Interrelation of stromal NAD(P)ase and human erythrocytic 6-phosphogluconic dehydrogenase.

Pentose phosphate metabolism of human red blood cells may be altered by inherited deficiency in cytoplasmic enzymes like glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP oxidoreductase, E.C. 1.1.1.49), resulting in increased hemolytic susceptibility.' This deficiency may be due either to a mutation at a structural gene or to an alteration in the rate of synthesis or of destruction of the enzyme. Two electrophoretic variants of glucose-6-phosphate dehydrogenase not accompanied by enzyme deficiency have been demonstrated to differ by a single amino acid substitution, and therefore a mutation at a structural gene has been postulated.2 On the other hand, interaction of stromal NAD(P)ase (NAD(P) glycohydrolase, E.C. 3.2.2.6) with glucose-6-phosphate dehydrogenase has been described as a possible mechanism for regulation of the stability of the enzyme.3' 4 Furthermore, two other cytoplasmic enzymes, glutathione reductase (reduced NAD(P):oxidized glutathione oxidoreductase, E.C. 1.6.4.2) and 6-phosphogluconic dehydrogenase (6-phosphoD-gluconate: NAD(P) oxidoreductase, E.C. 1.1.1.44) can be modified in their activity by incubation of hemolysates in the presence of red cell membranes (stromata), suggesting a more general regulatory function of the membranes on the cytoplasmic enzymes.5 6 These modifications consist of activation of glutathione reductase and, when NADP is added to the incubation mixture, inactivation of 6-phosphogluconic dehydrogenase accompanied by change in electrophoretic pattern.' This last effect of the membranes has now been further investigated because of its specificity in the requirement of the coenzyme and because it was accompanied by a structural change in the enzyme. The present data provide evidence that (1) the stromal factor required to induce this effect on 6-phosphogluconic dehydrogenase is NAD(P)ase and (2) a product of the NAD(P)ase reaction with NADP, P-ADPR, interacts with 6-PGD to cause its molecular alteration. Methods.-Freshly drawn heparinized human blood was used in all our experiments. After the washed red cells had been frozen and thawed, stroma-free hemolysate was prepared by centrifugation as previously described6 and, in addition, was passed through a Millipore filter (pore size, 0.80 1A) and checked with a particle counter (Coulter Counter B) for absence of stromata. In some experiments, stroma-free hemolysate was dialyzed by gel filtration through Sephadex G25 eluted with 0.162 M NaCI, and is referred to as dialyzed hemolysate. Partial purification of 6-PGD was accomplished by gel filtration through Sephadex G200 eluted with 0.05 M Tris-HCl buffer and 0.1 M KCl, pH 7.4. The fractions containing 6-PGD activity collected with a Gilson fraction collector formed a peak preceding hemoglobin and are referred to as purified 6-PGD. The activity of 6-PGD was measured at 370C by the method of Glock and McLean.8 Unless otherwise stated, the data refer to the most common human variant, Pd A, as determined by starch gel electrophoresis.9