Determination of NAD Malic Enzyme in Leaves of C4 Plants

Malate dehydrogenase may interfere with the assay of NAD malic enzyme, as NADH is formed during the conversion of malate to oxaloacetate. During the present study, two additional effects of malate dehydrogenase were investigated; they are evident only if the malate dehydrogenase reaction is allowed to reach equilibrium prior to initiating the malic enzyme reaction. One of these (Outlaw, Manchester 1980 Plant Physiol 65: 1136-1138) might cause an underestimation of NAD reduction by malic enzyme due to the oxidation of NADH during reversal of the malate dehydrogenase reaction. A second effect may result in overestimation of malic enzyme activity, as Mn(2+)-catalyzed oxaloacetate decarboxylation causes continuing net NADH formation via malate dehydrogenase. These effects were studied by assaying the activity of a partially purified preparation of Amaranthus retroflexus NAD malic enzyme in the presence or absence of purified NAD malate dehydrogenase.A model was developed which allowed the generation of theoretical curves describing the influence of malate dehydrogenase on the assay of malic enzyme activity. The experimental data obtained agreed closely with these curves. The conditions included 5 millimolar malate, 2 millimolar NAD, and 4 millimolar Mn(2+) (pH 7.2 to 7.8 at 30 degrees C). At low activities of malic enzyme (1 nanomole per minute per milliliter or less), the presence of malate dehydrogenase leads to a substantial overestimation of malic enzyme activity due to the Mn(2+)-catalyzed decarboxylation of oxaloacetate having a dominant effect. When the level of malic enzyme is greater than 1 nanomole per minute per milliliter, reversal of malate dehydrogenase has a dominant transient effect, causing a lag of up to several minutes, after which the change in absorbance reflects the true rate of malic enzyme. Independent of this effect is a lag in the activator-dependent rate, which could be eliminated by preincubating the enzyme with activator (coenzyme A).An assay procedure designed to minimize the influence of these effects is described. New data are presented on the activity of NAD malic enzyme in leaves of different subgroups of C(4) plants.