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Staffan Eriksson - One of the best experts on this subject based on the ideXlab platform.

  • human Deoxycytidine Kinase as a deoxyribonucleoside phosphorylase
    Journal of Molecular Biology, 2004
    Co-Authors: Elena V Usova, T V Maltseva, Andras Foldesi, Jyoti Chattopadhayaya, Staffan Eriksson
    Abstract:

    Human Deoxycytidine Kinase (dCK) is a key enzyme in the 5′-phosphorylation of purine and pyrimidine deoxynucleosides with Deoxycytidine as the most efficient substrate. The ability of dCK to degrade 2′-deoxyribonucleosides to free nucleobases and 2-deoxy-α- d -ribofuranose-1-phosphate was demonstrated by 1H–31P correlation spectroscopy and by isotope enzyme kinetic methods. The reaction depended on inorganic phosphate, and dCK showed maximum cleavage activity between pH 7 and pH 8. In this pH range, [ HPO 4 2 − ] is the dominant phosphate species, most likely being the phosphate donor. All natural deoxyribonucleosides could be cleaved and the Vmax of the phosphorylytic reaction compared to the Kinase reaction was about 2–10%. The formation of free nucleobases occurred only with reduced dCK, because the reaction was highly dependent on the presence of reducing agents such as dithiotreitol. Thus, recombinant dCK can act as a phosphorylase, similar to the nucleoside phosphorylase family of enzymes. This catalytic activity is important for the design of in vitro experiments with dCK, such as crystallization and NMR spectroscopy.

  • activation of Deoxycytidine Kinase by inhibition of dna synthesis in human lymphocytes
    Biochemical Pharmacology, 2001
    Co-Authors: Zsolt Csapo, Staffan Eriksson, Tatjana Spasokoukotskaja, Maria Sasvariszekely, Iannis Talianidis, Maria Staub
    Abstract:

    Abstract Deoxycytidine Kinase (dCK, EC.2.7.1.74) is a key enzyme in the intracellular metabolism of 2-chlorodeoxyadenosine, 1-β- d -arabinofuranosylcytosine, difluoroDeoxycytidine, and other drugs used in chemotherapy of different leukaemias and solid tumours. Recently, stimulation of dCK activity was shown by these analogues and by other genotoxic agents such as etoposide and NaF, all of which cause severe inhibition of DNA synthesis in cell cultures. Here we describe that direct inhibition of DNA polymerases by aphidicolin stimulated dCK activity in normal lymphocytes and acute myeloid leukaemic cells, as well as in HL 60 promyelocytic cell cultures. Increased dCK activity was not due to new protein synthesis under our conditions, as measured by immunoblotting. Partial purification by diethylaminoethyl–Sephadex chromatography revealed that the activated form of dCK survived purification procedure. Moreover, it was possible to inactivate purified dCK preparations by recombinant protein phosphatase with Ser/Thr/Tyr dephosphorylating activity. These data suggest that the activation of dCK may be due to phosphorylation, and that deoxynucleoside salvage is promoted during inhibition of DNA synthesis in human lymphocytes.

  • activation of Deoxycytidine Kinase during inhibition of dna synthesis by 2 chloro 2 deoxyadenosine cladribine in human lymphocytes
    Biochemical Pharmacology, 1998
    Co-Authors: Maria Sasvariszekely, Staffan Eriksson, Tatjana Spasokoukotskaja, Zsolt Csapo, Melinda Szoke, Agnes Turi, Ildiko Szanto, Maria Staub
    Abstract:

    Deoxycytidine Kinase (dCK, EC.2.7.1.74), a key enzyme in intracellular metabolism of many antileukemic drugs, was shown to be activated during treatment of lymphocytes by 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine), a potent inhibitor of DNA synthesis. While 5-[3H]-thymidine (TdR) incorporation into DNA was decreased by 80-90%, dCK activity was doubled as a consequence of incubating the cells with 1 microM 2-chloro-2'-deoxyadenosine. Thymidine Kinase (dTK, EC.2.7.1.21) activity was slightly decreased under the same conditions, similarly to 5-[3H]-thymidine incorporation. dCK activation could not be prevented by cycloheximide, and neither the amount of dCK protein nor its mRNA level was increased after 2-chloro-2'-deoxyadenosine treatment. These results suggest a post-translational activation of dCK protein during inhibition of DNA synthesis.

