Characterization of dicarboxylate stimulation of ammonia, glutamine, and 2-oxoglutarate-dependent o(2) evolution in isolated pea chloroplasts

Abstract

Intact isolated chloroplasts from pea (Pisum sativum) leaves carried out light-dependent (NH(3), 2-oxoglutarate) and (glutamine, 2-oxoglutarate)-dependent O(2) evolution at rates of 3.3 +/- 0.7 (n = 7) and 6.0 +/- 0.4 (n = 5) micromoles per milligram chlorophyll per hour, respectively. Malate stimulated the rate of (NH(3), 2-oxoglutarate)-dependent O(2) evolution 2.1 +/- 0.5 (n = 7)-fold in the absence of glutamine, and 3.3 +/- 0.4 (n = 11)-fold in the presence of glutamine. Malate also stimulated (glutamine, 2-oxoglutarate)-dependent O(2) evolution in the presence of high concentrations of glutamine. The affinity (K(1/2)) of (NH(3), glutamine, 2-oxoglutarate)-dependent O(2) evolution for 2-oxoglutarate was estimated at 200 to 250 micromolar in the absence of malate and 50 to 80 micromolar when malate (0.5 millimolar) was present. In contrast to malate and various other dicarboxylates, aspartate, glutarate, and glutamate did not stimulate (NH(3), glutamine, 2-oxoglutarate)-dependent O(2) evolution in isolated pea chloroplasts. Using both in vitro assays and reconstituted chloroplast systems, malate was shown to have no effect on the activities of either glutamine synthetase or glutamate synthase.The concentration of malate required for maximal stimulation of O(2) evolution was dependent on the concentration of 2-oxoglutarate present. However, the small extent of the competition between malate and 2-oxoglutarate for uptake was not consistent with that predicted by the current ;single carrier' model proposed for the uptake of dicarboxylates into chloroplasts.