The effects of ions and freeze-thawing on supernatant and mitochondrial malate dehydrogenase.

Abstract

The effects of various ions added to the assay system for purified mitochondrial and supernatant pig-heart malate dehydrogenase have been studied. Phosphate, arsenate, and zinc ions strongly stimulate the mitochondrial enzyme. Phosphate and arsenate ions have little effect on the supernatant enzyme whereas zinc inhibits it. The stimulation by phosphate is relieved by the addition of magnesium ion, to the assay system.The effects on enzymatic activity were studied when the supernatant and mitochondrial malate dehydrogenase were subjected to freezing and thawing in various media. Freezing and thawing of the mitochondrial enzyme in Tris or citrate buffers with or without mercaptoethanol results in small activity loss. Supernatant malate dehydrogenase displays a marked loss in activity when frozen quickly in the presence of phosphate and mercaptoethanol; if mercaptoethanol is omitted, activity loss is small. NADH, oxalacetic acid (OAA), NAD, and malate protect against activity loss in the phosphate–mercaptoethanol system. Repeated freeze–thawing of mitochondrial malate dehydrogenase in the presence of both phosphate and magnesium results in extensive losses in activity. In all cases slow freezing produces little activity loss for either enzyme.