The isolation and characterization of a variant cell line deficient in adenine uptake

A procedure involving treatment of Chinese hamster V-79A cells with ³H-adenine was tested for its ability to enrich for mutants defective in the uptake and utilization of adenine. A clone (Kc62) surviving radiolytic decay was isolated which displayed approximately a 70% reduction in the uptake of adenine, hypoxanthine or guanine as compared to V-79A.

When cells depleted of ATP and 5-phosphoribosyl pyrophosphate (PRPP) were labelled for 30 seconds with ³ H-adenine, the intracellular adenine pool size was the same for V-79A and KC62. These experiments provided evidence for normal adenine transport in KC62.

Activities of the purine salvage enzymes adenine phosphoribosyltransferase (ApRT) and hypoxanthine phosphoribosyltransferase (HPRT) were determined in 110,000 × G extracts. The kinetic parameters, Km and Vmax, were unaltered in KC62. The apparent Km values for APRT of both V-79A and KC62 were 3.6µM and 18.6µM for adenine and PRpp, respectively. The apparent Vmax for APRT was 1.03 picomoles min ^-1µg ^-1 of protein. The apparent Km values for HPRT of both V-79A and KC62 were 31.OuM and 25.5uM for hypoxanthine and PRPP, respectively. The apparent Vmax for HPRT was 1.00 picomoles min ^-1µg ^-1 of protein.

Labelling of KC62 cells with either ³ H-adenine or ³ H-hypoxanthine resulted in a uniform reduction of the ³H-ATP, ³H-ADP, and ³H-AMP pool sizes to 30% of those found in V-79A as determined by thin layer chromatography. Uniform reduction of labelled adenine nucleotide pools was consistent with a block at the level of the salvage enzymes.