Intracellular restraint: a new basis for the limitation in response to oxidative stress in human erythrocytes containing low-activity variants of glucose-6-phosphate dehydrogenase.
Several mechanisms recently proposed for regulation of the hexose monophosphate shunt require the concentration of NADP to be low or that of NADPH to be high. The present study indicates that the first enzyme of the hexose monophosphate shunt of human erythrocytes is under severe restraint even when these conditions do not exist. In human erythrocytes containing low-activity variants of this enzyme, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP(+) 1-oxidoreductase; EC 1.1.1.49), measurements of the rate of oxidation of C-1 labeled glucose show that the enzyme is operating at a rate much closer to its maximum than in normal cells. This requires that the ratio of inhibitory NADPH to NADP be much lower in the variant cells than in normal cells. A small increase in oxidative rate, induced by naphthol, then causes a disappearance in reduced glutathione in the variant cells, presumably because a significant further decrease in NADPH occurs in these cells, whereas the same oxidative stress in normal cells would not lower the NADPH level appreciably. A low NADPH/NADP ratio in unstressed cells deficient in glucose-6-phosphate dehydrogenase is confirmed by direct measurement. The maximum activity of the variant enzyme in the cell, as measured with methylene blue to keep most of the NADP in the oxidized form, is only about 1/60 of that found in hemolysates, thus accounting for the failure to compensate for a relatively small oxidative stress in vivo in spite of an apparent sufficiency of enzyme. The reason for the limitation on maximum intracellular activity is unknown. A similar limitation is seen with normal cells incubated with methylene blue, where the maximum intracellular rate is also only about 1/60 of that found in hemolysates.
D002250:Carbon Radioisotopes; D004912:Erythrocytes / Q000201:enzymology / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D005955:Glucosephosphate Dehydrogenase Deficiency / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D005980:Glutathione Reductase / Q000378:metabolism; D006801:Humans; D008297:Male; D008751:Methylene Blue / Q000494:pharmacology*; D009249:NADP / Q000378:metabolism; D009284:Naphthols / Q000494:pharmacology*; D010084:Oxidation-Reduction
D002250:Carbon Radioisotopes; D009284:Naphthols; D009249:NADP; D005954:Glucosephosphate Dehydrogenase; D005980:Glutathione Reductase; D005978:Glutathione; D005947:Glucose; D008751:Methylene Blue