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Purification and properties of the human erythrocyte malic dehydrogenase

Abstract An approximate hundredfold purification of the malic dehydrogenase (decarboxylating nicotinamide adenine dinucleotide phosphate [NADP + or NADPH] malic enzyme) which generates pyruvate from malic acid has been prepared from human erythrocytes. A rather broad pH optimum in the range of 7.5 to 8.5 was found. The apparent Michaelis constant (K m ) for the forward reaction at pH 7.5 employing 5 mM MnCl 2 , was 96 ± 5 μM for L-malate, and 2.2 ± 0.2 μM for NADP + . These results are similar to those found for pigeon liver at pH 7. However, the Km for the backward reaction was somewhat different in the erythrocyte: 2.01 ± 0.05 μM for NADPH, 0.75 ± 0.003 mM for pyruvate, and 12.5 ± 0.10 mM for bicarbonate. Stoichiometry of the malic enzyme preparation revealed that for each mole of malate utilized, 1 mole of pyruvate was produced. After incubation of uniformly labeled L-malic acid with washed erythrocytes in a Krebs-Ringer phosphate buffer, radioactivity was demonstrated in lactate by thin-layer chromatography. Human glucose-6-phosphate dehydrogenase (G-6-PD)-deficient erythrocytes showed a 60 per cent increase in utilization of L-malate in the first hour, following incubation with L-(4 14 C) malate, as compared to normal erythrocytes. The possibility is presented that the G-6-PD-deficient erythrocyte utilized malate more efficiently than normal red cells as a compensatory measure to increase production of NADPH.