  • decrease in thymidylate Kinase activity in peripheral blood mononuclear cells from hiv infected individuals
    Biochemical Pharmacology, 1998
    Co-Authors: Bengt Jacobsson, Sven Britton, Yvonne Tornevik, Staffan Eriksson
    Abstract:

    Nucleosides and nucleoside analogs are anabolised to their triphosphates by intracellular Kinases. The anti-HIV analogue zidovudine (AZT) is phosphorylated by cytosolic thymidine Kinase 1 (TK1), thymidylate Kinase (dTMPK), and nucleoside diphosphate Kinase. It is known that dTMPK is one of the rate-limiting steps in the activation of zidovudine. The activities of TK1, dTMPK, and Deoxycytidine Kinase (dCK) were determined in extracts of in vitro activated peripheral blood mononuclear cells from HIV-infected patients and healthy noninfected individuals. dTMPK activity was 10-fold lower and TK1 activity was five-fold lower in extracts from infected as compared to uninfected persons. Deoxycytidine Kinase activities in the extracts from both groups were very similar. Differences in in vitro activation, as determined by flow cytometry, of the peripheral lymphocytes were not responsible for the decreased TK1 and dTMPK activities. A reduced level of intracellular azido-dideoxythymidinetriphosphate in activated mononuclear cells from HIV-infected patients was also observed. The low levels of TK1 and dTMPK in lymphocytes from HIV-infected patients may be related to the anergy phenomenon observed as a result of HIV infection. This effect should also be considered in the development of new anti-HIV drugs.

  • substrate specificities of mitochondrial thymidine Kinase and cytosolic Deoxycytidine Kinase against 5 aryl substituted pyrimidine 2 deoxyribose analogues
    Nucleosides Nucleotides & Nucleic Acids, 1995
    Co-Authors: Staffan Eriksson, Jianghai Wang, Salo Gronowitz, Nils Gunnar Johansson
    Abstract:

    Abstract Some 5-aryl-2′-deoxyuridine and -Deoxycytidine analogues, many with known antiviral activity, were evaluated as substrates for pure Deoxycytidine Kinase (dCK) and pure mitochondrial thymidine Kinase (TK2). Some of the deoxyuridine compounds were also tested with pure cytosolic thymidine Kinase (TK1). TK2 showed the highest activity with this type of analogues.

Maria Staub - One of the best experts on this subject based on the ideXlab platform.

  • activation of Deoxycytidine Kinase by inhibition of dna synthesis in human lymphocytes
    Biochemical Pharmacology, 2001
    Co-Authors: Zsolt Csapo, Staffan Eriksson, Tatjana Spasokoukotskaja, Maria Sasvariszekely, Iannis Talianidis, Maria Staub
    Abstract:

    Abstract Deoxycytidine Kinase (dCK, EC.2.7.1.74) is a key enzyme in the intracellular metabolism of 2-chlorodeoxyadenosine, 1-β- d -arabinofuranosylcytosine, difluoroDeoxycytidine, and other drugs used in chemotherapy of different leukaemias and solid tumours. Recently, stimulation of dCK activity was shown by these analogues and by other genotoxic agents such as etoposide and NaF, all of which cause severe inhibition of DNA synthesis in cell cultures. Here we describe that direct inhibition of DNA polymerases by aphidicolin stimulated dCK activity in normal lymphocytes and acute myeloid leukaemic cells, as well as in HL 60 promyelocytic cell cultures. Increased dCK activity was not due to new protein synthesis under our conditions, as measured by immunoblotting. Partial purification by diethylaminoethyl–Sephadex chromatography revealed that the activated form of dCK survived purification procedure. Moreover, it was possible to inactivate purified dCK preparations by recombinant protein phosphatase with Ser/Thr/Tyr dephosphorylating activity. These data suggest that the activation of dCK may be due to phosphorylation, and that deoxynucleoside salvage is promoted during inhibition of DNA synthesis in human lymphocytes.

