Characterisation of the two malate dehydrogenases from Phytomonas sp. : Purification of the glycosomal isoenzyme

Abstract

Two NAD(H)-dependent malate dehydrogenase (MDH) isoenzymes were detected in Phytomonas isolated from the lactiferous tubes of Euphorbia characias. The total specific activity in crude extracts using oxaloacetate as substrate was 3.3 U mg −1 of protein. The two isoenzymes had isoelectric points of 6.0 and 7.2, respectively. The acidic isoform represented 80% of the total activity in the cell and was present in the glycosome. It was purified to homogeneity by a method involving hydrophobic interaction chromatography on Phenyl-Sepharose followed by ionic exchange on CM-Sepharose and affinity chromatography on Blue-Sepharose. The purified glycosomal MDH is a homodimeric protein with a subunit molecular mass of 37 kDa and it has a low substrate specificity, since it was able to reduce both aromatic and aliphatic α-ketoacids as substrate, including oxaloacetate, phenyl pyruvate, α-keto iso-caproate and pyruvate. The apparent K ms for oxaloacetate and NADH were 166 and 270 μM, respectively and for l-malate and NAD +, 3000 and 246 μM, respectively. The basic isoform was present in the mitochondrion. It has a high substrate specificity and an apparent K m of 132 and 63 μM for oxaloacetate and NADH, respectively, and of 450 and 91 μM, respectively, with l-malate and NAD +.