Genetic analysis of 2',3'-dideoxycytidine incorporation into cultured human T lymphoblasts

In order to analyze the cellular determinants that mediate the action of 2',3'-dideoxycytidine, the growth inhibitory and cytotoxic effects and the metabolism of the dideoxynucleoside were examined in wild type human CEM T lymphoblasts and in mutant populations of CEM cells that were genetically deficient in either nucleoside transport or deoxycytidine kinase activity. Whereas 2',3'-dideoxycytidine at a concentration of 5 microM inhibited growth of the wild type CEM parental strain by 50%, two nucleoside transport-deficient clones were 4-fold resistant to the pyrimidine analog. The deoxycytidine kinase-deficient cell line was virtually completely resistant to growth inhibition by the dideoxynucleoside at a concentration of 1024 microM. An 80% diminished rate of 2',3'-[5,6-3H]dideoxycytidine influx into the two nucleoside transport-deficient lines could account for their resistance to the dideoxynucleoside, while the resistance of the deoxycytidine kinase-deficient cells to 2',3'-dideoxycytidine toxicity could be explained by a virtually complete failure to incorporate 2',3'-[5,6-3H]dideoxycytidine in situ. Two potent inhibitors of mammalian nucleoside transport, 4-nitrobenzylthioinosine and dipyridamole, mimicked the effects of a genetic deficiency in nucleoside transport with respect to 2',3'-dideoxycytidine toxicity and incorporation. These data indicate that the intracellular metabolism of 2',3'-dideoxycytidine in CEM cells is initiated by the nucleoside transport system and the cellular deoxycytidine kinase activity.     

Metabolism and anti-human immunodeficiency virus-1 activity of 2-halo-2',3'-dideoxyadenosine derivatives

Both 2',3'-dideoxyadenosine and 2',3'-dideoxyinosine have been shown (Mitsuya, H., and Broder, S. (1987) Nature 325, 773-778) to have in vitro activity against the human immunodeficiency virus-1 (HIV). However, these dideoxynucleosides may be catabolized by human T cells, even when adenosine deaminase is inhibited by deoxycoformycin. To overcome this problem, we have synthesized the 2-fluoro-, 2-chloro-, and 2-bromo-derivatives of 2',3'-dideoxyadenosine. The metabolism and anti-HIV activity of the 2-halo-2',3'-dideoxyadenosine derivatives and of 2',3'-dideoxyadenosine were compared. The 2-halo-2',3'-dideoxyadenosine derivatives were not deaminated significantly by cultured CEM T lymphoblasts. Experiments with 2-chloro-2',3'-dideoxyadenosine showed that the T cells converted the dideoxynucleoside to the 5'-monophosphate, 5'-diphosphate, and 5'-triphosphate metabolites. At concentrations lower than those producing cytotoxicity in uninfected cells (3-10 microM), the 2-halo-2',3-dideoxyadenosine derivatives inhibited the cytopathic effects of HIV toward MT-2 T lymphoblasts, and retarded viral replication in CEM T lymphoblasts. Experiments with a deoxycytidine kinase-deficient mutant CEM T cell line showed that this enzyme was necessary for the phosphorylation and anti-HIV activity of the 2-chloro-2',3'-dideoxyadenosine. In contrast, 2',3'-dideoxyadenosine was phosphorylated by the deoxycytidine kinase-deficient mutant and retained anti-HIV activity in this cell line. Thus, the 2-halo derivatives of 2',3'-dideoxyadenosine, in contrast to 2',3'-dideoxyadenosine itself, are not catabolized by T cells. Their anti-HIV and anti-proliferative activities are manifest only in cells expressing deoxycytidine kinase. The in vivo implications of these results for anti-HIV chemotherapy are discussed.     

