The influence of pH on the contributions of the Embden-Meyerhof and of the oxidative pentose-phosphate pathways in the utilization of glucose by human erythrocytes in the presence and absence of methylene blue were determined from yields of 14C0, and [14C]lactate.I n the absence of methylene blue both the NADP+ concentration and the phosphofructokinase are controlling factors. At p H 7.0 the oxidative pentose-phosphate pathway accounts for nearly one-half of the metabolic flux. Its contribution nearly vanishes at pH 8.2.I n the presence of methylene blue the limitation by the NADP+ concentration disappears. At p H values of 7.4 and below the phosphofructokinase is completely inhibited and is circumvented for the formation of lactate. The limitation of the glucose influx resides in the hexokinase. At pH 8.2 both the Embden-Meyerhof and the oxidative pentose-phosphate pathway participate in the metabolic flux, since the inhibition of the phosphofructokinase is released.With methylene blue metabolic pools are formed. They are derived exclusively from the oxidative pentose-phosphate pathway up to pH 7.5-7.6 and entirely from the Embden-Meyerhof pathway a t pH 8.2.In mature erythrocytes of man the oxidative pentose-phosphate pathway plays a subordinate role in the utilization of glucose. It shares with glycolysis glucose-6-P as a common intermediate and merges with the Embden-Meyerhof pathway a t the level of triose-phosphates. Thus a loop is formed, by which the phosphofructokinase is circumvented. The rate of the oxidative pentose-phosphate pathway is primarily limited by the NADPf concentration [l]. Methylene blue increases the share of the oxidative pentose-phosphate pathway by changing the NADP+/ NADPH ratio in favour of the oxidized form. With this shift are connected changes in the rate of the Embden-Meyerhof pathway which are reflected in the concentrations of intermediates [l -31. The pronounced p H dependence of the key enzymes of the glycolysis, hexokinase and phosphofructokinase, and of the NADP+/NADPH ratio would lead one to expect an influence of pH on the interactions between the pathways of glucose utilization.Enzymes. ATP : ~-fructose-6-phosphate l-phosphotransferase or phosphofructokinase (EC 2.7.1.11); glucose-6-phosphate dehydrogenase or ~-glucose-6-phosphate : NADP oxidoreductase (EC 1.1.1.49); D-glyceraldehyde-3-phosphate : NAD oxidorcductase (phosphorylating) or glyceraldehydephosphate dehydrogenase or triosephosphate dehydrogenase (EC 1.2.1.12); ATP : u-hexose 6-phosphotransferase or hexokinase (EC 2.7.1.1); L-lactate: NAD oxidoreductase or lactate dehydrogenase (EC 1.1.1.27); 6-phospho-D-ghconate : NAD oxidoreductase (decarboxylating) or phosphogluconate dehydrogenase (decarboxylating) (EC 1.1.1.44).The present study had as its purpose the determination of the contributions of the oxidative pentosephosphate pathway and the Embden-Meyerhof pathway from the yields of 14C0, and [l4C]1actate from I4C-labelled glucose a t different pH values in the presence and absence of methylene blue. MATERIALS...

1. I n erythrocytes of man and rabbits methylene blue catalysis is limited by the following factors at the pH optimum 7.5; primarily by the cellular concentration of NADP+, next by that of glucose-6-P. Possible further limiting steps are glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase and reduced-NADP dehydrogenase.2. Calculation of the intracellular NADPf concentration with the two-substrate equation for the glucose-6-phosphate dehydrogenase-reaction does not yield reasonable results. The existence of inhibiting factors of the methylene blue catalysis is postulated. Their amount is presumably lower in intact cells as compared with hemolysates.3. There is no pH dependence of the glucose-6-P concentration in presence of methylene blue. There exists a competition between the oxidative pentose-phosphate-pathway and the EmbdenMeyerhof pathway, in which the phosphofructokinase plays a key role.4. The proportions of the two pathways a t different pH values are calculated from data on oxygen and glucose consumption. At pH values below 7.6 glucose is utilized practically exclusively via the oxidative pentose phosphate pathway, whereas the Embden-Meyerhof pathway is inhibited. At pH 8.2 the participation of the oxidative pentose-phosphate pathway amounts to 40°/, of the total glucose utilization, while the Embden-Meyerhof pathway is little if any reduced as compared to cells withouth methylene blue.5 . With increased degree of oxidation of the pyridine nucleotides there occur decreases in the levels of hexose-and triose-phosphates. With these changes and the decreased flow to lactate an increased formation of 2,3-diphosphoglycerate occurs.

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