Deoxycytidine Kinase

Durham, John P.; Ives, David H.

doi:10.1016/s0021-9258(18)63149-7

Cited by 118 publications

(17 citation statements)

References 41 publications

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“…The molecular weight of native deoxycytidine kinase was estimated to be 60000. This value is similar to that for deoxycytidine kinase from human leukemic spleen (Bohman & Eriksson, 1985), calf thymus (Durham & Ives, 1970a), and LI210 cells (Kessel, 1968) and in the range of 47 000-68 000 previously observed for deoxycytidine kinase from Bacillus subtilis, human myeloblasts, Lactobacillus acidophilus, and calf thymus (Diebel & Ives, 1977;Krygier & Momparler, 1971; Martin & Ives, 1978; Meyers & Kreis, 1976;Mollgaard, 1980). With a subunit molecular weight of 30500, the enzyme appears to be a dimer consisting of two identical subunits (Figure 6).…”

Section: Discussionsupporting

confidence: 79%

“…In S-49 lymphoma cells, deoxyguanosine kinase (dGK) activity is associated with deoxycytidine kinase (dCK) (Gudas et al, 1978), and deoxyadenosine kinase (dAK) is associated with adenosine kinase (AK) and deoxycytidine kinase (Ullman et al, 1978). In cultured human lymphoblasts, thymus, and LI210 cells, deoxyadenosine is phosphorylated by adenosine kinase and deoxycytidine kinase (Bacter et al, 1978;Carson et al, 1980; Chang et al, 1982; Durham & Ives, 1970aHershfield et al, 1982;Kessel, 1968; Ullman et al, 1981;Verhoef et al, 1981). These conclusions concerning substrate specificity have been derived largely from studies with mutant cell lines, and direct biochemical verification of the results has been limited by the use of partially purified or crude cell lysates which could contain more than one enzyme activity.…”

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confidence: 99%

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Human T-lymphoblast deoxycytidine kinase: purification and properties

Datta¹,

Shewach²,

Hurley³

et al. 1989

Biochemistry

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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.

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Section: Discussionsupporting

confidence: 79%

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confidence: 99%

Human T-lymphoblast deoxycytidine kinase: purification and properties

Datta¹,

Shewach²,

Hurley³

et al. 1989

Biochemistry

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“…The human lymphoblast or myeloblast dCyd kinase reported here is similar to bovine lymphoid dCyd kinase (Durham & Ives, 1970;Krenitsky et al, 1976), since the enzymes from these sources have similar molecular weights, pH optima, metal ion requirements, and substrate specificity. The inability of myloblast dCyd kinase to phosphorylate dAdo and dGuo reported (Cheng et al, 1977;Coleman et al, 1975) may be due to use of indirect assay (Cheng et al, 1977) and due to purification of dCyd kinase in the absence of the stabilizing agent DTT (Coleman et al, 1975).…”

Section: Discussionmentioning

confidence: 71%

“…This study is the first demonstrate that dCyd kinase shows substrate activation with both purine and pyrimidine deoxyribonucleosides. Previous investigators (Durham & Ives, 1970) may have failed to observe this because they did not use as wide a range of substrate concentrations in their kinetic studies. The substrate activation is unlikely to be due to artifacts of dCyd kinase assay, since kinetics with ara-A performed under the same conditions were linear with time and with protein concentration used.…”

Section: Discussionmentioning

confidence: 98%

“…but not from HEp-2 carcinoma or murine leukemia cells phosphorylates dAdo and ara-A (Schnebli et al, 1967;Chang et al, 1980). Similarly, partially purified dCyd kinase from calf thymus (Durham & Ives, 1970; Krenitsky et al, 1976) but not from human myeloid or murine leukemia cell phosphorylates dAdo and dGuo (Coleman et al, 1975;Cheng et al, 1977;Brockman et al, 1980). In agreement with this, Meyers and Kreis (1976) chromatographically separated dGuo-dAdo phosphorylating activity from dCyd kinase using murine leukemia cell extracts.…”

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Identification of purine deoxyribonucleoside kinases from human leukemia cells: substrate activation by purine and pyrimidine deoxyribonucleosides

Sarup¹,

Fridland²

1987

Biochemistry

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Cell extracts from human leukemic T lymphoblasts and myeloblasts were chromatographed on DEAE-cellulose columns to separate purine deoxyribonucleoside, deoxyadenosine (dAdo) and deoxyguanosine (dGuo), phosphorylating activities. Three distinct purine deoxyribonucleoside kinases, a deoxycytidine (dCyd) kinase, an adenosine (Ado) kinase, and a deoxyguanosine (dGuo) kinase (the latter appears to be localized in mitochondria), were resolved. dCyd kinase contained the major phosphorylating activity for dAdo, dGuo, and 9-beta-D-arabinofuranosyladenine (ara-A). Ado kinase represented a second kinase for dAdo and ara-A while a third kinase for dAdo was found in mitochondria. dCyd kinase was purified about 2000-fold with ion-exchange, affinity, and hydrophobic chromatographies. On gel electrophoresis, both dCyd and dAdo phosphorylating activities comigrated, indicating that the activities are associated with the same protein. The enzyme showed a broad pH optimum ranging from pH 6.5 to pH 9.5. Divalent cations Mg2+, Mn2+, and Ca2+ stimulated dCyd kinase activity; Mg2+ produced the maximal activity. dCyd kinase from either lymphoid or myeloid cells showed broad substrate specificity. The enzyme used several nucleoside triphosphates, but ATP, GTP, and dTTP were the best phosphate donors. dCyd was the best nucleoside substrate, since dCyd kinase had an apparent Km of 0.3, 85, 90, and 1400 microM for dCyd, dAdo, dGuo, and ara-A, respectively. The enzyme exhibited substrate activation with both pyrimidine and purine deoxyribonucleosides, suggesting that there is more than one substrate binding site on the kinase. These studies show that, in lymphoblasts and myeloblasts, purine deoxyribonucleosides and their analogues are phosphorylated by dCyd kinase, Ado kinase, and dGuo kinase.

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Radiometric Methods of Enzyme Assay

Oldham

1973

Methods of Biochemical Analysis

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Deoxycytidine Kinase

Cited by 118 publications

References 41 publications

Human T-lymphoblast deoxycytidine kinase: purification and properties

Human T-lymphoblast deoxycytidine kinase: purification and properties

Identification of purine deoxyribonucleoside kinases from human leukemia cells: substrate activation by purine and pyrimidine deoxyribonucleosides

Radiometric Methods of Enzyme Assay

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