INHIBITION OF PURINE PHOSPHORIBOSYLTRANSFERASES OF EHRLICH ASCITES-TUMOUR CELLS BY 6-MERCAPTOPURINE

Abstract

The formation of adenosine 5[image]-phosphate, guanosine 5[image]-phosphate and inosine 5[image]-phosphate from [8-14C]adenine, [8-14C]guanine and [8-14]hypo-xanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumor cells was assayed by a method involving liquid-scintillation counting of the radioactivity nucleo-tides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5[image]-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. At pH 7.8 and 25[degree] the Michaelis constants for adenine, guanine and hypo-xanthine were 0.9 [mu]M, 2.9 [mu]M and 11.0 [mu]M in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2.6 [mu]M. At pH 7.9 the Michaelis constant for 6-mercaptopurine was 10.9 [mu]M. 25 [mu]M-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (Ki 4.7 [mu]M) and hypoxanthine phosphoribosyltransferase (Ki 8.3 [mu]M). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (Ki 3.4 [mu]M). Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. From the low values of Ki for 6-mercaptopurine, and from published evidence that ascites-tumor cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.