Spin ECHO proton NMR studies of the metabolism of malate and fumarate in human erythrocytes. Dependence on free NAD levels

Abstract

The NAD-dependent conversion of malate to lactate in human erythrocytes was studied by spin echo proton NMR. A pathway involving the decarboxylation of oxaloacetate catalysed by haemoglobin is proposed to account for the observed reaction. NADP-dependent reaction was negligible. The rate of the reaction was measured in intact erythrocytes under controlled conditions. This rate correlates with that obtained with lysates at 30 microM free NAD and that obtained with purified human erythrocyte enzymes at about 15 microM NAD. The total extractable NAD in the intact cells was 70-90 microM. Experiments with cells containing elevated NAD levels could be explained by a significant fraction of the NAD being weakly bound (Kd about 1 mM) to haemoglobin.