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

Cheng, Yung‐Chi; Domin, Barbara A.; Lee, Lih-Syng

doi:10.1016/0005-2744(77)90281-9

Cited by 71 publications

(32 citation statements)

References 13 publications

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“…due to an inefficient conversion of ddI to ddATP coupled with a substantial increase of dATP. In this work, it was also noted that in stimulated PBM, activities of two enzymes, uridine kinase and adenosine kinase, were slightly lower and that of thymidine kinase was higher than such enzymic activities previously reported for certain tumor cells of human or murine origin (28,29), resulting in that activities of the enzymes listed in Fig. 4 were in the same range.…”

Section: Discussionsupporting

confidence: 61%

Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral blood mononuclear cells.

et al. 1993

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The antiviral activity of azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyinosine (ddI) against HIV-1 was comparatively evaluated in PHA-stimulated PBM. The mean drug concentrations which yielded 50% p24 Gag negative cultures were substantially different: 0.06, 0.2, and 6 ,uM for AZT, ddC, and ddI, respectively. We found that AZT was preferentially phosphorylated to its triphosphate (TP) form in PHA-PBM rather than unstimulated, resting PBM (R-PBM), producing 10-to 17-fold higher ratios of AZTTP/dTTP in PHA-PBM than in R-PBM. The phosphorylation of ddC and ddI to their TP forms was, however, much less efficient in PHA-PBM, resulting in -5-fold and -15-fold lower ratios of ddClP/ dCIP and ddATP/dATP, respectively, in PHA-PBM than in R-PBM. The comparative order of PHA-induced increase in cellular enzyme activities examined was: thymidine kinase > uridine kinase > deoxycytidine kinase > adenosine kinase > 5'-nucleotidase.We conclude that AZT, ddC, and ddI exert disproportionate antiviral effects depending on the activation state of the target cells, i.e., ddI and ddC exert antiviral activity more favorably in resting cells than in activated cells, while AZT preferentially protects activated cells against HIV infection. Considering that HIV-1 proviral DNA synthesis in resting lymphocytes is reportedly initiated at levels comparable with those of activated lymphocytes, the current data should have practical relevance in the design of anti-HIV chemotherapy, particularly combination chemotherapy. (J. Clin. Invest. 1993Invest. .91:2326Invest. -2333

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Section: Discussionsupporting

confidence: 61%

Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral blood mononuclear cells.

et al. 1993

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“…No such comparative study has to our knowledge been performed previously. TK2 from the different sources studied here was similar to the enzyme purified earlier from human lymphoblasts [3] or human adult liver [S]. However, our TK2 preparation has 5-fold and 18-fold higher specific activity, respectively, then the two other preparations.…”

