Human placental deoxyadenosine and deoxyguanosine phosphorylating activity

We studied deoxyadenosine and deoxyguanosine phosphorylating activities in human placental cytosol. The specific activities of nucleoside kinase enzymes in nanomoles per h per mg +/- SD were as follows: adenosine kinase, 30 +/- 14; deoxyadenosine kinase, 12 +/- 2; deoxycytidine kinase, 0.30 +/- 0.04; and deoxyguanosine kinase, 27 +/- 16. Three major activities were resolved by ion exchange and affinity chromatography: deoxyguanosine-deoxycytidine kinase, deoxycytidine-deoxyadenosine kinase, and adenosine-deoxyadenosine kinase. Two other activities contained significant quantities of deoxyadenosine kinase. Deoxyguanosine-phosphorylating activity eluted as a single peak in association with deoxycytidine kinase. This deoxyguanosine-deoxycytidine kinase had an apparent molecular weight of 54,000, a Stokes radius of 31 A, and apparent Km values of 10, 130, and 14 microM for deoxyguanosine, deoxycytidine, and ATP, respectively. Four peaks of deoxyadenosine phosphorylating activity were resolved by affinity chromatography with AMP-Sepharose 4B. Adenosine-deoxyadenosine kinase had an apparent molecular weight of 38,000, a Stokes radius of 27.4 A, and apparent Km values of 0.4, 510, and 75 microM for adenosine, deoxyadenosine, and ATP, respectively. Attempts to distinguish whether adenosine-deoxyadenosine kinase was one enzyme with these two activities or two separate enzymes suggested that the former was the case. Deoxycytidine-deoxyadenosine kinase had apparent Km values of 0.7, 670, and 12 microM for deoxycytidine, deoxyadenosine, and ATP, respectively. Its apparent molecular weight was estimated to be 49,000 and its Stokes radius 30 A. Two other minor peaks of deoxyadenosine-phosphorylating activity had characteristics different from either deoxycytidine kinase or adenosine kinase-associated deoxyadenosine kinase. Our studies indicate that human placental cytosol contains a complex mixture of nucleoside kinase enzymes.     

Purine nucleoside kinases in human T- and B-lymphoblasts

Purine nucleoside kinases in human B- and T-lymphoblasts were fractionated by DEAE-cellulose chromatography. Human B-lymphoblast cell extracts showed three peaks of nucleoside kinase activities, adenosine kinase (EC 2.7.1.20), deoxyguanosine kinase and deoxycytidine kinase (EC 2.7.1.74). However, T-lymphoblast cell extracts showed a nucleoside kinase activity which phosphorylates deoxycytidine, deoxyadenosine and deoxyguanosine, similar to deoxycytidine kinase, in addition to the three nucleoside kinases. The Km values of T-lymphoblast-specific nucleoside kinase for deoxyadenosine and deoxyguanosine, 15 and 26 microM, respectively, were smaller than those of deoxycytidine kinase, 150 and 330 microM, respectively. Deoxyadenosine phosphorylation by deoxycytidine kinase was strongly inhibited by dCTP, but the phosphorylation by T-lymphoblast-specific nucleoside kinase was only weakly inhibited by dCTP. Deoxyadenosine phosphorylating activity in B-lymphoblast extracts was more distinctly inhibited by dCTP than that in T-lymphoblast extracts.     

Deoxyadenosine metabolism and cytotoxicity in cultured mouse T lymphoma cells: a model for immunodeficiency disease.