Inhibition of various stages of DNA replication in sea urchin embryos

The embryos of the sea urchin Strongylocentrotus intermedius possess the ability to incorporate into their DNAs 2'-deoxynucleosides together with all their bases, i.e., adenine, guanine, cytosine and thymine. This incorporation is inhibited by 3'-amino-2',3'-dideoxynucleosides with the same bases. 5'-Amino-5'-deoxynucleosides and 5'-amino-2',5'-dideoxynucleosides moderately inhibit the incorporation of [3H]2'-deoxynucleosides into the DNAs by competing with the latter, presumably at the phosphorylation stage. The most potent inhibiting effect is exerted by 2'-amino-2'-deoxynucleosides and 2'-asido-2'-deoxynucleosides; the mechanism of this inhibition is still obscure, however.     

Cytotoxic activity of 2',2'-difluorodeoxycytidine, 5-aza-2'-deoxycytidine and cytosine arabinoside in cells transduced with deoxycytidine kinase gene

Deoxycytidine nucleoside analogs must be first phosphorylated to become active anticancer drugs. The rate-limiting enzyme in this pathway is deoxycytidine kinase (dCK). Cells deficient in this enzyme are resistant to these analogs. To evaluate the potential of dCK to be used as suicide gene for deoxycytidine nucleoside analogs, we transduced both human A-549 lung carcinoma and murine NIH3T3 fibroblast cell lines with this gene. The dCK-transduced cells showed an increase in cytotoxicity to the analogs, cytosine arabinoside (ARA-C), and 5-aza-2'-deoxycytidine (5-AZA-CdR). Unexpectedly, the related analog, 2',2'-difluorodeoxycytidine (dFdC), was less cytotoxic to the dCK-transduced cells than the wild-type cells. For the A-549-dCK cells, the phosphorylation of dFdC by dCK was much greater than control cells. In accord with the elevated enzyme activity, we observed a 6-fold increased dFdC incorporation into DNA and a more pronounced inhibition of DNA synthesis in the A-549-dCK cells. In an attempt to clarify the mechanism of dFdC, we investigated its action on A549 and 3T3 cells transduced with both cytidine deaminase (CD) and dCK. We reported previously that overexpression of CD confers drug resistance to deoxycytidine analogs. In this study, when the CD-transduced cells were also transduced with dCK they became relatively more sensitive to dFdC. In addition, we observed that dFdU, the deaminated form of dFdC, was cytotoxic to the A-549-dCK cells, but not the wild-type cells. Our working hypothesis to explain these results is that the mitochondrial thymidine kinase (TK2), an enzyme reported to phosphorylate dFdC, acts as an important modulator of dFdC-induced cell toxicity. These findings may further clarify the action of dFdC and the mechanism by which it induces cell death.     

The role of cytoplasmic deoxycytidine kinase in the mitochondrial effects of the anti-human immunodeficiency virus compound, 2',3'-dideoxycytidine.

2',3'-Dideoxycytidine (ddC) is a potent inhibitor of human immunodeficiency virus replication in vitro and shows beneficial effects in AIDS therapy. The compound inhibits mitochondrial DNA (mtDNA) synthesis at a clinically relevant concentration, which could be responsible for the side effects of ddC observed in the clinic. Thymidine (dThd), one of the substrates of mitochondrial deoxypyrimidine kinase (dPyd kinase), was not able to reverse the mitochondrial toxicity of ddC in CEM cells. Furthermore, the cytoplasmic deoxycytidine kinase (dCyd kinase)-deficient CEM cells were highly resistant to the mitochondrial toxicity of ddC. These data suggest a critical role for cytoplasmic dCyd kinase in the mitochondrial toxicity of ddC. The metabolites of ddC, but not ddC itself, were able to inhibit mtDNA synthesis in isolated mitochondria. The potency of the inhibitory effect was in the order of ddCTP greater than ddCDP greater than ddCMP greater than ddC. The lack of inhibition by ddC of mtDNA synthesis could be due to the inefficient ddC phosphorylation in mitochondria. Although the mitochondrial dPyd kinase was reported to phosphorylate ddC, the phosphorylation of ddC in isolated mitochondria was not detectable. The data suggest that ddC is phosphorylated to ddCTP in the cytoplasm and then transported into mitochondria to exert its inhibitory effect on mtDNA synthesis.     

