Department of Biochemistry, University of London King's College, Strand, WC2R 2LS, London, UK.
Planta. 1978 Jan;139(1):9-17. doi: 10.1007/BF00390803.
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 Zn(2+). 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.
谷胱甘肽还原酶（EC 1.6.4.2）通过 ADP-Sepharose 亲和层析从菠菜（Spinacia oleracea L.）叶片中纯化得到。纯化后的酶具有 246 个酶单位/毫克蛋白的比活性，并且通过天然和 SDS-凝胶的聚丙烯酰胺凝胶电泳标准呈现均一性。该酶的分子量为 145,000，由两个大小相似的亚基组成。菠菜谷胱甘肽还原酶的 pH 最适值为 8.5-9.0，这与其在叶绿体基质中的功能有关。它对氧化型谷胱甘肽（GSSG）具有特异性，但以 NADH 作为电子供体时活性较低。NADH 依赖的 GSSG 还原的 pH 最适值低于 NADPH 依赖的还原的 pH 最适值。该酶对还原型谷胱甘肽（GSH）和 NADP(+) 的亲和力较低，但添加二硫苏糖醇可刺激 GSH 依赖的 NADP(+)还原。谷胱甘肽还原酶在与巯基反应的试剂或重金属离子（如 Zn(2+)）孵育时会受到抑制。GSSG 可保护酶免受抑制，但 NADPH 则不能。酶与 NADPH 预孵育会降低其活性，因此进行了动力学研究，其中通过添加 NADPH 或酶来启动反应。GSSG 的 Km 值约为 200 μM，NADPH 的 Km 值约为 3 μM。NADP(+) 以竞争性方式抑制 GSSG 还原方向的酶，对 NADPH 的抑制作用具有竞争性，对 GSSG 的抑制作用具有非竞争性。相比之下，GSH 以非竞争性方式抑制 NADPH 和 GSSG。照光的叶绿体或在黑暗中保存的叶绿体均含有等量的谷胱甘肽还原酶。该酶的动力学特性（如上所述）表明，在光照和黑暗条件下，叶绿体中的 GSH/GSSG 比值将非常高。这一预测得到了实验的证实。GSH 或 GSSG 均不在光诱导的叶绿体果糖二磷酸酶或 NADP(+)-甘油醛-3-磷酸脱氢酶的激活中发挥作用。我们认为，GSH 有助于稳定叶绿体酶，并且可能在去除 H2O2 方面发挥作用。葡萄糖-6-磷酸脱氢酶活性可能在黑暗中在叶绿体中是必需的，以便为谷胱甘肽还原酶提供 NADPH。