The inherited absence of either adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with severe immunological impairment. We have developed a cell culture model using a mouse T cell lymphoma to simulate ADA deficiency and to study the relationship between purine salvage enzymes and immune function. 2′-deoxyadenosine triphosphate (deoxyATP) levels have been shown to be greatly elevated in erythrocytes of immunodeficient, ADA-deficient patients, suggesting that deoxyadenosine is the potentially toxic substrate in ADA deficiency. Using a potent ADA inhibitor, we have demonstrated that deoxyadenosine is growth-inhibitory and cytotoxic to S49 cells, and that deoxyATP accumulates in these cells. Cell variants, unable to transport or phosphorylate deoxyadenosine, are much less sensitive to deoxyadenosine, indicating that intracellular phosphorylation of deoxyadenosine is required for the lethal effects.We have partially reversed the cytotoxic effects of deoxyadenosine with deoxycytidine in wild-type cells, but we cannot show any reversal in cell lines lacking deoxycytidine kinase. Adenosine (ado) kinase-deficient cells are extremely resistant to deoxyadenosine in the presence of deoxycytidine. This deoxycytidine reversal of deoxyadenosine toxicity is consistent with an inhibition of ribonucleotide reductase by deoxyATP, and we have shown that incubation of S49 cells with deoxyadenosine markedly reduces intracellular levels of deoxyCTP, deoxyGTP and TTP.Kinetics data in wild-type cells and in cell variants are consistent with the presence of two deoxyadenosine-phosphorylating activities — one associated with ado kinase and another associated with deoxycytidine kinase.The S49 cells appear to be a valid model for the simulation of ADA deficiency in cell culture, and from our results, we can suggest administration of deoxycytidine as a pharmacological regimen to circumvent the clinicopathologic symptoms in ADA deficiency.     

Bystander effects of nucleoside analogs phosphorylated in the cytosol or mitochondria

The efficiency of nucleoside kinase suicide gene therapy for cancer is highly dependent on "bystander" cell killing, i.e., the transfer of cytotoxic phosphorylated nucleoside analogs to cells adjacent to those expressing the suicide enzyme. We have recently studied the possible use of mitochondrial nucleoside kinases as suicide genes. In the present study, we investigated if nucleoside analogs phosphorylated in the mitochondrial matrix cause bystander killing. We used deoxycytidine kinase-deficient Chinese hamster ovary cells reconstituted with deoxycytidine kinase targeted to either the cytosol or mitochondria matrix and determined the bystander cell killing when these cells were incubated with the nucleoside analogs 1-beta-D-arabinofuranosylcytosine and 2',2'-difluorodeoxycytidine. A bystander effect occurred when nucleoside analogs were phosphorylated in the cytosol, but not when these compounds were phosphorylated in the mitochondria. These findings suggest that nucleoside kinases targeted to the mitochondrial matrix have limited use in suicide gene therapy when efficient bystander cell killing is required.     

Nonenzymatic labeling of proteins by preparations of [35S]methionine

A rapid, simple, and sensitive radiochemical assay for the measurement of purine or pyrimidine nucleoside kinases (EC 2.7.1.-) is described. The substrate (thymidine, deoxyuridine, deoxycytidine, deoxyguanosine, deoxyadenosine, uridine, cytidine, and adenosine) is separated from the product (the respective 5′-nucleotide) on neutral alumina columns which retain the nucleotides but not the nucleosides. The nucleotides are recovered by elution with 0.4 m sodium phosphate buffer, pH 7.6.     

Purine ribonucleoside and deoxyribonucleoside kinase activities in thymocytes. Specificity and optimal assay conditions for phosphorylation

The optimal assay conditions and specificity for the principal reactions of purine nucleoside phosphorylation were studied in mouse thymocytes. The following relative activities were obtained for the nucleoside substrates: adenosine, 100; deoxyguanosine, 24; and deoxyadenosine, 14. The phosphorylation of adenosine, 45 microM, was optimal between pH 5.8 and 6.0 with a millimolar Mg:ATP ratio of 1:5. This activity was insensitive to inhibition by other nucleosides and dCTP. Optimal phosphorylation of deoxyguanosine, 350 microM, occurred at pH 8.4 with a millimolar Mg:ATP ratio of 10:3.5. Phosphorylation of 80 microM deoxyguanosine was inhibited approximately 90% by 10 microM deoxycytidine or dCTP and was inhibited 70% by 200 microM deoxyadenosine but unaffected by adenosine. Deoxyadenosine, 450 microM, phosphorylation was optimal between pH 6.5 and 8.5 with a millimolar Mg:ATP ratio of 5:1. Phosphorylation of deoxyadenosine, 100 microM, was partially inhibited by 200 microM adenosine, 34%; 200 microM deoxyguanosine, 10%; and 100 microM deoxycytidine or dCTP, 33%. Only deoxyadenosine phosphorylation was inhibited by 200 microM deoxyinosine, 10%. These results and those obtained from isokinetic sucrose density gradient analysis are consistent with there being a specific adenosine kinase, a faster sedimenting deoxycytidine kinase of broad specificity which also catalyzes the phosphorylation of deoxyguanosine and deoxyadenosine, and a specific deoxyguanosine kinase sedimenting more rapidly than either of the other activities.     

