Biochemical mechanisms of drug resistance. XII. Uridine kinases from mouse leukemic cells sensitive and resistant to 5-azacytidine.

Abstract

AbstractPartially purified uridine kinases from 5‐azacytidine‐sensitive and ‐resistant leukemic cells of AKR mice were separated on a Sephadex G‐200 column. The elution profile of both enzymes was similar although specific activity of uridine kinase from 5‐azacytidine‐resistant cells was more than 50% lower than that isolated from the parental cell line. The apparent Km constants with respect to the concentration of phosphate donor and acceptor were identical for both uridine kinases, while Vmax for enzymes from the resistant line were significantly decreased.The enzymes under study were tested for their sensitivity to SH‐blocking agents—p‐chloromercuribenzoate, N‐ethylmaleimide and iodoacetamide—and to urea, trypsin and cytidine 5′‐triphosphate. Inactivation following p‐chloromercuribenzoate affected the activity of uridine kinase from sensitive cells more markedly than that of uridine kinase from resistant ones. When the feedback effect of cytidine 5′‐triphosphate was examined, the results indicated, in both cases, a non‐competitive inhibition with respect to phosphate acceptor, Ki constant being the same (0.079 mM). Furthermore, vi/vo ratios for the intact and trypsin‐digested uridine kinases from both cell lines plotted against varying amounts of cytidine 5′‐triphosphate yielded a single curve in each instance. The significance of these findings is discussed.