Asparagine is present in the mature leaves of young pea (Pisum sativum cv Little Marvel) seedlings, and is synthesized in detached shoots. This accumulation and synthesis is greatly enhanced by darkening. In detached control shoots, '4Claspartate was metabolized predominantly to organic acids and, as other workers have shown, there was little labeling of asparagine (after 5 hours, 3.1% of metabolized label).Addition of the aminotransferase inhibitor aminooxyacetate decreased the flow of aspartate carbon to organic acids and enhanced (about 3-fold) the labeling of asparagine. The same treatment applied to darkened shoots resulted in a substantial conversion of I'4Claspartate to asparagine, over 10-fold greater than in control shoots (66% of metabolized label), suggesting that aspartate is the normal precursor of asparagine.Only traces of glutamine-dependent asparagine synthetase activity could be detected in pea leaf or root extracts; activity was not enhanced by sulfhydryl reagents, oxidizing conditions, or protease inhibitors. Asparagine synthetase is readily extracted from lupin cotyledons, but yield was greatly reduced by extraction in the presence of pea leaf tissue; pea leaf homogenates contained an inhibitor which produced over 95% inhibition of an asparagine synthetase preparation from lupin cotyledons. The inhibitor was heat stable, with a low molecular weight. Presence of an inhibitor may prevent detection of asparagine synthetase in pea extracts and in Asparagus, where a cyanide-dependent pathway has been proposed to account for asparagine synthesis: an inhibitor with similar properties was present in Asparagus shoot tissue.Asparagine is a major transport form of N in many plants, including legumes such as lupin (1) and pea (20). Asparagine synthetase (EC 6.4.5.4), catalyzing the glutamine-dependent amidation of aspartate, is active in germinating seedlings (9, 15, 17), but there is some question about the synthesis of asparagine in older, nongerminating tissue. '5N-Labeling patterns in pea leaves (2, 3) and spinach (21) for example in Asparagus (4). However, the enzymic properties and physiological concentrations of substrates have lead a number of workers to doubt that this can be a major route for asparagine synthesis (17-19). The principal argument in favor of the cyanide pathway is the inability to detect asparagine synthetase in Asparagus tissue (4).Although the level of asparagine decreases in mature pea leaves, presumably due to selective export (20), labeling data suggests that pea leaf tissue is capable of asparagine synthesis (2). This has been confirmed here by analysis of detached shoots; aspartate can be shown to be an asparagine precursor if a transaminase inhibitor is used to prevent the rapid equilibration of aspartate carbon with the organic acid pool. The presence of a potent asparagine synthetase inhibitor, detected in pea leaves and Asparagus shoots, may account for the inability to detect the enzyme in these and other tissues. MATERIALS AND METHODSPlants of Pisum sativum ...
In leaf pieces from nodulated soybean (Glycine max ILI Meff cv Maple Arrow) plants, '4Cqurea-dependent NH3 and "CO2 production in the dark showed an approximately 2:1 stoichiometry and was decreased to less than 11% of the control (12-19 micromoles NH3 per gram fresh weight per hour) in the presence of 50 millimolar acetohydroxamate, a urease inhibitor. NH3 and CO2 production from the utilization of 12-'4Cj allantoin also exhibited a 2:1 stoichiometry and was reduced to a similar extent by the presence of acetohydroxamate with a concomitant accumulation of urea which entirely accounted for the loss in NH3 production. The almost complete sensitivity of NH3 and CO2 production from allantoin and urea metabolism to acetohydroxamate, together with the observed stoichiometry, indicated a path of ureide assimilation (2.0 micromoles per gram leaf fresh weight per hour) via allantoate, ureidoglycolate, and glyoxylate with the production of two urea molecules yielding, in turn, four molecules of NH3 and two molecules of CO2.The ureides, allantoin and allantoate, constitute 60 to 80% of xylem-borne nitrogen in nitrogen-fixing tropical legumes (1,3,8,9,14,16) and apparently provide most of the nitrogen used in amino acid and protein synthesis during plant growth (1,2,16). Although the pathway of ureide assimilation in plant tissues has not been critically examined, five metabolic sequences, each giving rise to glyoxylate, NH3, and CO2 are possible based on bacterial metabolism ( Fig. 1; adapted from Refs.
Asparagine, a major transport compound, is metabolized in Pisum sativum by two enzymes, asparaginase (EC 3.5.1.1) and asparagine-pyruvate aminotransferase. The relative amount of the two enzymes varies between tissues. In developing seeds, there are very high levels of asparaginase but only trace amounts of the aminotransferase. Asparaginase is high in young leaves but falls rapidly during leaf growth; the aminotransferase remains high throughout development. Inhibitor studies with aminooxyacetate and methionine sulfoximine confirm that the aminotransferase is the main enzyme involved in asparagine utilisation in the leaf. Root tissue has low levels of asparaginase and only trace amounts of the aminotransferase. The asparaginase is potassium dependent, but is also partially activated by ammonium ions. The leaf aminotransferase has a lower K m for asparagine (4.5 mM) than the leaf asparaginase (8 mM). The seed asparaginase has a lower K m for asparagine (3 mM) than the leaf asparaginase.
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