphates as weil as by the phosphorylated derivatives of cytosine arabinoside and to a 1imited extent by those of cytidine. The most potent inhibitor was dCTP which at a concentration of 0.5 ~M resulted

in a 60% inhibition of the enzyme. 4 The Km values for dAR, dGR and CR were 7.4 x 10- M,

1.1 x 10-3 M and 6.3 x 10 -4 M respectively. dAR was found to be a

competitive inhibitor of dGR and CR phosphorylation. dGR also competitively

interfered with the phosphorylation of dAR. The inhibition produced by dA TP, dGTP, dCTP, CTP and araCTP appeared to be competitive with respect to the phosphate donor, A TP, and non competitive with respect to the phosphate acceptor, dAR, the Ki values being 2.7 x 10-5 M, 4.5 x 10-5 M, 2.0 x 10-7 M, 8.0 x 10-5 M and 4.0 x 10-6 M respectively. In the presence of the phosphate acceptor, dGR, dCTP was still a competitive inhibitor with respect to ATP and the Ki value, 1.3 x 10-7 M, was also similar. Studies on the initial velocity indi cate that the enzyme reaction follows a ping-pong mechanism. -135-

CLAIMS TO ORIGINAL RESEARCH

1. Deoxyadenosine kinase was purified about 140 fold from calf thymus.

A purification procedure involving fractionation by streptomycin,

protamine sulfate, Sephadex G-150 and DEAE-cellulose was employed.

Purity of the final enzyme fraction was checked by polyacrylamide

disc electrophoresis.

2. The enzyme was shown te catalyze the transfer of a phosphate group

from specifie nucleaside 51-triphosphate donors to the 51 position of

dAR.

3. The enzyme was found te require the presence of a divalent cation,

Mg ++, and a phosphate donor, such as A TP, for activity. The presence

of mercuric ions effectively inhibited the enzyme indicating the

importance of sulfhydryl groups at the catalytic site.

4. The molecular weight of dAR kinase was determined by gel filtration

and found to be about 63,000.

5. Among the nucleotides tested for their ability to serve as phosphate donors ATP and GTP were the most effective followed by UTP and

dTTP. -136-

6. dAR kinase was shown to catalyze the phosphorylation of dAR, dGR

and CR by experiments involving inhibition and stability studies as

weil as kinetic data.

7. The enzyme was found to be subject to inhibition by adenine, guanine

and cytosine deoxynucleoside 5'-mono-di- and triphosphates

as weil as by the phosphorylated derivatives of cytosine arabinoside and to

a limited extent by those of cytidine.

-4 -3 8. The Km values for dAR, dGR and CR were 7.4 x 10 M, 1.1 x 10 M and 6.3 x 10-4 respectively.

9. dAR was found to be a competitive inhibitor of dGR and CR

phosphorylation. dGR also competitively interfered with the phosphorylation

of dAR.

10. The inhibition produced by dATP, dGTP, dCTP and araCTP

appeared to be competitive with respect to the phosphate don or , A TP,

and noncompetitive with respect to the phosphate acceptor, dAR, the 7 5 Ki values being 2.7 x 10-5 M, 4.5 x 10-5 M, 2.0 x 10- M, 8.0 x 10- M -6 and 4.0 x 10 M respectively.

11. In the presence of the phosphate acceptor, dGR, dCTP was still a 7 competitive inhibitor with respect to ATP and the Ki value, 1.3 x 10- M, -137-

was also similar.

12. Studies on the initial velocity indicate that the enzyme reaction

follows a ping-pong mechanism. -138-

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