Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061.
Plant Physiol. 1992 Sep;100(1):138-45. doi: 10.1104/pp.100.1.138.
Glutathione reductase was purified from pea seedlings using a procedure that included 2',5'-ADP Sepharose, fast protein liquid chromatography (FPLC)-anion exchange, and FPLC-hydrophobic interaction chromatography. The purified glutathione reductase was resolved into six isoforms by chromatofocusing. The isoform eluting with an isoelectric point of 4.9 accounted for 18% of the total activity. The five isoforms with isoelectric points between 4.1 and 4.8 accounted for 82% of the activity. Purified glutathione reductase from isolated, intact chloroplasts also resolved into six isoforms after chromatofocusing. The isoform eluting at pH 4.9 constituted a minor fraction of the total activity. By comparing the chromatofocusing profile of the seedling extract with that of the chloroplast extract, we inferred that the least acidic isoform was extraplastidic and that the five isoforms eluting from pH 4.1 to 4.8 were plastidic. Both the plastidic (five isoforms were pooled) and extraplastidic glutathione reductases had a native molecular mass of 114 kD. The plastidic glutathione reductase is a homodimer with a subunit molecular mass of 55 kD. Both glutathione reductases had optimum activity at pH 7.8. The K(m) for the oxidized form of glutathione (GSSG) was 56.0 and 33.8 mum for plastidic and extraplastidic glutathione reductase, respectively, at 25 degrees C. The K(m) for NADPH was 4.8 and 4.0 mum for plastidic and extraplastidic isoforms, respectively. Antiserum raised against the plastidic glutathione reductase recognized a 55-kD polypeptide from purified antigen on western blots. In addition to the 55-kD polypeptide, another 36-kD polypeptide appeared on western blots of leaf crude extracts and the purified extraplastidic isoform. The lower molecular mass polypeptide might represent GSSG-independent enzyme activity observed on activity-staining gels of crude extracts or a protein that has an epitope similar to that in glutathione reductase. Fumigation with 75 nL L(-1) ozone for 4 h on 2 consecutive days had no significant effect on glutathione reductase activity in peas (Pisum sativum L.). However, immunoblotting showed a greater level of glutathione reductase protein in extracts from ozone-fumigated plants compared with that in control plants at the time when the target concentration was first reached, approximately 40 min from the start of the fumigation, and 4 h on the first day of fumigation.
用 2'，5'-ADP 琼脂糖、快速蛋白质液相色谱（FPLC）-阴离子交换和 FPLC-疏水相互作用色谱法从豌豆幼苗中纯化谷胱甘肽还原酶。通过等电聚焦将纯化的谷胱甘肽还原酶分为六个同工型。等电点为 4.9 的同工型占总活性的 18%。等电点在 4.1 到 4.8 之间的五个同工型占活性的 82%。用分离的完整叶绿体进行纯化时，谷胱甘肽还原酶在用等电聚焦后也分为六个同工型。在 pH 4.9 洗脱的同工型构成总活性的一小部分。通过比较幼苗提取物和叶绿体提取物的等电聚焦图谱，我们推断出最酸性的同工型是质外体的，而 pH 4.1 到 4.8 洗脱的五个同工型是质体的。质体（五个同工型合并）和质外体谷胱甘肽还原酶的天然分子质量均为 114 kD。质体谷胱甘肽还原酶是一种具有 55 kD 亚基分子质量的同源二聚体。两种谷胱甘肽还原酶在 pH 7.8 时活性最佳。在 25°C 时，对于氧化型谷胱甘肽（GSSG），质体和质外体谷胱甘肽还原酶的 K(m)分别为 56.0 和 33.8 µm。对于 NADPH，质体和质外体同工型的 K(m)分别为 4.8 和 4.0 µm。针对质体谷胱甘肽还原酶的抗血清在 Western 印迹中从纯化抗原上识别出 55 kD 的多肽。除了 55 kD 的多肽外，在叶片粗提取物和纯化的质外体同工型的 Western 印迹中还出现了另一种 36 kD 的多肽。较低的分子质量多肽可能代表粗提取物的活性染色凝胶上观察到的 GSSG 非依赖性酶活性，或者是一种具有与谷胱甘肽还原酶相似表位的蛋白质。在连续两天每天用 75 nL L(-1) 臭氧熏蒸 4 小时对豌豆（Pisum sativum L.）的谷胱甘肽还原酶活性没有显著影响。然而，免疫印迹显示，与对照植物相比，臭氧熏蒸植物的提取物中谷胱甘肽还原酶蛋白水平在目标浓度首次达到时（大约从熏蒸开始 40 分钟）和熏蒸的第一天 4 小时更高。
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