An Apparent Oligomer of Malate Dehydrogenase from Bean Leaves

Abstract

Two forms of malate dehydrogenase of widely differing molecular weight have been examined from primary leaves of Phaseolus vulgaris. In addition to the normal 69,000 molecular weight enzyme, an unusual form of 280,000 molecular weight may be detected by sucrose density gradient centrifugation or gel filtration with Sephadex G-200. Isopycnic density gradient centrifugation showed that both forms of malate dehydrogenase differed markedly from the bulk of the leaf protein by their low bouyant density of 1.261 g/cm(3).High molecular weight (280,000) malate dehydrogenase could be converted to active low molecular weight (69,000) malate dehydrogenase by treatment with 2.5 m CsCl, 1.0 m NaCl, 6 m urea, pH 6.5 or below, or one freeze-thaw cycle. Simple removal of salt or raising the pH were not effective in reforming the high molecular weight malate dehydrogenase after dissociation. The high molecular weight enzyme was not dissociated during prolonged dialysis against 0.1 m NaCl or 0.05 m phosphate, pH 7.0. Calcium at concentrations up to 0.1 m produced no activation or differential response in the two MDH forms.High and low molecular weight malate dehydrogenase were nearly identical in susceptibility to inhibition by various unreactive substrate analogs. However, there was a marked difference in the ability of the two forms of malate dehydrogenase to reduce 3-acetylpyridine-deamino-NAD. This difference in activity was the basis of a convenient assay for determining the ratio of high to low molecular weight malate dehydrogenase in crude extracts. The pH activity profiles and Michaelis constant for malate were nearly identical for the two molecular weight forms.Analysis by polyacrylamide gel electrophoresis revealed one high molecular weight and two low molecular weight malate dehydrogenase zones. Dissociation of high molecular weight malate dehydrogenase resulted in formation of low molecular weight enzyme whose electrophoretic properties differed from the normal low molecular weight forms.