The photorespiratory hydrogen shuttle. Synthesis of phthalonic acid and its use in the characterization of the malate/aspartate shuttle in pea (Pisum sativum) leaf mitochondria.
A method is presented for the preparation of pure phthalonic acid (PTA) in high yields. This PTA was used to determine the capacity of the malate/aspartate shuttle in pea (Pisum sativum) leaf mitochondria. The inhibition of glycine-dependent O2 uptake in the combined presence of 5 mM-aspartate and 5 mM-2-oxoglutarate (2-OG) was decreased by 55 +/- 22% (n = 13) in washed and 50 +/- 2% (n = 11) in purified mitochondria by 0.23 mM-PTA. This concentration of PTA had no effect on the oxidation of 5 mM-2-OG, suggesting that part of the observed inhibition of O2 uptake in the presence of aspartate and 2-OG was due to the production of oxaloacetate (OAA) by aspartate aminotransferase external to the mitochondrial inner membrane. Levels of external aspartate aminotransferase were estimated to be 24 +/- 1% (n = 4) and 13 +/- 1% (n = 4) of the total mitochondrial activity in washed and purified mitochondria respectively. Malate/aspartate-shuttle activity was estimated directly by measuring rates of malate efflux from isolated mitochondria and was found to match estimates of shuttle activity based on the PTA-insensitive inhibition of O2 uptake. Comparisons of malate/aspartate- and malate/OAA-shuttle activities indicated potentially similar rates of NADH export from pea leaf mitochondria under conditions in vivo. These extrapolated to whole-tissue rates of 5-11 mumol of NADH.h-1.mg of chlorophyll-1. The potential role of the malate/aspartate shuttle in the support of photorespiratory glycine oxidation in leaf tissue is discussed.
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Department of Botany, University of Adelaide, Australia.
Dimitriadis
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Ward
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Wiskich
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JT
D001219:Aspartate Aminotransferases / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism*; D007887:Fabaceae / Q000187:drug effects / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008293:Malates / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption / Q000187:drug effects; D010946:Plants, Medicinal
D007656:Ketoglutaric Acids; D008293:Malates; D001224:Aspartic Acid; C030298:malic acid; D001219:Aspartate Aminotransferases; D005998:Glycine