Glutamate Dehydrogenase from Pumpkin Cotyledons

Abstract

GDH2 from a number of plant sources has been investigated (2,3,6,15,18,20,27). The enzyme appears to have a sulfhydryl (3) and metal (27) requirement, has a number of isoenzymes (25, 28), and has been found entirely (5) or partially in the mitochondrial fraction (2,15,18,20,22,28). This is a report on the localization, characterization, and isoenzymes of GDH from pumpkin cotyledons during germination. Enzyme Isolation. GDH was isolated at 2 C from 100 cotyledons ground in a mortar with 20 ml of 0.25 M sucrose in 50 mM tris-HCl buffer, pH 7.5. The slurry was passed through four layers of cheesecloth and centrifuged at 1,000g to remove debris. Additional grinding and extraction of the debris did not yield additional enzyme activity. Centrifugation of the debris-free solution at 10,000g for 15 min yielded the particulate fraction. The pale yellow supernatant fraction contained the soluble enzyme. The particulate enzyme was solubilized by osmotic shock in 10 mm phosphate, pH 7.5 (27). Attempts to detect residual enzyme activity in the organelle debris were unsuccessful. The particulate and soluble enzymes were then purified in a separate but similar manner. The extracts were heated 5 min at 60 C (1), and the precipitate was discarded. The enzymes were precipitated from the supernatant solutions with 40 to 60% saturated ammonium sulfate and then dissolved in 10 mm sodium phosphate buffer, pH 7.0. The solutions were dialyzed against this buffer for 16 hr at 4 C. This gave a 6-fold purification of the soluble enzyme and a 40-fold purification of the particulate enzyme. Before purification, particulate GDH was entirely inactivated in 6 days at 0 C while soluble GDH had lost only 10% of its activity. After the above purification, both enzymes retained their activity for at least 10 days at 0 C. Protein was determined by the Lowry method. MATERIALS AND METHODSEnzyme Assay. The 3-ml reductive amination assay contained in ,umoles: ammonium chloride, 300; a-ketoglutarate, 30; NADH or NADPH, 0.25; tris-HCl, pH 8.0, 130. The oxidative amination assay contained in ,umoles: glutamate, 100; NAD or NADP, 3; tris-HCl, pH 8.0, 130. One unit of enzyme activity is defined as that amount which will cause a decrease in absorbance of 0.01 unit per min at 340 nm. Generally, 3 units of enzyme activity (60 ,tg of protein) were used, and this gave a reaction rate which was linear for 25 min. The initial reaction velocities were directly proportional to the enzyme concentration over a 10-fold range. The enzyme activity was corrected for NADH oxidase activity which never exceeded 10% of the total activity.The interference of phosphatase with GDH activity toward NADP was considered. Phosphatase activity toward p-nitrophenyl phosphate was assayed by measuring, at 410 nm, the amount of p-nitrophenol liberated (11). Phosphatase activity toward NADP was assayed by coupling the assay with alcohol dehydrogenase and ethanol (14), and the NADH produced was measured at 340 nm. Phosphatase activity never exceeded 10% of the soluble or 2.4% of the particu...

Properties of glutamate dehydrogenase from Beta vulgaris

“…These findings are again comparable with data of GDH from other plant sources and bacteria (Bulen 1956, LeJohn and McCrea 1968, Chou and Splittstoesser 1972, Davies and Teixeira 1975, Ehmke and Hartmann 1976. The relation of 2:1 between NADH and NADPH as substrates resembles most closely the dala of Alekhina and Sokolova (1975) for GDH from grain roots.…”

“…A further increase in a-KGA concentration results in a clearly noticeable substrate inhibition. The apparent K^, determined according to Lineweaver and Burk (1934), has a value of 6.7 x 10^ M. This is lower than usually found in GDH from plants, where values around 1-8 x 10~^ M are common (Bulen 1956, Sanval and Lata 1961, King and Wu 1971, Chou and Splittstoesser 1972, Errel etal. 1973, Postius 1975, Ehmke and Hartmann 1976, Fawole and Boulter 1977.…”

Characterization and Expression of NAD(H)-Dependent Glutamate Dehydrogenase Genes in Arabidopsis

“…The activity of severa1 NAD(H)-GDH isoenzymes has been shown to be stimulated by divalent cations, and is particularly sensitive to Ca2+ (Chou and Splittstoesser, 1972;Joy, 1973;Garland and Dennis, 1977;Yamaya et al, 1984;Itagaki et al, 1988;Loulakakis and Roubelakis-Angelakis, 1990a; F. J. Turano, unpublished results). In most cases, e.g.…”

Regulation of glutamate dehydrogenase activity by amino acids in maize seedlings

“…1974,, 1975,1976, Tsenova 1975, Stewart and Rhodes 1977, Bielawski and Rafalski 1979, Singh and Srivastava 1982. The activity of this enzyme in relation to several endogenous and exogenous factors has been studied in higher plants (Joy 1969, 1973, Chou and Splittstoesser 1972, Jain and Srivastava 1981.…”