F. M. Huennekens

ERYTHROCYTE METABOLISM. V. LEVELS OF GLYCOLYTIC ENZYMES AND REGULATION OF GLYCOLYSIS

In contrast to many other cell types in which a variety of metabolic pathways may contribute to energy production, the human erythrocyte derives its energy almost exclusively from the breakdown of glucose to lactate via the glycolytic sequence. Lactate can arise also from glucose by an alternate route, namely the hexose monophosphate shunt and the pentose cycle, but this latter pathway is relatively inoperative in the erythrocyte under normal conditions, owing to the unexplained preferential conversion of glucose-6-phosphate to fructose-6-phosphate, rather than to 6-phosphogluconate. The red cell, therefore, offers a unique opportunity to correlate physiological function, or malfunction, with enzymatic activity, since the number of metabolic pathways, fortunately, is somewhat restricted. In the present investigation a detailed study has been undertaken to define the optimal conditions for the conversion of glucose to lactate in the intact erythrocyte and in hemolysates. In addition, levels of the individual glycolytic enzymes have been determined and this information has been used to discuss regulatory mechanisms of glycolvsis in the ervthrocyte.

Studies on the interaction of tritium-labeled aminopterin with dihydrofolic reductase☆

Abstract The inhibition of leucocyte dihydrofolic reductase by aminopterin, amethopterin, and dichloroamethopterin was measured at inhibitor concentrations ranging from 10 −6 to 10 −9 M . Tritium-labeled aminopterin was used to prepare the enzyme-inhibitor (E-I) complex of both the leucocyte and chicken liver dihydrofolic reductases. Dialysis of the leucocyte E-I complex resulted in a loss of radioactivity which corresponded to the reappearance of enzyme activity. The chicken liver E-I complex, however, was not dissociated by dialysis. The leucocyte E-I complex was readily dissociated by chromatography on DEAE-cellulose using stepwise desorption with KCl. The reactivated enzyme and the free inhibitor appeared in the 0.05 and 0.15 M fractions, respectively. The chicken liver and leucocyte E-I complexes were readily dissociated by fractionation with ammonium sulfate. A turnover number of 50–100 at pH 8.5 was determined for the chicken liver reductase from data on the amount of inhibitor bound to the enzyme in various ammonium sulfate fractions.

Patterns of dihydrofolic reductase and tetrahydrofolate-dependent enzymes in the developing chick embryo☆

Abstract In the whole embryo and liver of the developing chick the levels of dihydrofolic reductase, formate-activating enzyme, N 5 ,N 10 -methylene tetrahydrofolic dehydrogenase, and serine hydroxymethylase reach a maximum at 11–13 days of incubation, and thereafter decline. On the other hand, the levels, of those enzymes, with respect to each other, remain constant throughout the development of the embryo. Formate-activating enzyme and dihydrofolic reductase have been purified from embryonic chick liver, and the pH optima, kinetic constants, and substrate requirements of these enzymes are similar to those of their counterparts in other tissues. The administration of aminopterin to the chick embryo does not cause a rise in dihydrofolic reductase activity, contrary to results in other tissues. Under these conditions, the activity of dihydrofolic reductase is actually decreased when measured at low pH values, although no inhibition is detected when the assay is performed at higher pH values.