Deoxycytidine kinase from human leukemic spleen: preparation and characteristics of homogeneous enzyme

Deoxycytidine kinase from human leukemic spleen has been purified 6000-fold to apparent homogeneity with an overall yield of 10%. The purification was achieved by using DEAE chromatography, hydroxylapatite chromatography, and affinity chromatography on dTTP-Sepharose. Only one form of deoxycytidine kinase activity was found during all the chromatographic procedures. The subunit molecular mass, as judged by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, was 30 kilodaltons. The pure enzyme phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, demonstrating for the first time that the same enzyme molecule has the capacity to use these three nucleosides as substrates. The apparent molecular weight of the active enzyme, determined by gel filtration and glycerol gradient centrifugation, was 60,000. Thus, the active form of human deoxycytidine kinase is a dimer. The kinetic behavior of pure human deoxycytidine kinase was studied in detail with regard to four different phosphate acceptors and two different phosphate donors. The apparent Km values were 1, 20, 150, and 120 microM for deoxycytidine, arabinosylcytosine, deoxyguanosine, and deoxyadenosine, respectively. The Vmax values were 5-fold higher for the purine nucleosides as compared to the pyrimidine substrates. We observe competitive inhibition of the phosphorylation of one substrate by the presence of either of the three other substrates, but the apparent Ki values differed greatly from the corresponding Km values, suggesting the existence of allosteric effects. The double-reciprocal plots for ATP-MgCl2 as phosphate donor were convex, indicating negative cooperative effects. In contrast, plots with varying dTTP-MgCl2 concentration as phosphate donor were linear with an apparent Km of 2 microM. The enzyme activity was strongly inhibited by dCTP, in a noncompetitive way with deoxycytidine and in a competitive way with ATP-MgCl2.     

Specific selection of deoxycytidine kinase mutants with tritiated deoxyadenosine

We have shown previously that a low concentration of tritiated deoxyadenosine, i.e., 1 µCi/ml, selectively kills wild-type S49 murine lymphoma cells. Mutant cells resistant to [³H]deoxyadenosine lacked adenosine kinase completely but retained a significant level of deoxyadenosine phosphorylating activity. To study further the specificity of [³H]deoxyadenosine selection, lymphoma cell clones resistant to 15 µCi/ml [³H]deoxyadenosine have been derived. The resistant line, S49-dA15, is also resistant to high levels of nonradioactive deoxyadenosine and to deoxyguanosine but remains sensitive to thymidine. The thymidine inhibition of the growth of the mutant, in contrast to that of the wild-type cells, cannot be prevented by deoxycytidine. The mutant line lacks deoxycytidine kinase that also phosphorylates deoxyadenosine. In addition, the mutant cells excrete a large amount of deoxycytidine into culture medium, consistent with a failure of salvage of the nucleoside in the absence of an appropriate kinase, i.e., deoxycytidine kinase. In contrast, a deoxycytidine kinase-deficient cell line that was selected with arabinosylcytosine does not excrete deoxycytidine and contains high deoxycytidine deaminase activity. [³H]Deoxyadenosine can be used as a selective agent for specific selection of deoxycytidine kinase-negative mutants.     

Nucleoside kinase activities of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells and appropriate drug-resistant mutants derived from them have been analyzed for nucleoside kinase activities relevant to the phosphorylation of adenosine, deoxyadenosine, deoxyguanosine and deoxycytidine and for resistance to a variety of nucleoside analogs. Fractionation of extracts by DEAE-cellulose chromatography revealed three major peaks of activity. Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20), the first to elute from the column is responsible for the majority of the deoxyadenosine phosphorylation in cell extracts and, according to resistance data, appears to phosphorylate most adenosine analogs tested, including 9-beta-D-arabinosyladenine (ara-A). A deoxyguanosine kinase, the second enzyme to elute from the column, was responsible for the majority of deoxyguanosine and deoxyinosine phosphorylation in cell extracts. The function of this enzyme in cell metabolism is unclear. 2-Chlorodeoxyadenosine, on the other hand, appeared from resistance data to be phosphorylated, at least in part, by deoxycytidine kinase (ATP:deoxycytidine 5'-phosphotransferase, EC 2.7.1.74), which in cell extracts could also phosphorylate deoxyguanosine and deoxyadenosine, though much less efficiently than deoxycytidine.     

