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A Critical Role for Uridine Nucleotides in the Regulation of Deoxycytidine Kinase and the Concentration Dependence of 1-ß-D-Arabinofuranosylcytosine Phosphorylation in Human Leukemia Cells¹

Departments of Biochemistry [J. C. W.] and Medicine [R. L. C.], Pharmacology and Experimental Therapeutics Program of the Comprehensive Cancer Center of Wake Forest University, Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103

The intracellular concentration of 1-ß-D-arabinofuranosylcytosine (ara-C) for half-maximal phosphorylation by leukemic blasts obtained directly from patients was 2.1 ± 2.5 µM (median, 1.3 µM, N = 25), and the rate of ara-C accumulation actually declined at concentrations above 20 µM in 35% of these cell populations. These apparent K_m values for cellular phosphorylation were an order of magnitude lower than the K_m of deoxycytidine (dCyd) kinase for ara-C with ATP as phosphate donor. dCyd kinase was purified from human leukemia cells and assayed for [³H]ara-C kinase activity with a mixture of 7 nucleotides at their approximate cellular concentrations or with a single nucleotide deleted. At low or high ara-C concentrations, ATP, GTP, CTP, or dTTP could be eliminated without significantly altering the rate. The only potential phosphate donor that was clearly important was UTP, since its deletion reduced the rate to only 25% of that with the complete mix. As anticipated, eliminating dCTP, the end product of this salvage pathway, moderately increased the rate by 50% at 0.4 µM ara-C or by 26% at 40 µM ara-C. At 40 µM ara-C, deleting UDP from the mix increased the rate more than deleting dCTP. dCTP was less inhibitory against 1 mM UTP (50% inhibitory concentration, 26 µM) than against 4 mM ATP (50% inhibitory concentration, 2.2 µM). In kinetic assays with 4 mM ATP and variable ara-C, UDP was a potent uncompetitive inhibitor with a K_i of 4 µM; the K_i for ADP was 1000-fold higher. Direct fit of kinetic data to the Michaelis equation yielded a K_m for ara-C of 49 µM with 4 mM ATP as the phosphate donor; however, there was evidence of negative cooperativity with a Hill coefficient of 0.7. High ara-C K_m values were also obtained with GTP and CTP, but with no evidence of cooperativity. With 1 mM UTP, the K_m was 1.5 µM with moderate substrate inhibition; thus the kinetic data with UTP were similar to those for ara-C phosphorylation by intact cells. UDP was less potent versus UTP than versus ATP. It lowered the V_max and enhanced the ara-C substrate inhibition without altering the K_m. When 1 mM UTP and 4 mM ATP were mixed, the kinetic pattern was similar to that for UTP alone. The K_m for UTP with [³H]dCyd as the phosphate acceptor of 0.8 µM was 25-fold lower than the K_m for ATP of 20 µM. These data indicate that dCyd kinase has a strong preference for uridine nucleotides, and UTP rather than ATP, is the principal intracellular phosphate donor. The apparent K_m for cellular phosphorylation of ara-C is low because the K_m for UTP-mediated phosphorylation of ara-C by dCyd kinase is low.

¹ Supported by Grants CH289C and CH289D from the American Cancer Society. This work was also supported in part by the Leukemia Cell Distribution and Pharmacology Core Laboratories of the Cancer Center of Wake Forest University (Grant CA12197).

² Leukemia Society of America Scholar. To whom requests for reprints should be addressed.

Received 10/12/90. Accepted 3/ 8/91.

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