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Stimulation of Ammonia and 2-Oxoglutarate-Dependent O₂ Evolution in Isolated Chloroplasts by Dicarboxylates and the Role of the Chloroplast in Photorespiratory Nitrogen Recycling

Department of Environmental Biology, Research School of Biological Sciences, Australian National University, Canberra City, A.C.T. 2601, Australia

Intact chloroplasts isolated from spinach (Spinacia oleracea L.) leaves showed a light-dependent O₂ evolution (5.5 ± 0.75 micromoles per milligram chlorophyll per hour) when supplied with ammonia and 2-oxoglutarate. This (ammonia, 2-oxoglutarate)-dependent O₂ evolution was stimulated 2- to 4-fold by the dicarboxylates, malate, succinate, fumarate, glutarate, and L-tartarate. Evolution of O₂ in the presence of malate was dependent on the presence of both 2-oxoglutarate and NH₄Cl; malate with only either 2-oxoglutarate and NH₄Cl alone did not support O₂ evolution. Furthermore, in the presence of malate, the amount of O₂ evolved was solely dependent on the amount of NH₄Cl or 2-oxoglutarate added and malate did not affect the ratio of O₂ evolved to NH₄Cl or 2-oxoglutarate consumed. Studies with inhibitors (2-(3,4-dichlorophenyl)-1,1-dimethyl urea, methionine sulfoximine, and azaserine) indicated that the above activity was directly linked to glutamine synthetase and glutamate synthase activity in the chloroplast and was not caused by the metabolism of malate. The V_max/2 of (ammonia, 2-oxoglutarate)-dependent O₂ evolution was reached at 32 micromolar NH₄Cl and 6 millimolar (approximately) 2-oxoglutarate in the absence of malate, and at 22 micromolar NH₄Cl and 73 micromolar 2-oxoglutarate when malate (3 millimolar) was present.

Intact chloroplasts isolated from pea (Pisum sativum) leaves also showed a stimulation of (ammonia, 2-oxoglutarate)-dependent O₂ evolution by malate. However glutamine was required for this activity even though glutamine with only either NH₄Cl or 2-oxoglutarate did not respond to malate stimulation.

The measured rates of (ammonia, 2-oxoglutarate)-dependent O₂ evolution in isolated spinach chloroplasts in the presence of malate were about 19.5 ± 4.5 micromoles O₂ evolved per milligram chlorophyll per hour. This is adequate to sustain photorespiratory NH₃ recycling and the refixation of NH₃ arising from NO₃ under ambient conditions in the light. The role of the chloroplast in photorespiratory NH₃ recycling and the nature of the associated transport of 2-oxoglutarate into the chloroplast is discussed.

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				A. Weber and U.-I. Flugge Interaction of cytosolic and plastidic nitrogen metabolism in plants J. Exp. Bot., April 15, 2002; 53(370): 865 - 874. [Abstract] [Full Text] [PDF]