OVEREXPRESSION OF THE NORMAL PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE-1ISOFORM UNDERLIES CATALYTIC SUPERACTIVITY OF HUMAN PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE

To define the enzymatic and genetic basis of X-linked phosphoribosylpy rophosphate synthetase (PRS) catalytic superactivity, we measured conc entrations of X-linked PRS1 and PRS2 isoforms in cultured fibroblasts and lymphoblasts by immunoblotting after separation by polyacrylamide- urea isoelectric focusing. PRS1 comprised >80% of measurable PRS isofo rms in all fibroblast strains, but PRS1 concentrations in cells from s ix affected males exceeded those in normal cells by 2-6-fold, PRS abso lute specific activities (activity per mg of PRS isoforms) were compar able in all fibroblast strains and in purified recombinant normal PRS1 , confirming selectively increased levels of PRS1 isoform as the enzym atic basis of PRS catalytic superactivity, Cloning, sequencing, and ex pression of normal subject- and patient-derived PRS cDNAs predicted no rmal translated region sequences for both PRS isoforms and revealed no differences in catalytic properties of recombinant PRS1. Normal and p atient PRPS1 transcribed but untranslated DNA sequences were also iden tical. Northern blot analysis showed selective increase in relative co ncentrations of PRS1 transcripts in patient fibroblasts. In PRS cataly tic superactivity, overexpression of the normal PRS1 isoform thus appe ars to result from an altered pretranslational mechanism of PRPS1 expr ession. In lymphoblasts, however, expression of this alteration is att enuated, explaining the absence of phenotypic expression of PRS cataly tic superactivity in these cells.