PURIFICATION OF MITOCHONDRIAL GLUTAMATE-DEHYDROGENASE FROM DARK-GROWNSOYBEAN SEEDLINGS

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 gl utamate dehydrogenase (GDH) were resolved and visualized in gels stain ed for GDH activity. Two isoforms with high electrophoretic mobility, GDH1 and GDH2, were in protein extracts from cotyledons and a third is oform with the lowest electrophoretic mobility, GDH3, was identified i n protein extracts from root and hypocotyls. Subcellular fractionation of dark-grown soybean tissues demonstrated that GDH3 was associated w ith intact mitochondria. GDH3 was purified to homogeneity, as determin ed by native and sodium dodecyl sulfate-polyacrylamide gels. The isoen zyme was composed of a single 42-kD subunit. The pH optima for the red uctive amination and the oxidative deamination reactions were 8.0 and 9.3, respectively. At any given pH, GDH activity was 12- to 50-fold hi gher 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 knowled ge, this is the first biochemical and physical characterization of a p urified mitochondrial NAD(H)-dependent GDH isoenzyme from soybean.