Biochemistry and molecular biology of glutathione in higher plants

对高等植物光合作用组织中谷胱甘肽(GSH)和高谷胱甘肽(HGSH)的分布进行了研究。采用固相法合成了真品hGSH。大豆植株只含有hGSH，而不含GSH，大豆叶片中的hGSH合成酶与萝卜中的GSH合成酶是不同的。HGSH合成酶的底物β-丙氨酸在大豆叶片中检测到，而在萝卜叶片中未检测到。然后，我们尝试从萝卜子叶中分离纯化谷氨酰半胱氨酸合成酶(GluCys Synthetase，GluCys Synthetase)和谷胱甘肽合成酶(GSH Synthetase，GSH)，从大豆叶片中分离纯化hGSH合成酶。我们可以用硫酸铵分级沉淀和各种库曼层析对这些酶进行部分纯化。GSH合成酶是一种相对分子质量为60,000的单体蛋白。这些酶的最适pH为8，反应依赖于ATP和镁离子。构建了大豆叶片的cDNA文库，用于分离编码谷氨酸合成酶和hGSH合成酶的基因。以拟南芥gsh1和gsh2基因片段为探针，可以从文库中分离出多个克隆。GluCys合成酶的编码基因被分离出来，不幸的是没有全长。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个克隆不是编码hGSH合成酶的基因，因为分离的一个克隆与拟南芥gsh2的序列没有同源性，我们分离的3个克隆之间也没有同源性。我们没有试图分离编码hGSH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.