Activation of kinase pathways in MCF-7 cells by 17beta-estradiol and structurally diverse estrogenic compounds

17beta-Estradiol (E2) activates non-genomic pathways in MCF-7 cells, and this study investigates the effects of structurally-diverse estrogenic compounds on activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3-K), protein kinase C (PKC), PKA, and calcium calmodulin-dependent kinase IV (CaMKIV). Activation of kinases was determined by specific substrate phosphorylation and transactivation assays that were diagnostic for individual kinases. The compounds investigated in this study include E2, diethylstilbestrol (DES), the phytoestrogen resveratrol, and the following synthetic xenoestrogens, bisphenol-A (BPA), nonylphenol, octylphenol, endosulfan, kepone, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and 2',3',4',5'-tetrachloro-4-biphenylol (HO-PCB-Cl(4)). With the exception of resveratrol, all the compounds activated PI3-K and MAPK. Activation of PKC by the xenoestrogens was structure-dependent since resveratrol, kepone and HO-PCB-Cl(4) were inactive and only minimal activation of PKA was observed. CaMKIV was activated only by E2 and DES, and HO-PCB-Cl(4) was a potent inhibitor of CaMKIV-dependent activity. These results demonstrate that activation of estrogen receptor-alpha-mediated non-genomic pathways by estrogenic compounds in MCF-7 cells is structure-dependent and can result in activation or inhibition of kinase activities.     

Deoxypyrimidine nucleoside metabolism in varicella-zoster virus-infected cells.

Noninfected and varicella-zoster virus (VZV)-infected human foreskin fibroblasts were examined for thymidine kinase activity. The specific activity of VZV-infected cell extracts was approximately 7.5-fold greater than that of mock-infected cells and 3-fold greater than that of actively growing cells. The pH optimum of VZV-infected cell thymidine kinase activity was found to be 8.0, whereas thymidine kinase activity in noninfected cells exhibited a sharp pH optimum at 7.4. Electrophoretic analysis of cellular enzymes involved in pyrimidine nucleoside phosphorylation revealed at least three enzymes distinguishable by electrophoretic mobility and substrates used. These enzymes were presumed to be thymidine kinase, deoxycytidine kinase, and uridine kinase. The relative mobilities of these enzymes on 5% polyacrylamide gels were 0.18, 0.91, and 0.54, respectively. In VZV-infected cells, a single band of activity catalyzing the phosphorylation of thymidine, deoxyuridine, deoxycytidine, and cytidine was observed with a relative mobility of 0.48. Cellular pyrimidine-phosphorylating enzymes were not detected in VZV-infected cells. The molecular weight of the VZV-induced enzyme was determined to be 72,000 +/- 7%.     

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.     

Role of salvage and phosphorylation in the immunostimulatory activity of C8-substituted guanine ribonucleosides

