Regulation of glucose-6-phosphate dehydrogenase. I. Intact red cells

The intracellular levels of substates and products of G6PD were measured in normal human red cells that were incubated in a Krebs-Ringer buffer without methylene blue and with increasing amounts of methylene blue. The use of [1-14C]glucose and [2-14C]glucose allowed estimates of the rate at which the G6PD was functioning within the cell. Kinetic characteristics of the purified enzyme were determined in the same Krebs-Ringer buffer. G6PD of normal red cells exhibits two unusual properties. (1) The intracellular enzyme is under unexplained intracellular restraint that ranges from 200-fold (if most of the NADP is in the reduced form) to five-fold (if most of the NADP is in the oxidized form). When coupled with earlier reports, these findings suggest that unexplained intracellular restraint is common to both G6PD-normal and G6PD-deficient (A-) red cells, (2) A highly sigmoid curve is obtained when the rate of the intracellular enzyme is plotted against the ratio of oxidized NADP to total NADP. In contrast, a nearly straight line is obtained with the corresponding plot of the pure enzyme under similar conditions of buffer and substrate concentration.