Light-dependent Reduction of Oxidized Glutathione by Ruptured Chloroplasts

Abstract

Crude extracts of pea shoots (Pisum sativum) catalyzed oxidized glutathione (GSSG)-dependent oxidation of NADPH which was attributed to NADPH-specific glutathione reductase. The pH optimum was 8 and the K(m) values for GSSG and NADPH were 23 mum and 4.9 mum, respectively. Reduced glutathione (GSH) inhibited the reaction. Crude extracts also catalyzed NADPH-dependent reduction of GSSG; the ratio of the rate of NADPH oxidized to GSH formed was 0.49. NADH and various substituted mono- and disulfides would not substitute for NADPH and GSSG respectively. Per mg of chlorophyll, enzyme activity of isolated chloroplasts was 69% of the activity of crude extracts.Illuminated sonicated pea chloroplasts, in the presence of catalytic amounts of NADPH, catalyzed GSSG-dependent O(2) evolution (mean of 10 determinations, 10.4 mumol per mg chlorophyll per hour, sd 1.4) with the concomitant production of GSH. The molar ratio of GSH produced to O(2) evolved was 3.8 and the highest ratios for O(2) evolved to GSSG added were 0.46 and 0.44. The K(m) value for GSSG was 26 mum. GSH inhibited the reaction. The reaction was attributed to photosynthetically coupled glutathione reductase.Ruptured chloroplasts, in the presence of catalytic amounts of GSSG and NADPH, did not catalyze sustained O(2) evolution in the presence of substrate amounts of hydrogen peroxide, dehydroascorbate, l-cystine, sulfite, or sulfate.