Lymphokine-like activity and selective stimulation of B cell growth is exerted by a group of synthetic ribonucleosides derivatized at C8 and exemplified by 8-bromoguanosine (8BrGuo), 8-mercaptoguanosine, and 7-methyl 8-oxoguanosine. However, relatively little is known about their molecular mechanism of action. Like naturally occurring nucleosides, 8BrGuo is taken up into lymphocytes by a process of facilitated diffusion. Naturally occurring nucleosides are then reclaimed by a well characterized salvage pathway, involving sequential phosphorolysis and phosphoribosylation. The studies reported in this communication demonstrate that, in contrast to naturally occurring nucleosides, 8BrGuo is not a substrate for salvage by purine nucleoside phosphorylase. The base that would be produced by putative phosphorolysis, 8-bromoguanine, is biologically inactive and is not a substrate for hypoxanthine-guanine phosphoribosyl-transferase. Accordingly, inhibitors of purine nucleoside phosphorylase-mediated salvage fail to inhibit nucleoside-induced immunostimulation selectively. Examination of the metabolism of 8BrGuo provides no direct evidence that 8BrGuo is phosphorylated by B lymphocytes. Direct enzymatic phosphorylation does not seem to be essential to the mechanism of action of the nucleoside insofar as competitive inhibition of deoxycytidine kinase (an enzyme that directly phosphorylates purines as well as pyrimidines) or of deoxyguanosine kinase fails to inhibit 8BrGuo stimulation selectively. Moreover, studies with synthetic nucleosides in which 3' and/or 5' hydroxyl groups were irreversibly blocked, precluding their phosphorylation, demonstrated that immunobiologic activity can occur in the absence of 3' and/or 5' phosphorylation. Finally, experiments with radiolabeled nucleosides provide no evidence to support the hypothesis that they are incorporated into cellular nucleic acid. These data, together with previous studies, suggest that it is the unmetabolized nucleoside that is active and, as such, is most likely to act in a regulatory capacity.     

Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase

The kinetic properties of 50,000-fold purified cultured human T lymphoblast (MOLT-4) deoxycytidine kinase were examined. The reaction velocity had an absolute requirement for magnesium. Maximal activity was observed at pH 6.5-7.0 with Mg:ATP for 1:1. High concentrations of free Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both deoxycytidine and MgATP2-. dCMP was a competitive inhibitor with respect to deoxycytidine and ATP. ADP was a competitive inhibitor with respect to ATP and a mixed inhibitor with respect to deoxycytidine. dCTP, an important end product, is a very potent inhibitor and was a competitive inhibitor with respect to deoxycytidine and a non-competitive inhibitor with respect to ATP. TTP reversed dCTP inhibition. The data suggest that (a) MgATP2- is the true substrate of deoxycytidine kinase; (b) the kinetic mechanism of deoxycytidine kinase is consistent with rapid equilibrium random Bi Bi; (c) deoxycytidine kinase may be regulated by its product ADP and its end product dCTP as well as the availability of deoxycytidine. While many different nucleotides potently inhibit deoxycytidine kinase, their low intracellular concentrations make their regulatory role less important.     

Selective Phosphorylation of South and North-Cytidine and Adenosine Methanocarba-Nucleosides by Human Nucleoside and Nucleotide Kinases Correlates with Their Growth Inhibitory Effects on Cultured Cells

Here bicyclo[3.1.0]hexane locked deoxycytidine (S-MCdC, N-MCdC), and deoxyadenosine analogs (S-MCdA and N-MCdA) were examined as substrates for purified preparations of human deoxynucleoside kinases: dCK, dGK, TK2, TK1, the ribonucleoside kinase UCK2, two NMP kinases (CMPK1, TMPK) and a NDP kinase.

dCK can be important for the first step of phosphorylation of S-MCdC in cells, but S-MCdCMP was not a substrate for CMPK1, TMPK, or NDPK.

dCK and dGK had a preference for the S-MCdA whereas N-MCdA was not a substrate for dCK, TK1, UCK2, TK2, dGK nucleoside kinases. The cell growth experiments suggested that N-MCdC and S-MCdA could be activated in cells by cellular kinases so that a triphosphate metabolite was formed.

List of abbreviations: ddC, 2′, 3′-didioxycytosine, Zalcitabine; 3TC, β-L-(-)-2′,3′-dideoxy-3′-thiacytidine, Lamivudine; CdA, 2-cloro-2′-deoxyadenosine, Cladribine; AraA, 9-β-D-arabinofuranosyladenine; hCNT 1–3, human Concentrative Nucleoside Transporter type 1, 2 and 3; hENT 1–4, human Equilibrative Nucleoside Transporter type 1, 2, 3, and 4.     