T-lymphoblast-specific nucleoside kinase: characterization and comparison with deoxycytidine kinase

The results of Sephadex G-100 gel filtration and sucrose density gradient centrifugation showed that T-lymphoblast-specific nucleoside kinase (TSK) purified from MOLT 4FT cell extract has a molecular weight of 26,500, while deoxycytidine kinase (dCK) 56,000. The pI value of TSK (pH 8.2) is quite different from that of dCK (4.8). TSK phosphorylated deoxycytidine, deoxyadenosine, deoxyguanosine and arabinocytidine, similar to dCK, but the respective kinetic properties were quite different. In the phosphate donor specificity and metal ion requirement, some differences were observed between TSK and dCK. dTTP was a good phosphate donor for dCK but no effect at all as phosphate donor for TSK. dCK was inhibited at very low concentration of dCTP, but TSK at much higher concentration of dCTP.     

Deoxyadenosine/deoxycytidine kinase from Bacillus subtilis. Purification, characterization, and physiological function

Three different deoxyribonucleoside kinases with specificities toward thymidine, deoxyguanosine, and deoxyadenosine/deoxycytidine, respectively, are identified in Bacillus subtilis. The deoxyadenosin/deoxycytidine kinase is purified 950-fold employing blue Sepharose CL-6B column chromatography. The two deoxyribonucleoside kinase activities copurify and are present in the same band after polyacrylamide gel electrophoresis. The molecular weight is determined by gel filtration to be 47,000. Cytidine, adenosine, arabinosylcytosine, and arabinosyladenine are substrates for the enzyme. The activities toward these substrates are less than 20% of the activities obtained with deoxyadenosin and deoxycytidine. The deoxycytidine and deoxyadenosine saturation curves are hyperbolic with Km values for both nucleosides around 5 microM. The maximal velocities for the two deoxyribonucleosides are nearly identical with GTP as phosphate donor. GTP is the best donor showing hyperbolic saturation curves and Km values around 150 microM depending on the deoxyribonucleoside concentration. dATP and dCTP are inhibitors when GTP is the phosphate donor. They may both act as phosphate donors themselves. A divalent metal ion is required, Mg2+ giving the highest activity. A spontaneous mutant, selected as resistant to 5-fluorodeoxycytidine, lacks both deoxycytidine and deoxyadenosine kinase activity, while it retains normal activities toward deoxyguanosine, deoxyuridine, and thymidine.     

Adenosine kinase deficiency in tritiated deoxyadenosine-resistant mouse S49 lymphoma cell lines

Mutant sublines were derived of S49 mouse T-lymphoma cells that were resistant to tritiated deoxyadenosine. Twenty-five isolates that were selected in 1 microCi/ml of the nucleoside were cross-resistant to 6-thioguanine, were sensitive to HAT (hypoxanthine, aminopterin, and thymidine), and contained less than 1% of hypoxanthine phosphoribosyltransferase activity in wild-type cells. One of the mutant clones, S49-dA2, was further subjected to selection in a medium containing 2 microCi/ml tritiated deoxyadenosine and 1 microgram/ml deoxycoformycin, an inhibitor of adenosine deaminase. All resistant subclones were cross-resistant to tubercidin, 6-methylmercaptopurine riboside, and arabinosyladenine. One of the subclones, S49-12, was completely devoid of adenosine kinase and was partially deficient in deoxyadenosine kinase. This subclone, however, contained wild-type levels of deoxycytidine kinase. DEAE chromatography of the wild-type cell extracts revealed two deoxyadenosine phosphorylating activities, one of which coeluted with adenosine kinase and was the enzyme missing in S49-12. The other species phosphorylated both deoxyadenosine and deoxycytidine, of which deoxycytidine was the preferred substrate.     