  • activation of Deoxycytidine Kinase during inhibition of dna synthesis by 2 chloro 2 deoxyadenosine cladribine in human lymphocytes
    Biochemical Pharmacology, 1998
    Co-Authors: Maria Sasvariszekely, Staffan Eriksson, Tatjana Spasokoukotskaja, Zsolt Csapo, Melinda Szoke, Agnes Turi, Ildiko Szanto, Maria Staub
    Abstract:

    Deoxycytidine Kinase (dCK, EC.2.7.1.74), a key enzyme in intracellular metabolism of many antileukemic drugs, was shown to be activated during treatment of lymphocytes by 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine), a potent inhibitor of DNA synthesis. While 5-[3H]-thymidine (TdR) incorporation into DNA was decreased by 80-90%, dCK activity was doubled as a consequence of incubating the cells with 1 microM 2-chloro-2'-deoxyadenosine. Thymidine Kinase (dTK, EC.2.7.1.21) activity was slightly decreased under the same conditions, similarly to 5-[3H]-thymidine incorporation. dCK activation could not be prevented by cycloheximide, and neither the amount of dCK protein nor its mRNA level was increased after 2-chloro-2'-deoxyadenosine treatment. These results suggest a post-translational activation of dCK protein during inhibition of DNA synthesis.

Tatjana Spasokoukotskaja - One of the best experts on this subject based on the ideXlab platform.

  • activation of Deoxycytidine Kinase by inhibition of dna synthesis in human lymphocytes
    Biochemical Pharmacology, 2001
    Co-Authors: Zsolt Csapo, Staffan Eriksson, Tatjana Spasokoukotskaja, Maria Sasvariszekely, Iannis Talianidis, Maria Staub
    Abstract:

    Abstract Deoxycytidine Kinase (dCK, EC.2.7.1.74) is a key enzyme in the intracellular metabolism of 2-chlorodeoxyadenosine, 1-β- d -arabinofuranosylcytosine, difluoroDeoxycytidine, and other drugs used in chemotherapy of different leukaemias and solid tumours. Recently, stimulation of dCK activity was shown by these analogues and by other genotoxic agents such as etoposide and NaF, all of which cause severe inhibition of DNA synthesis in cell cultures. Here we describe that direct inhibition of DNA polymerases by aphidicolin stimulated dCK activity in normal lymphocytes and acute myeloid leukaemic cells, as well as in HL 60 promyelocytic cell cultures. Increased dCK activity was not due to new protein synthesis under our conditions, as measured by immunoblotting. Partial purification by diethylaminoethyl–Sephadex chromatography revealed that the activated form of dCK survived purification procedure. Moreover, it was possible to inactivate purified dCK preparations by recombinant protein phosphatase with Ser/Thr/Tyr dephosphorylating activity. These data suggest that the activation of dCK may be due to phosphorylation, and that deoxynucleoside salvage is promoted during inhibition of DNA synthesis in human lymphocytes.

  • activation of Deoxycytidine Kinase during inhibition of dna synthesis by 2 chloro 2 deoxyadenosine cladribine in human lymphocytes
    Biochemical Pharmacology, 1998
    Co-Authors: Maria Sasvariszekely, Staffan Eriksson, Tatjana Spasokoukotskaja, Zsolt Csapo, Melinda Szoke, Agnes Turi, Ildiko Szanto, Maria Staub
    Abstract:

    Deoxycytidine Kinase (dCK, EC.2.7.1.74), a key enzyme in intracellular metabolism of many antileukemic drugs, was shown to be activated during treatment of lymphocytes by 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine), a potent inhibitor of DNA synthesis. While 5-[3H]-thymidine (TdR) incorporation into DNA was decreased by 80-90%, dCK activity was doubled as a consequence of incubating the cells with 1 microM 2-chloro-2'-deoxyadenosine. Thymidine Kinase (dTK, EC.2.7.1.21) activity was slightly decreased under the same conditions, similarly to 5-[3H]-thymidine incorporation. dCK activation could not be prevented by cycloheximide, and neither the amount of dCK protein nor its mRNA level was increased after 2-chloro-2'-deoxyadenosine treatment. These results suggest a post-translational activation of dCK protein during inhibition of DNA synthesis.