Selective assays for thymidine kinase 1 and 2 and deoxycytidine kinase and their activities in extracts from human cells and tissues.

Human cells salvage pyrimidine deoxyribonucleosides via 5'-phosphorylation which is also the route of activation of many chemotherapeutically used nucleoside analogs. Key enzymes in this metabolism are the cytosolic thymidine kinase (TK1), the mitochondrial thymidine kinase (TK2) and the cytosolic deoxycytidine kinase (dCK). These enzymes are expressed differently in different tissues and cell cycle phases, and they display overlapping substrate specificities. Thymidine is phosphorylated by both thymidine kinases, and deoxycytidine is phosphorylated by both dCK and TK2. The enzymes also phosphorylate nucleoside analogs with very different efficiencies. Here we present specific radiochemical assays for the three kinase activities utilizing analogs as substrates that are by more than 90 percent phosphorylated solely by one of the kinases; i.e. 3'-azido-2',3'-dideoxythymidine (AZT) as substrate for TK1, 1-beta-D-arabinofuranosylthymidine (AraT) for TK2 and 2-chlorodeoxyadenosine (CdA) for dCK. We determined the fraction of the total deoxycytidine and thymidine phosphorylating activity that was provided by each of the three enzymes in different human cells and tissues, such as resting and proliferating lymphocytes, lymphocytic cells of leukemia patients (chronic lymphocytic, chronic myeloic and hairy cell leukemia), muscle, brain and gastrointestinal tissue. The detailed knowledge of the pyrimidine deoxyribonucleoside kinase activities and substrate specificities are of importance for studies on chemotherapeutically active nucleoside analogs, and the assays and data presented here should be valuable tools in that research.     

Cellular phosphorylation of 2',3'-dideoxyadenosine-5'-monophosphate, a key intermediate in the activation of the antiviral agent DDI, in human peripheral blood mononuclear cells

2',3'-dideoxyadenosine 5-monophosphate (ddAMP), is a key intermediate in the metabolism of the antiviral agent 2',3'-dideoxyinosine (ddI) to its active triphosphate derivative, 2',3'-dideoxyadenosine-5'-triphosphate (ddATP). The potential role of adenylate kinase in the phosphorylation of ddAMP was studied in human peripheral blood mononuclear cells (PBMC) and a human T cell line, CEMss. Subcellular distribution, sulfhydryl inhibitor, and substrate specificity studies support the hypothesis that the mitochondrial adenylate kinase (AK2) is a major route of cellular activation of these compounds in human lymphocytes.     

Substrate/inhibitor properties of human deoxycytidine kinase (dCK) and thymidine kinases (TK1 and TK2) towards the sugar moiety of nucleosides, including O'-alkyl analogues.

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono- and di-O'-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower Ki values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3'-hexanoylamino-2',3'-dideoxythymidine, with a Ki of approximately 600 microM for TK1 and approximately 0.1 microM for TK2. 3'-OMe-dC was a superior inhibitor of dCK to its 5'-O-methyl congener, consistent with possible participation of the oxygen of the (3')-OH or (3')-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly alpha-dT was a good substrate of both TK1 and TK2, with Ki values of 120 and 30 microM for TK1 and TK2, respectively; and a 3'-branched alpha-L-deoxycytidine analogue proved to be as good a substrate as its alpha-D-counterpart. Several 5'-substituted analogues of dC were good non-substrate inhibitors of dCK and, to a lesser extent, of TK2. Finally, some ribonucleosides are substrates of the foregoing enzymes; in particular C is a good substrate of dCK, and 2'-OMe-C is an even better substrate than dC.     

