Measured and calculated NAD+-NADH ratios in human erythrocytes.

Abstract

Abstract 1. 1. The NAD + /NADH ratios associated with lactate dehydrogenase and with glyceraldehyde 3- P dehydrogenase coupled with 3- P -glycerate kinase were calculated from equilibrium constants and measured substrate concentrations in human erythrocytes. These ratios were similar in freshly drawn cells as well as in cells glycolyzing at different rates as a results of incubation in media containing different P i concentrations. These values appear, therefore, to be reasonable approximations of the true redox state. 2. 2. The NAD + /NADH ratio measured directly was only 1/100th of the calculated ratios in freshly drawn red cells. Since the measured and calculated ratios both increased with elevation in medium P i , it appears that (i) changes in the total NAD + /total NADH ratio reflect the changes in the free NAD + /free NADH ratio and (ii) the free and bound pyridine nucleotides are in equilibrium. 3. 3. The (ATP)/(ADP)(HPO 4 2− ) ratio determined by direct measurement was similar to the ratio calculated from an equation based on the equilibrium states of glyceraldehyde-3- P dehydrogenase, 3- P -glycerate kinase, and lactate dehydrogenase. The direct relationship found between the redox and phosphorylation states as medium P i was raised may be explained mainly by the influence of the P i level on the glycolytic rate. 4. 4. The decrease in pyruvate as other intermediates increased indicates that the redox state may modulate glycolytic rate.