Akira Omachi

Pyridine nucleotides in human erythrocytes in different metabolic states

Abstract 1. 1. NAD + , NADH, NADP + , and NADPH concentrations in intact erythrocytes 2. 2. When acid extracts were prepared in the presence of ascorbic acid, NADP + was markedly lower as observed by Burch, Bradley and Lowry . An influence of this agent on NAD + content was not readily apparent but was demonstrable under appropriate conditions. Accordingly, ascorbic acid treatment appears to be necessary in order to obtain valid figures for NAD + as well as for NADP + . 3. 3. In incubation studies, NADP + increased and both NADPH and NADH decreased when medium glucose was omitted. With 0.5 mM iodoacetate and 10 mM glucose, only a decrease in NADH was noted. Thus, glucose lack alters nucleotide concentrations associated with both glycolytic adn phosphogluconate pathways, whereas iodoacetate only modifies the reduced nucleotide level related to glycolysis. 4. 4. In contrast to glucose-free controls, 10 mM lactate increased NADPH as well as NADH levels. Both NADPH and NADH were decreased in the presence of 10 mM pyruvate and glucose. These findings suggest the presence of a transhydrogenase activity. 5. 5. Change in NAD + was not generally observed except for the decrease noted with 5 mM fluoride. It appears that NAD + content may be influenced by factors other than those affecting glycolysis directly.

Measured and calculated NAD+/NADH ratios in human erythrocytes

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 Pi 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 Pi, 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)(HPO42−) 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 Pi was raised may be explained mainly by the influence of the Pi level on the glycolytic rate. 4. 4. The decrease in pyruvate as other intermediates increased indicates that the redox state may modulate glycolytic rate.

Intratubular fluid movement in dog kidney during stop flow.

Summary The movement of intratubular fluid during stop flow has been investigated by injecting 2 or more filtration indicators at different intervals during period of ureteral occlusion. Appearance of these indicators in different samples collected upon release of the occlusion demonstrates that appreciable fluid movement takes place during stop flow.

Sulfate Transport in Human Red Cells: Inhibition by Some Uncouplers of Oxidative Phosphorylation

Release of inorganic sulfate from human erythrocytes is depressed in the presence of 2,4-dinitrophenol (5 x 10-4M) or dicumarol (5 x 10-4M). This effect cannot be readily attributed to uncoupling of phosphorylation from respiration, since the study was conducted with cells that metabolize principally by anaerobic means and since the effect was not influenced by iodoacetic acid. A more reasonable explanation may be that permeability of the erythrocyte membrane to anions may be reduced by these agents.

Inhibition of Chloride Binding to the Anion Transport Site by Diethylpyrocarbonate Modification of Band 3

SummaryThe line widths of35Cl− nuclear magnetic resonances were used to measure chloride binding by Band 3. Since this procedure related directly to binding, the data obtained may be interpreted more unequivocally than affinities derived from kinetic data which could be related to either translocation or binding. Chloride binding to the active sites in Band 3 was assessed from that portion of the total line width which was sensitive to 4,4′-dinitrostilbene-2,2′-disulfonic acid. These sites appeared to be completely inhibited by treatment of erythrocyte membranes with diethylpyrocarbonate. This result is consistent with our previous observation that this reagent inhibits anion transport in resealed erythrocyte ghosts (Izuhara, Okubo & Hamasaki, 1989,Biochemistry28:4725–4728). Hydroxylamine could not reverse the diethylpyrocarbonate inhibition of chloride binding to Band 3. The pH-dependence of diethylpyrocarbonate reactivity suggests that the modified residues may be those of histidine.

Phosphate release from human erythrocytes

Abstract 1. 1. 32 P i -labeled erythrocytes were suspended in balanced saline media and P i release was estimated from changes in medium 32 P i ([ 32 P i ] m ), cellular 32 P i ([ 32 P i ] c ) and cellular P i concentration ([P i ] c ). 2. 2. The outward transfer of P i from the human red cell may be due to a passive process because (i) metabolic inhibition with iodoacetate did not reduce P i exit and (ii) the temperature coefficient for 32 P i release was similar to that found for 35 SO 4 2− release. Also, P i release did not appear to be saturable over a 0.5 mM range of cellular P i concentration. 3. 3. These transport studies were associated with metabolic interactions. When [P i ] c was elevated by raising medium P i concentration ([P i ] m ), [ 32 P 1 ] c also increased. Related studies with iodoacetate suggest that this was due to decreased esterification of 32 P i in favor of P i as [P i ] c was elevated, simultaneous with continuous liberation of 32 P i from an ester P pool into the cellular P i pool.

Erythrocyte membrane lysophospholipase activity in muscular dystrophy

The membrane lysophospholipase activity of erythrocytes obtained from Duchenne muscular dystrophy patients was lower than that of erythrocytes from age-matched normal boys. On the other hand, the membrane enzyme activity of erythrocytes from myotonic dystrophy patients was not different from that of their age- and sex-matched controls. Dipyridamole (0.1 mM) and glycerol 3-phosphorylcholine (2 mM) had no significant effect on these enzyme activities. These results suggest that membrane lysophospholipid metabolism may be altered in Duchenne muscular dystrophy but not in myotonic dystrophy.

2,4-dinitrophenol inhibition of P32 release from human red cells.

Inkubiert man P32-vorbeladene menschliche Erythrozyten unter Zusatz von 5 × 10−4 M Dinitrophenol, so lässt sich eine Reduktion der Phosphatabgabe feststellen. Mit Erhöhung des extrazellulären Orthophosphatgehalts kann diese Wirkung besser gesehen werden.