The enzymatic mechanisms for deoxythymidine synthesis in human leukocytes. 3. Inhibition of deoxythymidine phosphorylase by purines.

(1) Synthesis of deoxythymidine by either direct transfer of deoxyribosyl to thymine (pyrimidine deoxyribosyltransferase) or by a coupled deoxynucleoside phosphorylase mechanism is approximately twofold greater with normal leukocyte extracts (55 to 88% granulocytes) than with extracts prepared from leukocytes obtained from patients with chronic myelogenous leukemia. Activities in lymphocytes (normal or leukemic) are one-fifth the activity of normal granulocytes.(2) The lower activity in chronic myelogenous leukemia remains at 50% of normal even when patients are in hematologic remission with a normal per cent mature granulocytes in the peripheral blood.(3) The leukemic enzyme could not be distinguished from the normal by pH optima, thermal stability, or kinetic properties. The Km's for the deoxyribosyl acceptor and deoxyribosyl donors were identical for both enzymes. Both are subject to substrate inhibition by thymine and to inhibition by purine bases with similar Ki's. In addition, the transferase component of both the leukemic and the normal cell enzyme is activated by phosphate and arsenate. It appears, therefore, that there is no qualitative difference between the enzyme obtained from leukocytes of patients with chronic myelogenous leukemia and the enzyme obtained from normal leukocytes, suggesting that the difference in total cell activity is due to an actual decrease in amount of enzyme in chronic myelogenous leukemia or to a mixed cell population, one with a normal quantity of enzyme and the other with little or no active enzyme.(4) In both the normal cell and the leukemic cell extracts, transferase and phosphorylase activities could not be separated. The ratio of the two activities remained constant over a 140- and a 230-fold purification in normal and leukemic cell extracts, respectively. These and other observations indicate that transferase and phosphorylase activities are associated with the same protein.(5) The metabolism of pyrimidine and purine deoxynucleosides is similar for normal and leukemic cells. Catabolism of all deoxynucleosides tested was by direct phosphorolysis, except for deoxyadenosine which required initial deamination to deoxyinosine before phosphorolysis. In contrast to the greater rates of pyrimidine deoxynucleoside synthesis and cleavage with normal leukocyte extracts, the rates of purine deoxynucleoside synthesis and cleavage were approximately twofold greater with extracts prepared from cells of patients with chronic myelogenous leukemia. There was no significant difference in the rate of phosphorolytic cleavage of pyrimidine nucleosides (uridine) between the CML and normal leukocyte extracts.