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ARS Home » Research » Publications at this Location » Publication #63498
Title: PURIFICATION OF MITOCHONDRIAL GLUTAMATE DEHYDROGENASE FROM DARK GROWN SOYBEAN SEEDLINGS

Author
item Turano, Frank
item DASHNER, RALPH - AMVAX INC
item UPADHYAYA, ABHA - UNIVERSITY OF MARYLAND
item Caldwell, Charles

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/23/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Ammonia, which is toxic to cells, has been shown to accumulate in plants grown under different types of environmental stress. There is a correlation between a plant's ability to detoxify (reassimilate) ammonia efficiently and tolerance to various types of environmental stress. Glutamate dehydrogenase (GDH) has been reported to be one of the enzymes involved in the reassimilation of ammonia when plants are environmentally challenged. In this study, we report the isolation of GDH from an agronomically important crop, soybean. The present study focuses on the cellular factors that control enzymatic activity. In addition, we identified where the enzyme is in germinating soybean seedlings. We also provide evidence to show that we purified the enzyme to homogeneity. Now that we have obtained the purified enzyme, we can initiate molecular studies to find the gene and study how the gene is controlled in plants. In the future, with a more thorough understanding of the factors that control enzymes and genes involved in nitrogen metabolism, we could increase the efficiency of nitrogen metabolism in plants and in return improve crop quality and production.

Technical Abstract: Protein 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 visualized in gels specifically stained for GDH activity. Two isoforms with high electrophoretic mobility (GDH1 and GDH2) were in protein extracts from cotyledons and the isoform with the lowest electrophoretic mobility (GDH3) was identified in root and hypocotyl extracts. Subcellular fractionation of dark grown soybean tissues demonstrated that GDH3 was associated with intact mitochondria.The pH optima for the reductive amination and the oxidative deamination reactions were 8.0 and 9.3, respectively. Estimates of GDH activity were 12- to 50-fold higher in the direction of reductive amination when compared to estimates of enzyme activity in the direction of the oxidative deamination reaction conducted at the same pH. The enzyme, GDH3 had a cofactor preference for NAD(H) over NADP(H). The Km for glutamate and NAD were 15 and 0.1mM at pH 9.3, respectively. The Km for a-ketoglutarate, ammonia and NADH at pH 8.3 were 3.6, 17.7, and 0.07 mM, respectively. GDH3 was purified to homogeneity, as verified by native and SDS gels, the isoform was composed of a single 42 kD subunit.