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

Abstract

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.