Human T-lymphoblast deoxycytidine kinase: purification and properties

Previous observations present tremendous variations in the properties of deoxycytidine kinase. To clarify the properties and physiologic role of deoxycytidine kinase, we have undertaken its purification. Deoxycytidine kinase was purified from cultured human T-lymphoblasts (MOLT-4) to 90% purity with an estimated specific activity of 8 mumol min-1 (mg of protein)-1. The purification procedure included ammonium sulfate precipitation, Superose-12 HPLC gel filtration chromatography, DE-52 ion-exchange chromatography, AMP-Sepharose 4B affinity chromatography, and dCTP-Sepharose-4B affinity chromatography. Deoxyguanosine, deoxyadenosine, and cytidine phosphorylating activities copurified with deoxycytidine kinase to final specific activities of 7.2, 13.5, and 4 mumol min-1 (mg of protein)-1, respectively. The enzyme is very unstable at low protein concentration and is stabilized by storage at -85 degrees C with 1 mg/mL bovine serum albumin, 20% glycerol (v/v), 200 mM potassium chloride, and 25 mM dithiothreitol. The molecular weight was 60,000, and the Stokes radius was 32 A by gel filtration chromatography. The subunit molecular weight was 30,500. This enzyme had apparent Km values of 1.5, 430, 500, 450, and 40 microM for deoxycytidine, deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside, respectively. The pH optimum ranged from 6.5 to 9.0. Mg2+ and Mn2+ were the preferred divalent cations. ATP, GTP, dGTP, ITP, dITP, TTP, and XTP were substrates for the enzymes. Our study indicates that deoxycytidine kinase is a dimer with two subunits and has phosphorylating activity for deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside. This highly purified enzyme will facilitate the study of its regulation and phosphorylation of anticancer or antiviral nucleoside analogues.     

Deoxyguanosine kinase. Distinct molecular forms in mitochondria and cytosol.

Two distinct deoxyguanosine kinase activities have been identified in calf thymus tissue. They can be differentiated by subcellular location, electrophoretic mobility, chromatographic behavior, nucleoside specificity, apparent Km values, and end product inhibition. After a 130-fold purification from mitochondrial extract, the newly discovered kinase was specific primarily for deoxyguanosine and deoxyinosine. Unlike the cytosol enzyme, which proved to be the broadly specific deoxycytidine kinase studied previously, the mitochondrial enzyme does not phosphorylate deoxycytidine. Its apparent Km for deoxyguanosine, 6 micromolar, is 2 orders of magnitude lower than that of the cytosol enzyme. The mitochondrial enzyme is strongly inhibited by dGTP and dITP and activated up to 6-fold by dTDP and UDP, whereas neither dCTP nor dATP had much effect.     

Metabolic investigations of fibroblasts from horses, Equus caballus, with hereditary severe combined immunodeficiency

In an attempt to determine the metabolic defect causing severe combined immunodeficiency (SCID) in horses in which altered purine metabolism has been observed, various parameters of purine and pyrimidine metabolism were evaluated. The activities of nine purine enzymes (adenosine kinase, purine nucleoside phosphorylase, deoxyadenosine kinase, deoxycytidine kinase, 5'-nucleotidase, AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase, and adenine phosphoribosyl transferase were measured in fibroblasts. All activities determined for SCID horses were normal. Uptake of 10 microM adenosine or 2'-deoxyadenosine (a growth inhibitory concentration for SCID fibroblasts) by SCID fibroblasts was identical to that found for normal fibroblasts in the presence of both 1 and 50 microM phosphate. The Km determined for the transport of both adenosine and 2'-deoxyadenosine was 35 microM. In the presence of p-nitrobenzylthioguanosine (a nucleoside transport inhibitor), 2'-deoxyadenosine uptake was inhibited to the same extent in all fibroblast lines tested. To determine if the last step in pyrimidine biosynthesis might be altered in SCID fibroblasts, UMP synthase activities were evaluated but found to be normal (0.5 nmol UMP formed/min/mg protein).     

Activation of deoxycytidine kinase by deoxyadenosine: implications in deoxyadenosine-mediated cytotoxicity

The inborn deficiency of adenosine deaminase is characterised by accumulation of excess amounts of cytotoxic deoxyadenine nucleotides in lymphocytes. Formation of dATP requires phosphorylation of deoxyadenosine by deoxycytidine kinase (dCK), the main nucleoside salvage enzyme in lymphoid cells. Activation of dCK by a number of genotoxic agents including 2-chlorodeoxyadenosine, a deamination-resistant deoxyadenosine analogue, was found previously. Here, we show that deoxyadenosine itself is also a potent activator of dCK if its deamination was prevented by the adenosine deaminase inhibitor deoxycoformycin. In contrast, deoxycytidine was found to prevent stimulation of dCK by various drugs. The activated form of dCK was more resistant to tryptic digestion, indicating that dCK undergoes a substrate-independent conformational change upon activation. Elevated dCK activities were accompanied by decreased pyrimidine nucleotide levels whereas cytotoxic dATP pools were selectively enhanced. dCK activity was found to be downregulated by growth factor and MAP kinase signalling, providing a potential tool to slow the rate of dATP accumulation in adenosine deaminase deficiency.     