Human Thymidine Kinase Can Functionally Replace Herpes Simplex Virus Type 1 Thymidine Kinase for Viral Replication in Mouse Sensory Ganglia and Reactivation from Latency upon Explant

Herpes simplex virus type 1 thymidine kinase exhibits a strikingly broad substrate specificity. It is capable of phosphorylating deoxythymidine and deoxyuridine as does human thymidine kinase, deoxycytidine as does human deoxycytidine kinase, the cytosolic kinase whose amino acid sequence it most closely resembles, and thymidylate as does human thymidylate kinase. Following peripheral inoculation of mice, viral thymidine kinase is ordinarily required for viral replication in ganglia and for reactivation from latency following ganglionic explant. To determine which activity of the viral kinase is important for replication and reactivation in mouse ganglia, recombinant viruses lacking viral thymidine kinase but expressing individual human kinases were constructed. Each recombinant virus expressed the appropriate kinase activity with early kinetics following infection of cultured cells. The virus expressing human thymidine kinase exhibited thymidine phosphorylation activity equivalent to ~5% of that of wild-type virus in a quantitative plaque autoradiography assay. Nevertheless, it was competent for ganglionic replication and reactivation following corneal inoculation of mice. The virus expressing human thymidylate kinase was partially competent for these activities despite failing to express detectable thymidine kinase activity. The virus expressing human deoxycytidine kinase failed to replicate acutely in neurons or to reactivate from latency. Therefore, it appears that low levels of thymidine phosphorylation suffice to fulfill the role of the viral enzyme in ganglia and that this role can be partially fulfilled by thymidylate kinase activity alone.     

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.     

Regulation of urocaninase activity in the liver: role of 3',5'-AMP]

A dependence of rat liver urocaninase activity on the agents affecting the adenylate cyclase system was studied in vitro and in vivo. Urocaninase is considerably activated after the injection of glucagone, NaF, theophylline and 3',5'-AMP. Under conditions optimal for the protein kinase activity of phosphorylase the urocaninase of liver extracts was activated 7-fold on the average. The nezyme retains its activity after gel-filtration through Sephadex G-25 and is capable of inactivation in the presence of Mg2+ and of reactivation after addition of ATP and 3',5'-AMP. These data suggest a possibility of regulation of mammalian liver urocaninase activity by 3',5'-AMP-dependent phosphorylation of the enzyme. Derivatives of hypoxanthine (theophylline and caffeine) in concentration 10(-4) M activate urocaninase in liver extracts 2--3 and 1.5-fold respectively. The activation is probably not due to the 3',5'-AMP phosphodiesterase inhibition, since another phosphodiesterase inhibitor--papaverine--has no activating effect on urocaninase.     

Human deoxycytidine kinase. Purification and characterization of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia patients.

A procedure for purifying human cytoplasmic and mitochondrial deoxycytidine kinase (NTP:deoxycytidine 5'-phosphotransferase, EC 2.7.1.74) was developed. Both purified isozymes have a similar molecular weight, activation energy and catalyze the reaction by a sequential mechanism. These two isozymes differ with respect to their substrate specificities. With cytoplasmic deoxycytidine kinase, ATP, GTP and TTP have the highest reaction velocity. Pyrimidine nucleoside triphosphates have higher affinity but lower V than purine nucleoside triphosphates. Cytidine and arabinosylcytidine can serve as substrates. With mitochondrial isozyme only ATP gives the highest reaction velocity. ATP and dATP have the same Km but different V values. Besides deoxycytidine, also deoxythymidine but not cytidine or arabinosylcytidine can serve as substrates. There are also differences between these two isozymes with respect to their sensitivity to inhibition. For cytoplasmic enzyme, Br5dCyd and Iodo5dCyd are not inhibitory. Both dCTP and UTP are competitive inhibitors (Ki 0.25 and 0.5 micronM, respectively) with respect to ATP. For mitochondrial isozyme both Br5dCyd and Iodo5dCyd are inhibitory and dCTP and TTP are competitive inhibitors (Ki 2 and 10 micronM, respectively) with respect to ATP.