Showing papers by "Christine H. Foyer published in 1978"

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography.

Abstract: Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP+, but GSH-dependent NADP+ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn2+. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 μM and that for NADPH was about 3 μM. NADP+ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP+-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H2O2. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.

Subcellular Localisation and Identification of Superoxide Dismutase in the Leaves of Higher Plants

Abstract: 1 The subcellular location of superoxide dismutase in the leaves of spinach and other C3 plants has been investigated. 2 Most activity appeared to be located within chloroplasts. These organelles contain a cyanidesensitive (copper-zinc) superoxide dismutase, most of which is located in the stroma although some is bound to the thylakoids. 3 Intact chloroplast fractions also contain a cyanide-insensitive (manganese) superoxide dismutase, but this activity is located on the outside of the outside of the chloroplasts and may be adsorbed onto them during isolation. 4 Leaf mitochondrial fractions contain only a small percentage of total leaf superoxide dismutase activity, but there is more than can be accounted for by contamination with chloroplasts. 5 Mitochondria contain both a cyanide-sensitive dismutase, apparently located in the intermembrane space, and a cyanide-insensitive activity, apparently located in the matrix. 6 The microsomal fraction contains no superoxide dismutase activity.