Regulation of the synthesis of 5-phosphoribosyl- i-pyrophosphate in intact red blood cells and in cell-free preparations

Abstract

The metabolic regulation of the synthesis of 5-phosphoribosyl- i-pyrophosphate (PRPP) was investigated in intact erythrocytes and in cell-free enzyme preparations with the aid of specific enzymic assays described in the present communication. The synthesis of PRPP in erythrocytes incubated in saline-glucose medium was strikingly enhanced by increasing the P i content of the medium up to a 60 mM concentration. Incubation of the erythrocytes in the presence of methylene blue or inosine resulted in a marked increase of their steady-state level of Rib-5- P, which showed no relation to the P i concentration in the medium. On the other hand, the stimulation of PRPP formation attending the build-up of the Rib-5- P pool exhibited a strict dependence on an adequately high P 1 level. These results were taken as evidence that the synthesis of PRPP in the erythrocytes is governed primarily by the P i-dependent catalytic capacity of PRPP synthetase, wherease the rate-limiting role of the intracellular supply of Rib-5- P manifest itself only when the optimum requirement of the system for P i had been satisfied. The activity of PRPP synthetase, as measured in cell-free preparations, was found to be about 200-fold higher than the corresponding rate of PRPP synthesis observed in the intact cells. The catalytic rate of the enzyme was strongly inhibited by ADP, GDP and 2,3-diphosphoglycerate at concentrations close to the physiological levels found in erythrocytes. The inhibition was partially relieved by raising the P i concentration in the reaction mixture over and above the optimum level required in the absence of the inhibitors. The interrelations between P i and the inhibitory metabolites, their implications in the control of the intracellular synthesis of PRPP and the allosteric nature of the underlying molecular mechanism are discussed in the light of available data.