The subcellular location of nucleoside analog phosphorylation is a determinant of synergistic effects of hydroxyurea

The ribonucleotide reductase inhibitor hydroxyurea exhibits synergistic pharmacological activity with several nucleoside analogs used in antiviral and anticancer chemotherapy. We have used a cell model system where a deoxycytidine kinase (dCK)-deficient cell line was reconstituted with genetically engineered dCK targeted to the cytosol, the nucleus, or the mitochondria to investigate how the subcellular location of nucleoside analog phosphorylation affected the synergistic effects of a ribonucleotide reductase inhibitor. Hydroxyurea showed synergistic cytotoxicity with the nucleoside analogs 1-beta-d-arabinofuranosylcytosine and 2-chloro-2'-deoxyadenosine when dCK was expressed in the cytosol or in the nucleus, but not when dCK was expressed in the mitochondria. These data indicate that the synergistic effect of ribonucleotide reductase inhibition is limited to nucleoside analogs phosphorylated in the cytosol or the cell nucleus.     

A method for the enzymatic synthesis and purification of [alpha-32P] nucleoside triphosphates.

A simplified method is described for the enzymatic synthesis and purification of [alpha-32P]ribo- and deoxyribonucleoside triphosphates. The products are obtained at greater than 97% radiochemical purity with yields of 50--70% (relative to 32Pi) by a two-step elution from DEAE-Sephadex. All reactions are done in one vessel as there is no need for intermediate product purifications. This method is therefore suitable for the synthesis of these radioactive compounds on a relatively large scale. The sequential steps of the method involve first the synthesis of [gamma-32P]ATP and the subsequent phosphorylation of nucleoside 3' monophosphate with T4 polynucleotide kinase to yield nucleoside 3', [5'-32P]diphosphate. Hexokinase is used after the T4 reaction to remove any remaining [gamma-32P]ATP. Nucleoside 3',[5'-32P]diphosphate is treated with nuclease P-1 to produce the nucleoside [5'-32P]monophosphate which is phosphorylated to the [alpha-32P]nucleoside triphosphate with pyruvate kinase and nucleoside monophosphate kinase. Adenosine triphosphate used as the phosphate donor for [alpha-32P]deoxynucleoside triphosphate syntheses is readily removed in a second purification step involving affinity chromatography on boronate-polyacrylamide. [alpha-32P]Ribonucleoside triphosphates can be similarly purified when deoxyadenosine triphosphate is used as the phosphate donor.     

Deoxynucleoside Kinases of Bacillus megaterium KM

Dialyzed extracts of Bacillus megaterium KM contain thymidine, deoxyadenosine, and deoxyguanosine kinase activities. Thymidine kinase activity is best with deoxyadenosine triphosphate or deoxyguanosine triphosphate (dGTP) as the phosphoryl donor, whereas the best deoxyadenosine kinase activity is obtained with dGTP or adenosine triphosphate. Deoxyguanosine kinase activity functions optimally with deoxycytidine triphosphate as the donor. Although the thymidine kinase activity of crude extracts does not have a demonstrable divalent cation requirement, the addition of Mg(2+) or Mn(2+) is necessary for the formation of thymidine di- and triphosphates. The synthesis of thymidine kinase appears to be partially derepressed by thymine starvation. Incubation of extracts with deoxyadenosine and dGTP results in the substantial accumulation of deoxyadenosine di- and triphosphates. Extracts deaminate deoxycytidine to deoxyuridine, presumably as a consequence of the action of deoxycytidine deaminase, and then convert deoxyuridine to deoxyuridylic acid. B. megaterium extracts do not contain any detectable deoxycytidine kinase activity.     

Purification and properties of deoxyadenosine kinase from rat liver mitochondria. II. Kinetics and inhibition studies

Kinetic and inhibition studies of partially purified deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76, AdR kinase) from rat liver mitochondria were performed, including reaction properties, specificity of phosphate donors, phosphate acceptors, influence of nucleotides, nucleosides and their derivatives. The results were compared with those obtained from partially purified AdR kinase from rat liver cytosol.