Chloroplast glutathione reductase: Purification and properties

Glutathione reductase was partially purified from isolated pea chloroplasts (Pisum sativum L. cv. Progress #9). A 1600‐fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)−1 min−1. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The Km for oxidized glutathione was 11 μM and the Km for NADPH was 1.7 μM. Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 mM. The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H2O2 by reducing dehydroascorbate for ascorbate‐linked reduction of H2O2. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.