Metabolism of Arginine by Aging and 7 Day Old Pumpkin Seedlings

Splittstoesser, Walter E.

doi:10.1104/pp.44.3.361

Cited by 38 publications

(15 citation statements)

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“…The increase in water-soluble protein parallels the increase in GDH during germination (24), which may suggest that the isoenzymes are synthesized de novo. The increase in the number of isoenzyme bands in pumpkin cotyledons during germination may also suggest an induction of GDH by NH4+ or glutamate (13,17), both of which are produced in this tissue in high quantities (4,23). However, high amounts of NH4' or a-ketoglutarate inhibit enzyme activity (Figs.…”

Section: Enase From Pumpkins 553mentioning

confidence: 99%

Glutamate Dehydrogenase from Pumpkin Cotyledons

Chou

Splittstoesser

1972

Plant Physiol.

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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...

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Section: Enase From Pumpkins 553mentioning

confidence: 99%

Glutamate Dehydrogenase from Pumpkin Cotyledons

Chou

Splittstoesser

1972

Plant Physiol.

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“…As part of the overall reconfiguration of free and storage-protein-bound amino acid profiles to that of seedling protein (Thompson, 1980;Polacco and Holland, 1993), Arg breakdown to urea and Orn occurs by the action of arginase (l-Arg amidinohydrolase, EC 3.5.3.1). Arginase activity increases sharply during germination in several species, including pumpkin (Splittstoesser, 1969), broad bean (Kollö ffel and Van Dijke, 1975), soybean (Matsubara and Suzuki, 1984;Kang and Cho, 1990), Arabidopsis (Zonia et al, 1995), and loblolly pine (King and Gifford, 1997). After arginase action, Arg nitrogen contained in urea is recycled to ammonia by the action of urease (Polacco and Holland, 1993).…”

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Arginase Is Inoperative in Developing Soybean Embryos1

Goldraij

Polacco

1999

Plant Physiology

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Arginase (EC 3.5.3.1) transcript level and activity were measured in soybean (Glycine max L.) embryos from the reserve deposition stage to postgermination. Using a cDNA probe for a small soybean arginase gene family, no transcript was detected in developing embryos. However, arginase transcripts increased sharply on germination, reaching a maximum at 3 to 5 d after germination. There was low but measurable in vitro arginase specific activity in developing embryos (less than 6% of seedling maximum). During germination arginase specific activity increased in parallel with the sharply increasing arginase transcript level. Seedling arginase activity was largely localized in cotyledons. Arginase activity was assayed in vivo by measuring urea accumulation in a urease-deficient mutant. No urea was detected in developing embryos, whereas accumulated urea paralleled arginase specific activity and transcript level in germinating seedlings. As in planta embryos, cultured cotyledons did not accumulate urea when arginine (Arg) was provided with other amino acids in a "mock" seed-coat exudate. Arg as the sole nitrogen source was converted to urea but did not support cotyledon growth. There appeared to be a lack of recruitment of the low-level arginase activity to hydrolyze free Arg in developing embryos, thus avoiding a futile urea cycle.

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“…It has been shown with '4C-labeled arginine (9) that in broad bean cotyledons arginine is degraded mainly in situ by an arginasecatalyzed formation of ornithine and urea. Pumpkin cotyledons also contain appreciable amounts of protein-bound arginine which is metabolized primarily in situ (20); less than 1 % of the arginine is translocated to the axis tissue (5). In these pumpkin cotyledons (19), as in seeds of Dolichos lablab (21), the arginase activity increased during germination.…”

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Mitochondrial Arginase Activity from Cotyledons of Developing and Germinating Seeds of Vicia faba L

Kollöffel

Dijke²

1975

Plant Physiol.

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Differential and sucrose density gradient centrifugation established that about 80% of the total arginase activity (EC 3.5.3. 1) in cotyledons of germinating broad bean seeds (Vicia faba L.) was present in the mitochondrial fraction. The mitochondrial arginase activity was enhanced considerably by exposure to osmotic shock, by freezing and thawing, or by Triton X-100 treatment. About 10% of the total arginase activity was recovered from the 40,000g supernatant fraction. During seed maturation, arginase activity in the cotyledons decreased to about one-third of its maximal activity, while increasing over 10-fold during subsequent germination. The time courses of mitochondrial arginase, succinate oxidase, and succinate dehydrogenase activities differed considerably during germination.Cotyledons of developing Vicia faba (L.) seeds accumulate large amounts of arginine-rich protein (2, 13). During subsequent germination these storage proteins are hydrolyzed (1) and the amino acids released are either metabolized in situ or directly translocated to the growing axis tissue. It has been shown with '4C-labeled arginine (9) that in broad bean cotyledons arginine is degraded mainly in situ by an arginasecatalyzed formation of ornithine and urea. Pumpkin cotyledons also contain appreciable amounts of protein-bound arginine which is metabolized primarily in situ (20); less than 1 % of the arginine is translocated to the axis tissue (5). In these pumpkin cotyledons (19), as in seeds of Dolichos lablab (21), the arginase activity increased during germination. This paper shows that this enzyme is already present in developing broad bean cotyledons.Although arginase has been isolated from many plant tissues (3,6,9,14,19,21), its subcellular distribution is unknown. In mammalian tissues (7,8,15) and in Neurospora (22), arginase activity was located in the cytosol. This paper reports that in germinating broad bean cotyledons arginase activity was present mainly in the mitochondrial fraction. MATERIALS AND METHODSPlant Material. Seeds of Vicia faba L. cv. R 35 were planted in the field and 3 to 4 months later pods were taken from the plants and selected for equal thickness, color, and turgor. The seeds were removed from the pods; the cotyledons were separated and divided into two portions, one for the extraction of the enzymes and the other for the determination of fresh and dry weight (24 hr at 105 C.). The relative water content of the seeds, defined as mg water/mg fresh weight x 100%, was taken as the criterion for their physiological state. During the growth period studied, the relative water content decreased from about 70% to about 12% (air-dry mature seeds).Germination Conditions. Air-dry mature seeds were harvested and soaked in tap water under aeration for 18 to 24 hr. They were allowed to germinate further by transferring them subsequently to moist filter paper in large Petri dishes in darkness at 23 C for the appropriate time. The period between the beginning of soaking and the moment the cotyledons were used for...

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Metabolism of Arginine by Aging and 7 Day Old Pumpkin Seedlings

Cited by 38 publications

References 20 publications

Glutamate Dehydrogenase from Pumpkin Cotyledons

Glutamate Dehydrogenase from Pumpkin Cotyledons

Arginase Is Inoperative in Developing Soybean Embryos1

Mitochondrial Arginase Activity from Cotyledons of Developing and Germinating Seeds of Vicia faba L

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