Over a range of concentrations from less than 0.1 mM to more than 70 mM, sweet potato root mitochondria display a bimodal substrate saturation isotherm for malate. The high affinity portion of the isotherm has an apparent Km for malate of 0.85 mm and fits a rectangular hyperbolic function. The low affinity portion of the isotherm is sigmoid in character and gives an apparent S0.5 of 40.6 mm and a Hill number of 3.7.Extracts of sweet potato mitochondria contain both malate dehydrogenase and NAD malic enzyme. The malate dehydrogenase, assayed in the forward direction at pH 7.2, shows typical Michaelis-Menten kinetics with a Km for malate of 0.38 mm. The NAD malic enzyme shows pronounced sigmoidicity in response to malate with a Hill number of 3.5 and an S0 5 of 41.6 mM.On the basis of the normal kinetics, the Km, and the fact that oxaloacetate production from malate by mitochondria appears most active at low malate concentrations, the high affinity portion of the malate isotherm with mitochondria is attributed to malate dehydrogenase. The low affinity portion of the malate isotherm with mitochondria is thought, on the basis of the similarity of SO.5 values, the Hill numbers, and the greater production of pyruvate from malate at high malate concentrations, to represent the activity of the NAD malic enzyme.The response of plant mitochondria to malate is often variable and erratic (6,10,12,13,15). Saturation with the substrate is difficult to achieve, coupling with phosphorylation is sometimes less than with other tricarboxylic acid cycle substrates using NAD as a cofactor (16) and the response to exogenously added cofactors such as NAD may be at variance with that of other substrates (3,6,15). In part, these unexpected responses may arise from the characteristics of the enzyme, malate dehydrogenase, usually assumed to be responsible for the oxidation of malate in the tricarboxylic acid cycle. Malate dehydrogenase is strongly inhibited by its product, oxaloacetate, at pH values in the range likely to be found in the mitochondria (10,17,18), and is subject to allosteric regulation by a number of effectors (1).We have observed in mitochondria from a variety of plant tissues, including sweet potato, beet roots, cauliflower florets, and Arum spadix, a failure of 02 uptake rates to be saturated by levels of malate greatly in excess of the concentrations which would be expected to elicit no further response on the basis of the observed Km for malate of malate dehydrogenase in mitochondrial extracts. In addition, it is frequently difficult to obtain smooth curves in the 02 uptake response of plant mitochondria to malate, and where sufficient numbers of careful determinations are made, the malate response curve appears to be inflected. Similar results have been obtained by others (3, 13).This report details the characteristics of 02 uptake by sweet potato mitochondria in response to malate, and shows that the saturation isotherm for malate is bimodal, with an intermediary plateau, and provides evidence that this resp...
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