Section: Discussionsupporting

confidence: 51%

Mammalian thymidine kinase 2

Jansson¹,

Bohman²,

Munch‐Petersen³

et al. 1992

European Journal of Biochemistry

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Thymidine kinase 2 (TK2), also called mitochondria1 thymidine kinase, is a pyrimidine deoxyribonucleoside kinase expressed in all cells and tissues. It was recently purified to apparent homogeneity from human leukemic spleen and the active enzyme was shown to be a monomer of a 29-kDa polypeptide. The enzyme is feedback-inhibited by both end products, dCTP and dTTP. Here we show that TK2 purified from several different sources, including purified beef heart mitochondria, could be directly photoaffinity labeled with radioactive dTTP (approximately 18% of all TK2 molecules were cross-linked to dTTP after 20 min of ultraviolet irradiation) or to a lower extent with dCTP.Photo-incorporation was inhibited by the presence of the other effector but also the phosphate donor ATP blocked photolabeling, with dTTP. Addition of nucleoside substrates gave only a marginal inhibition of photo-incorporation. There were no detectable difference in the molecular size of photolabeled TK2 isolated from human spleen, brain or placenta, monkey liver, beef heart and beef heart mitochondria. Nor was there any significant differences in the enzyme kinetic properties of these enzymes. Cleavage of labeled TK2 with cyanogen bromide showed that dTTP was incorporated into a single 3-kDa peptide.TK2 was the only pyrimidine deoxynucleoside kinase expressed in liver, heart and brain. A detailed characterization of the subunit structure and substrate specificity of this enzyme is of importance for the design of new antiviral and cytostatic therapies based on nucleoside analogs.Thymidine kinase (ATP : thymidine 5'-phospho-transferase) catalyses the phosphorylation of dThd to dTMP. In eucaryotic cells two different forms of the enzyme have been found. One is the well characterized cytosolic and S-phaseexpressed enzyme, TK1, which is coded for by a gene on human chromosome 17 of determined sequence [l -61. The other form, TK2, is synthesized in the cytoplasm but predominantly found in the mitochondria and is expressed constitutively in all tissues [l -101. TK2 has been purified from many sources but most extensively from human liver [8] and recently to apparent homogeneity from leukemic spleen [9]. Detailed studies of the subcellular localization of TK2 was performed earlier by Kit et al. and different forms were found in the cytoplasm and the matrix and inner membrane of the mitochondria [I, 71. The TK2 gene is localized on chromosome 16 [2] but its sequence is not known.Active TKI and TK2 have different subunit structures since TKI is a tetramer of 25-kDa polypeptides whereas TK2 appears to be a monomer of 29 kDa [6, 91. The substrate specificities and kinetic properties of the two enzymes also differ. TK2 has a much broader specificity and it can efficiently

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“…In addition, only the deletion of UTP from NTP mixtures in dCK assays significantly reduced the rate of phosphorylation of dCyd and araC (White and Capizzi, 1991;Shewach et al, 1992). Other results with purified enzymes have indicated that the catalytic efficiencies of dCyd and several other nucleoside analogs were greater using UTP as the phosphate donor rather than ATP, and that UTP is preferred over ATP as a phosphate donor (Cheng et al, 1977;Shewach et al, 1992;Krawiec et al, 1995;Hughes et al, 1997).…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of 4′-thio-β-d-Arabinofuranosylcytosine and Its Analogs by Human Deoxycytidine Kinase

Someya¹,

Shaddix²,

Tiwari³

et al. 2002

J Pharmacol Exp Ther

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4Ј-thio-␤-D-Arabinofuranosylcytosine (T-araC) exhibits excellent in vivo antitumor activity against a variety of solid tumors despite its structural similarity to ␤-D-arabinofuranosylcytosine (araC), an agent which is poorly active against solid tumors in vivo. It is of great interest to elucidate why these compounds show a profound difference in antitumor activity. Because deoxycytidine kinase (dCK) is the critical enzyme in the activation of both compounds, here we report the differences in the substrate characteristics with human dCK between these compounds. The catalytic efficiency (V max /K m ) of araC was 100-fold higher than that of T-araC using either ATP or UTP as the phosphate donor. However, V max values of araC and T-araC were similar when UTP was the phosphate donor. Since UTP is believed to be the true phosphate donor for dCK in intact cells, these data indicated that the rates of phosphorylation of these two compounds at high pharmacologically relevant concentrations would be similar. This prediction was confirmed in intact cell experiments, which supported the hypothesis that UTP is the physiological phosphate donor for dCK phosphorylation in cells. The relative lack of importance of phosphate donor to the phosphorylation of T-araC by dCK revealed important insights into the activation of this compound in human cells at pharmacological doses. These studies indicated that replacement of the 4Ј-oxygen with sulfur significantly reduced the substrate activity of nucleoside analogs with dCK and that the superior activity of T-araC with respect to araC against solid tumors was not due to superior activity with dCK.

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

Cited by 71 publications

References 13 publications

Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral blood mononuclear cells.

Differential phosphorylation of azidothymidine, dideoxycytidine, and dideoxyinosine in resting and activated peripheral blood mononuclear cells.

Mammalian thymidine kinase 2

Phosphorylation of 4′-thio-β-d-Arabinofuranosylcytosine and Its Analogs by Human Deoxycytidine Kinase

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