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Activation Kinetics of NAD-Dependent Malic Enzyme of Cauliflower Bud Mitochondria ¹

Institute of Botany, University of Modena, Modena, Italy

NAD-dependent malic enzyme (EC 1.1.1.39) was obtained from isolated mitochondria of cauliflower buds (Brassica oleracea L., var. botrytis). The NAD-linked activity is accompanied by a minor NADP-linked activity. Some contaminant NADP-malic enzyme from the supernatant and the plasma membrane is usually present in crude mitochondrial preparations. NAD-dependent malic enzyme has been purified 38-fold by ammonium sulfate fractionation and gel permeation chromatography, to a specific activity up to 2 micromoles per minute per milligram.

The nature of the activating effect of coenzyme A and dithiothreitol has been investigated. Both compounds act by decreasing the apparent Michaelis constants for L-malate and NAD⁺ (and NADP⁺), V_max remaining approximately constant. However, enzyme fully activated by dithiothreitol can still be stimulated up to 2.4-fold by coenzyme A treatment.

Velocity versus substrate responses show hyperbolic kinetics under present assay conditions (pH 7.5, 2 millimolar Mn²⁺), but biphasic kinetics have been observed with enzyme purified in the presence of 10 millimolar dithiothreitol, suggesting enzyme heterogeneity with respect to an activated state. This condition is reverted to linearity by treatment with coenzyme A. K_m values do not vary with changing concentrations of the second substrate. Enzyme molecular weight is 400,000 in the completely activated state and 200,000 in the `inactivated' state; intermediate forms are also found. All coenzyme A derivatives tested are effective activators, showing activation constants lower than for coenzyme A itself. The concentration dependence of the activation is sigmoidal.