Characterisation of the two malate dehydrogenases from Phytomonas sp. Purification of the glycosomal isoenzyme.
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 alpha-ketoacids as substrate including oxaloacetate, phenyl pyruvate, alpha-keto iso-caproate and pyruvate. The apparent K(m)s for oxaloacetate and NADH were 166 and 270 microM, respectively and for L-malate and NAD+, 3000 and 246 microM, respectively. The basic isoform was present in the mitochondrion. It has a high substrate specificity and an apparent K(m) of 132 and 63 microM for oxaloacetate and NADH, respectively, and of 450 and 91 microM, respectively, with L-malate and NAD+.
Uttaro
A D
AD
Research Unit for Tropical Diseases, Catholic University of Louvain ICP-TROP/74.39, Brussels, Belgium.
Opperdoes
F R
FR
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D045022:Lactate Dehydrogenases; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification*; D008970:Molecular Weight; D015388:Organelles / Q000737:chemistry / Q000201:enzymology*; D015800:Protozoan Proteins / Q000737:chemistry / Q000302:isolation & purification; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D014351:Trypanosomatina / Q000201:enzymology*
D007527:Isoenzymes; D015800:Protozoan Proteins; D000429:Alcohol Oxidoreductases; D045022:Lactate Dehydrogenases; D007770:L-Lactate Dehydrogenase; C071195:D-lactate dehydrogenase; D008291:Malate Dehydrogenase; C021379:D-2-hydroxyacid dehydrogenase