Purification of mitochondrial glutamate dehydrogenase from dark-grown soybean seedlings

• Published in: Plant physiology (Bethesda) Vol. 112; no. 3; pp. 1357 - 1364
• Main Authors: Turano, F.J. (USDA, ARS, Climate Stress Laboratory, Beltsville, MD.), Dashner, R, Upadhyaya, A, Caldwell, C.R
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01-11-1996
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amination; Analytical, structural and metabolic biochemistry; Biochemistry and Enzymology; Biological and medical sciences; COENZIMAS; COENZYME; COTILEDONES; COTYLEDON; Cotyledons; Dehydrogenases; Enzymes; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Gels; GLUTAMATE DESHYDROGENASE; GLUTAMATO DEHIDROGENASA; GLYCINE MAX; HIPOCOTILOS; HYPOCOTYLE; Hypocotyls; ISOENZIMAS; ISOENZYME; MEDIO DE CULTIVO; Metabolism; MILIEU DE CULTURE; Mitochondria; MITOCHONDRIE; MITOCONDRIA; NUCLEOTIDE; NUCLEOTIDOS; OBSCURIDAD; OBSCURITE; Oxidoreductases; Plant physiology and development; Plants; PLANTULAS; PLANTULE; Protein isoforms; RACINE; RAICES; Soybeans; Glutamate dehydrogenase; Purification; Root; Enzyme; Isozyme; Glycine max; Characterization; Grain legume; Mitochondria; Hypocotyl; Leguminosae; Enzymatic activity; Dicotyledones; Angiospermae; Spermatophyta; Oxidoreductases; Kinetics; Optimum pH
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.112.3.1357

Proteins in extracts from cotyledons, hypocotyls, and roots of 5-d-old, dark-grown soybean (Glycine max L. Merr. cv Williams) seedlings were separated by polyacrylamide gel electrophoresis. Three isoforms of glutamate dehydrogenase (GDH) were resolved and visualized in gels stained for GDH activity. Two isoforms with high electrophoretic mobility, GDH1 and GDH2, were in protein extracts from cotyledons and a third isoform with the lowest electrophoretic mobility, GDH3, was identified in protein extracts from root and hypocotyls. Subcellular fractionation of dark-grown soybean tissues demonstrated that GDH3 was associated with intact mitochondria. GDH3 was purified to homogeneity, as determined by native and sodium dodecyl sulfate-polyacrylamide gels. The isoenzyme was composed of a single 42-kD subunit. The pH optima for the reductive amination and the oxidative deamination reactions were 8.0 and 9.3, respectively. At any given pH, GDH activity was 12- to 50-fold higher in the direction of reductive amination than in the direction of the oxidative deamination reaction. GDH3 had a cofactor preference for NAD(H) over NADP(H). The apparent Michaelis constant values for alpha-ketoglutarate, ammonium, and NADH at pH 8.0 were 3.6, 35.5, and 0.07 mM, respectively. The apparent Michaelis constant values for glutamate and NAD were 15.8 and 0.10 mM at pH 9.3, respectively. To our knowledge, this is the first biochemical and physical characterization of a purified mitochondrial NAD(H)-dependent GDH isoenzyme from soybean