Effect of NAD and Rotenone on the Partitioning of Malate Oxidation Between Malate Dehydrogenase and Malic Enzyme in Isolated Plant Mitochondria

Abstract

Our aim was to determine whether there is a specific link between NAD-malic enzyme and the rotenone- insensitive bypass of electron transport. Mitochondria were isolated from fresh beetroot tissue, aged beetroot slices, and turnips. Oxygen uptake and pyruvate production were measured in reactions where these mitochondria were metabolizing malate at pH 6.8 in the presence of glutamate, to facilitate the removal of oxaloacetate, and in its absence. In the absence of glutamate there was substantial activity of malic enzyme. NAD⁺ (577 µM) prevented a fall in oxygen uptake by stimulating malic enzyme. Rotenone (19 µM) reduced oxygen uptake. This inhibited rate was stimulated by NAD⁺ due, in particular, to a stimulation of malic enzyme. We conclude that the stimulation of malate metabolism by NAD⁺ is accounted for by malic enzyme due to the unfavourable equilibrium of malate dehydrogenase for malate oxidation and the resultant accumulation of oxaloacetate, and not to any specific link between malic enzyme and the rotenone-insensitive bypass. In the presence of glutamate, malate dehydrogenase was the predominant malate metabolizing enzyme. Oxygen uptake and malic enzyme were stimulated and inhibited by NAD⁺ and rotenone, respectively. In the presence of rotenone, NAD⁺ stimulated oxygen uptake and increased the percentage due to malic enzyme. This stimulation is accounted for by the higher Kin of the rotenone-insensitive dehydrogenase for NADH and the unfavourable equilibrium position of malate dehydrogenase resulting in activation of malic enzyme only. We conclude that malic enzyme is not specifically linked to the rotenone-insensitive pathway of electron transport. This has important implications for the regulation of energy metabolism in plants.