pentostatin and 2-6-diaminopurine

Variants of Chinese hamster ovary and Novikoff rat hepatoma cells resistant to tubercidin and 2,5-diaminopurine, or to both drugs, were isolated, and their ability to convert adenosine and various adenosine analogs to nucleotides was compared to that of wild-type cells, both in intact cells and cell-free extracts. Adenosine deamination, and thus its conversion to nucleotides via inosine-hypoxanthine-inosine monophosphate, was inhibited by pretreatment of the cells or cell extracts with 2-deoxycoformycin. Cell-free extracts of the tubercidin-resistant variants, as well as of two adenosine-resistant mutants of Chinese hamster ovary cells, phosphorylated adenosine, tubercidin, pyrazofurin, or tricyclic nucleoside in the presence of ATP at less than 1% of the rate of extracts of wild-type cells. However, addition of phosphoribosyl pyrophosphate stimulated the conversion of adenosine to nucleotides 40-fold. Similarly, intact adenosine kinase-deficient cells failed to phosphorylate the adenosine analogs, but still converted adenosine to nucleotides at 5-10% the rate observed with wild-type cells. Phosphorylation of adenosine and tubercidin in wild-type cells was inhibited by substrate at concentration above 5-10 microM. In contrast, the rate of conversion of adenosine to nucleotides by adenosine kinase-deficient cells increased linearly up to a concentration of 400 microM adenosine, with the consequence that, at this concentration, these cells took up adenosine almost as rapidly as wild-type cells. Adenosine uptake by these kinase-deficient cells was inhibited by adenine and 5'-deoxyadenosine, and was largely abolished in mutants devoid also of adenine phosphoribosyltransferase. We conclude that adenosine is converted to nucleotides in adenosine kinase-deficient cells via adenine. Indirect evidence implicates 5'-methylthioadenosine phosphorylase as the enzyme responsible for the degradation of adenosine to adenine.

Topics: 2-Aminopurine; Adenine Nucleotides; Adenosine; Adenosine Kinase; Animals; Cell Division; Cell Line; Cell-Free System; Coformycin; Cricetinae; Female; Kinetics; Liver Neoplasms, Experimental; Ovary; Pentostatin; Phosphorylation; Phosphotransferases; Purine-Nucleoside Phosphorylase; Rats; Tubercidin