Biochemistry and molecular biology of glutathione in higher plants

研究了高等植物光合组织中谷胱甘肽（GSH）和同型谷胱甘肽（hGSH）的分布。采用固态法合成了真正的高谷胱甘肽。大豆植株只含有高谷胱甘肽而不含谷胱甘肽，大豆叶片中的高谷胱甘肽合成酶与萝卜中的高谷胱甘肽合成酶有明显区别。在大豆叶片中检测到高谷胱甘肽合成酶的底物-丙氨酸，而在萝卜叶片中未检测到。然后，我们尝试从萝卜子叶中纯化γ -谷氨酰半胱氨酸合成酶（GluCys合成酶）和谷胱甘肽合成酶，从大豆叶片中纯化高谷胱甘肽合成酶。利用硫酸铵分馏法和各种柱层析法可以对这些酶进行部分纯化。谷胱甘肽合成酶是一种分子量为60000的单体蛋白。这些酶的最佳pH为ca 8，反应依赖于ATP和Mg^2+。构建大豆叶片cDNA文库，分离编码葡聚糖合成酶和高谷胱甘肽合成酶的cDNA。以拟南芥gshl和gsh2 cDNA片段为探针，从文库中分离出多个克隆。编码葡聚糖合成酶的cDNA被分离出来，可惜长度不完整。获得的序列与拟南芥和番茄的序列高度同源。以拟南芥gsh2为探针的3个克隆均与拟南芥gsh2序列无同源性，且3个克隆间也无同源性，结果表明该克隆并非编码hGSH合成酶的cDNA。我们没有尝试分离编码高谷胱甘肽合成酶的cDNA。

Distribution of glutathione (GSH) and homoglutathione (hGSH) in the photosynthetic tissues of higher plants were examined. Authentic hGSH was synthesized by the solid state method. Soybean plants exclusively contained hGSH but not GSH, and hGSH synthetase in soybean leaves was distinguished from GSH synthetase in radish. beta-Alanine, substrate for hGSH synthetase, was detected in soybean leaves but not in radish leaves. Then, we attempted to purify gamma-glutamylcysteine synthetase (GluCys synthetase) and GSH synthetase from radish cotyledons, and hGSH synthetase from soybean leaves. We could partially purify these enzymes using ammonium sulfate fractionation and various kinds of coumn chromatographies. GSH synthetase was indicated to be a monomeric protein with a MW of 60,000. Optimal pH for these enzymes was ca 8 and the reaction was an ATP and Mg^2+ dependent one. The cDNA library of soybean leaves was constructed to isolate cDNAs encoding GluCys synthetase and hGSH synthetase. We could isolate several clones from the library using Arabidopsis thaliana gshl and gsh2 cDNA fragments as probes. cDNA encoding for GluCys synthetase was isolated, unfortunately not complete length. The sequence obtained was highly homologous to A.thaliana and tomato ones. Three clones using A.thaliana gsh2 as a probe, were concluded not to be a cDNA encoding hGSH synthetase, since homology was not found between the sequences of isolated one and A.thaliana gsh2, and also between three clones we isolated. We are noy trying to isolate cDNA encoding hGSH synthetase.