  • selective assays for thymidine Kinase 1 and 2 and Deoxycytidine Kinase and their activities in extracts from human cells and tissues
    Biochemical and Biophysical Research Communications, 1992
    Co-Authors: Elias S.j. Arnér, Tatjana Spasokoukotskaja, Staffan Eriksson
    Abstract:

    Abstract 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-β-D-arabinofura-anosylthymidine (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.

Donna S. Shewach - One of the best experts on this subject based on the ideXlab platform.

  • metabolism in human cells of the d and l enantiomers of the carbocyclic analog of 2 deoxyguanosine substrate activity with Deoxycytidine Kinase mitochondrial deoxyguanosine Kinase and 5 nucleotidase
    Antimicrobial Agents and Chemotherapy, 1998
    Co-Authors: L. Lee Bennett, Paula W. Allan, G. Arnett, Donna S. Shewach, William S. Mason, Isabelle Fourel, Fulmer Y Shealy, William B. Parker
    Abstract:

    The carbocyclic analog of 2′-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the l enantiomer rather than, as expected, with the d enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for Deoxycytidine Kinase (EC 2.7.1.74) from MOLT-4 cells, 5′-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine Kinase (EC 2.7.1.113) from human platelets and CEM cells. For both Deoxycytidine Kinase and mitochondrial deoxyguanosine Kinase, the l enantiomer was the better substrate. Even though the d enantiomer was the preferred substrate with 5′-nucleotidase, the rate of phosphorylation of the l enantiomer was substantial. The phosphorylation of d-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of d-CdG was stimulated by mycophenolic acid and was not affected by Deoxycytidine suggested that 5′-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. d-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of d-CdG and related analogs and in the design of new biologically active analogs.

  • kinetic analysis of human Deoxycytidine Kinase with the true phosphate donor uridine triphosphate
    Biochemistry, 1997
    Co-Authors: Trudy L Hughes, Tina M Hahn, Kara K Reynolds, Donna S. Shewach
    Abstract:

    : Deoxycytidine Kinase is the rate-limiting process in the activation for several clinically important antitumor agents. Previous studies have focused on Deoxycytidine (dCyd) and adenosine triphosphate (ATP) as substrates for this enzyme. In view of recent data indicating that uridine triphosphate (UTP) is the physiologic phosphate donor for this enzyme, a study of the kinetic properties of dCyd Kinase with dCyd and UTP was undertaken. The results presented here demonstrate that UTP and ATP produce kinetically distinguishable differences in nucleoside phosphorylation by dCyd Kinase. At high dCyd concentrations, dCyd Kinase exhibited substrate activation with ATP. In contrast, in the presence of UTP, substrate inhibition was observed at concentrations of dCyd greater than 3 microM. Inhibition by dCyd was noncompetitive with respect to UTP and could not be reversed by a 200-fold increase in UTP concentration, indicating that the inhibition was not due to dCyd binding at the nucleotide binding site. The kinetic mechanism for dCyd Kinase was determined with dCyd and UTP as substrates. UTP was the preferred phosphate donor with a true Km value of 1 microM compared to 54 microM with ATP, resulting in a 50-fold greater substrate efficiency for UTP. Although the double-reciprocal plots with UTP produced parallel lines, initial velocity plots with other phosphate donors and product inhibition studies indicated that dCyd Kinase formed a ternary complex with its substrates. The parallel lines with UTP were apparently due to a low dissociation constant for UTP, which was calculated as more than 13-fold lower than its Km value. Analysis of product inhibition studies indicated that dCyd Kinase followed an ordered A-B random P-Q reaction sequence, with UTP as the first substrate to bind. In contrast, previous results demonstrated a random bi-bi sequence for dCyd Kinase in the presence of ATP. The combined results indicate that the enzyme can follow a random bi-bi reaction sequence, but with UTP as the phosphate donor, the addition of nucleotide prior to dCyd is strongly preferred. The noncompetitive substrate inhibition, which was independent of UTP concentration, indicates that high concentrations of dCyd promote addition of the nucleoside prior to UTP, resulting in a lower velocity.

