CHARACTERIZATION OF THE 2 MALATE-DEHYDROGENASES FROM PHYTOMONAS SP - PURIFICATION OF THE GLYCOSOMAL ISOENZYME

Two NAD(H)-dependent malate dehydrogenase (MDH) isoenzymes were detect ed in Phytomonas isolated from the lactiferous tubes of Euphorbia char acias. The total specific activity in crude extracts using oxaloacetat e as substrate was 3.3 U mg(-1) of protein. The two isoenzymes had iso electric points of 6.0 and 7.2, respectively. The acidic isoform repre sented 80% of the total activity in the cell and was present in the gl ycosome. It was purified to homogeneity by a method involving hydropho bic interaction chromatography on Phenyl-Sepharose followed by ionic e xchange on CM-Sepharose and affinity chromatography on Blue-Sepharose. The purified glycosomal MDH is a homodimeric protein with a subunit m olecular mass of 37 kDa and it has a low substrate specificity, since it was able to reduce both aromatic and aliphatic alpha-ketoacids as s ubstrate, including oxaloacetate, phenyl pyruvate, alpha-keto iso-capr oate and pyruvate. The apparent K(m)s for oxaloacetate and NADH were 1 66 and 270 mu M, respectively and for L-malate and NAD(+), 3000 and 24 6 mu M, respectively. The basic isoform was present in the mitochondri on. It has a high substrate specificity and an apparent K-m of 132 and 63 mu M for oxaloacetate and NADH, respectively, and of 450 and 91 mu M, respectively, with L-malate and NAD(+). (C) 1997 Elsevier Science B.V.