Light-dependent Reduction of Oxidized Glutathione by Ruptured Chloroplasts

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Abstract
Crude extracts of pea shoots (Pisum sativum) catalyzed oxidized glutathione (GSSG)-dependent oxidation of NADPH which was attributed to NADPH-specific glutathione reductase [EC 1.6.4.2]. The pH optimum was 8 and the Km values for GSSG and NADPH were 23 .mu.M and 4.9 .mu.M, 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 O2 evolution (mean of 10 determinations, 10.4 .mu.mol per mg chlorophyll/h, SD 1.4) with the concomitant production of GSH. The molar ratio of GSH produced to O2 evolved was 3.8 and the highest ratios for O2 evolved to GSSG added were 0.46 and 0.44. The Km value for GSSG was 26 .mu.M. 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 O2 evolution in the presence of substrate amounts of hydrogen peroxide, dehydroascorbate, L-cystine, sulfite, or sulfate.
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