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...

Ungerminated pumpkin (Cucurbita moschata Poir.) cotyledons contained 30 % of their dry weight as lipid and 26 % as protein, of which 93 % was globulin. There was a rapid degradation of these reserves 4 to 8 days after planting when the cotyledons had their maximum metabolic activity. About half of the mole percent of amino acids found in the globulin reserve was in arginine, glutamate, aspartate, and their amides. The cotyledons had a large soluble pool of arginine, glutamine, glutamate, and leucine. Most amino acids increased steadily in amount in the cotyledons during germination, except glutamine, ornithine, alanine, serine, glycine, and γ‐aminobutyrate and these appeared in large amounts in the translocation stream to the axis tissue. Little arginine or proline was translocated. By 10 days, when translocation had decreased, amino acids accumulated. Ornithine, γ‐aminobutyrate, and aspartate were rapidly utilized in the hypocotyl, while glutamine, glycine, and alanine accumulated there. Cysteine and methionine levels were low in the reserve, trans‐location stream and soluble fractions. γ‐Aminobutyrate‐U−14C injected into cotyledons or incubated with hypocotyls was utilized in a similar fashion. The label appeared in citric acid cycle acids and in the amino acids closely related to this cycle, but the bulk of the label appeared in CO2. The labeling pattern suggests that γ‐aminobutyrate was utilized via succinate, and thus entered the citric acid cycle. A close relationship between arginine, ornithine, glutamate, and γ‐aminobutyrate exists in the cotyledon with all but arginine being translocated rapidly to the axis tissue where these amino acids are rapidly metabolized.

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