  • affinity of the antiviral enantiomers of oxathiolane cytosine nucleosides for human 2 Deoxycytidine Kinase
    Biochemical Pharmacology, 1993
    Co-Authors: Donna S. Shewach, Dennis C Liotta, Raymond F Schinazi
    Abstract:

    Abstract The two enantiomers of 2′,3′-dideoxy-3′-thiacytidine (BCH-189) and their 5-fluoro analogs (FTC) were found to be good substrates for human 2′-Deoxycytidine Kinase with K m values in the 5.7 to 42.1 μM range. The affinity of the (−)-enantiomers was greater than that of the (+)-compounds. These results may explain the greater in vitro antiviral potency against human immunodeficiency virus and hepatitis B virus of the (−)-enantiomers when compared to their (+)-counterparts. The (+)- and (−)-enantiomers of FTC and BCH-189 are the first nucleoside analogs for which we have observed lower apparent kinetic constants for this enzyme in the presence of ATP compared to UTP.

Maria Sasvariszekely - One of the best experts on this subject based on the ideXlab platform.

  • activation of Deoxycytidine Kinase by inhibition of dna synthesis in human lymphocytes
    Biochemical Pharmacology, 2001
    Co-Authors: Zsolt Csapo, Staffan Eriksson, Tatjana Spasokoukotskaja, Maria Sasvariszekely, Iannis Talianidis, Maria Staub
    Abstract:

    Abstract Deoxycytidine Kinase (dCK, EC.2.7.1.74) is a key enzyme in the intracellular metabolism of 2-chlorodeoxyadenosine, 1-β- d -arabinofuranosylcytosine, difluoroDeoxycytidine, and other drugs used in chemotherapy of different leukaemias and solid tumours. Recently, stimulation of dCK activity was shown by these analogues and by other genotoxic agents such as etoposide and NaF, all of which cause severe inhibition of DNA synthesis in cell cultures. Here we describe that direct inhibition of DNA polymerases by aphidicolin stimulated dCK activity in normal lymphocytes and acute myeloid leukaemic cells, as well as in HL 60 promyelocytic cell cultures. Increased dCK activity was not due to new protein synthesis under our conditions, as measured by immunoblotting. Partial purification by diethylaminoethyl–Sephadex chromatography revealed that the activated form of dCK survived purification procedure. Moreover, it was possible to inactivate purified dCK preparations by recombinant protein phosphatase with Ser/Thr/Tyr dephosphorylating activity. These data suggest that the activation of dCK may be due to phosphorylation, and that deoxynucleoside salvage is promoted during inhibition of DNA synthesis in human lymphocytes.

  • activation of Deoxycytidine Kinase during inhibition of dna synthesis by 2 chloro 2 deoxyadenosine cladribine in human lymphocytes
    Biochemical Pharmacology, 1998
    Co-Authors: Maria Sasvariszekely, Staffan Eriksson, Tatjana Spasokoukotskaja, Zsolt Csapo, Melinda Szoke, Agnes Turi, Ildiko Szanto, Maria Staub
    Abstract:

    Deoxycytidine Kinase (dCK, EC.2.7.1.74), a key enzyme in intracellular metabolism of many antileukemic drugs, was shown to be activated during treatment of lymphocytes by 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine), a potent inhibitor of DNA synthesis. While 5-[3H]-thymidine (TdR) incorporation into DNA was decreased by 80-90%, dCK activity was doubled as a consequence of incubating the cells with 1 microM 2-chloro-2'-deoxyadenosine. Thymidine Kinase (dTK, EC.2.7.1.21) activity was slightly decreased under the same conditions, similarly to 5-[3H]-thymidine incorporation. dCK activation could not be prevented by cycloheximide, and neither the amount of dCK protein nor its mRNA level was increased after 2-chloro-2'-deoxyadenosine treatment. These results suggest a post-translational activation of dCK protein during inhibition of DNA synthesis.