Biochemistry and molecular biology of glutathione in higher plants

高等植物谷胱甘肽的生物化学和分子生物学

基本信息

批准号：
09660062
负责人：
SEKIYA Jiro
金额：
$ 2.05万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

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.

检查了较高植物的光合组织中谷胱甘肽（GSH）和均谷胱甘肽（HGSH）的分布。真实的HGSH是通过固态方法合成的。大豆植物仅包含HGSH但不包含GSH，而大豆叶中的HGSH合成酶则与萝卜中的GSH合成酶区分开。 β-丙氨酸是HGSH合成酶的底物，在大豆叶中检测到，但在萝卜叶中未检测到。然后，我们试图从萝卜共叶叶片中纯化γ-谷氨酰胺半胱氨酸合成酶（Glucys合成酶）和GSH合成酶，以及来自大豆叶的HGSH合成酶。我们可以使用硫酸铵分馏和各种COUMN色谱法部分纯化这些酶。 GSH合成酶表示为单体蛋白，MW为60,000。这些酶的最佳pH值为Ca 8，反应是ATP和Mg^2+依赖性的反应。构建了大豆叶的cDNA库，以分离编码Glucys合成酶和HGSH合成酶的cDNA。我们可以使用拟南芥GSHL和GSH2 cDNA片段从文库中分离几个克隆作为探针。不幸的是，分离出glucys合成酶的cDNA编码，不完整。获得的序列与A.thaliana和番茄的序列高度同源。三个使用A.thaliana gsh2作为探针的克隆得出的结论不是编码HGSH合成酶的cDNA，因为在分离的一个和A.thaliana gsh2的序列之间找不到同源性，也没有在我们分离的三个克隆之间找到同源性。我们正在尝试分离编码HGSH合成酶的cDNA。