Intracellular activity of HPRT Cape Town: purine uptake and growth of cultured cells in selective media

Abstract

The low activity of the human variant HPRT Cape Town is associated with substrate inhibition by hypoxanthine and guanine in vitro. The intracellular activity of this variant was investigated by study the relative uptake of radiolabelled purine nucleotide precursors and the growth in selective media of EBvirus-transformed lymphoblasts prepared from the proband (TK). These cells incorporated less than 10% of the [14C]hypoxanthine and [14C]guanine of the control cells; while their purine de novo synthesis was accelerated 8-fold. In selective media the HPRT Cape Town cells grew in a similar manner to HPRT-ve cells. These results indicate that if substrate inhibition is responsible for the low intracellular activity of HPRT Cape Town, the concentration of either hypoxanthine or guanine in the vicinity of the active site of the enzyme must be greater than the Ki(app) for these substrates, 118 and 28 mumol L(-1) respectively. Evidence is presented that the intracellular concentration of guanine, but not hypoxanthine, is well in excess of the Ki(app) in cultured lymphoblasts.