Isolation of a Chinese Hamster Cell Mutant with Low Intracellular Phosphoribosylpyrophosphate Concentration

Abstract

A tritium-adenine suicide procedure was used to select for mutants with reduced uptake of adenine from a population of Chinese hamster V79 cells mutagenized with ethyl methane sulfonate. In one of the mutant lines isolated, designated KC62, the uptake of adenine, hypoxanthine, and guanine was reduced by approximately 70%. The specific activities,Kmvalues, andVmaxvalues of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase were the same in extracts from KC62 and from the parental cell line. Metabolic fate studies of incorporated [3H]adenine and [3H]hypoxanthine revealed a metabolic block at the level of phosphoribosylation. Determination of phosphoribosylpyrophosphate pool size showed that the mutant contained only 25% of the phosphoribosylpyrophosphate found in the parent. Its reduced availability in KC62 appears to result in a decreased ability to salvage adenine, hypoxanthine, and guaninine via phosphoribosylation. Phosphoribosylpyrophosphate synthetase from KC62 was shown to have an increased sensitivity to inhibition by a variety of nucleotides.