Studies on Catabolism of Glutathione and Recycle System of Cysteine in Higher Plants

高等植物谷胱甘肽分解代谢及半胱氨酸回收系统的研究

基本信息

批准号：
12660059
负责人：
SEKIYA Jiro
金额：
$ 2.3万
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：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

In higher plants, the amount of cysteine accumulating in cells is small, and glutathione (GSH), tripeptide, with cysteine residue accumulates instead. For recycling of cysteine in GSH, catabolic metabolism of GSH is required. In this research, we analyzed γ-glutamyltransferase (γ-GT) and dipeptidase (GT) from higher plants. The soluble type of γ-GT was purified from radish cotyledons for the first time with higher plants and its properties were clarified. cDNA endocing soluble γ-GT was also cloned. Based on the results obtained from analysis of the enzyme protein and cDNA, the transcript for γ-GT was a single one and consequently the proteins immediately after translation might be a single polypeptide. Then a single polypeptide was cleaved into large and small polypeptides, which are constituents of the mature γ-GT enzyme protein, as seen in animals and E. coli. In additon to the soluble γ-GT, we confirmed occurrence of γ-GT bound to cell wall of radish cotyledons. We also confirmed occurrence of two types, soluble and bound, of γ-GT in mature leaves and roots. Thus we concluded all plant tissues have two types of γ-GT. The bound form of γ-GT was also purified to the homogeniety. The mature form of bound γ-GT was composed of a single polypeptide. Thus we can distinguish soluble and bund γ-GT each other. Both purified γ-GT utilized GSH as a good substrate and other properties were mostly similar. One of three DPs was purified for the first time from radish cotyledons to the homogeneity. Mw was ca 300,000, oligomer of identical subunits (55,000). DP was assumed to be metal enzyme specific to cysteinylglycine as a substrate. The results obtained here lead to a new hypothesis that catabolism of GSH occurred both in apoplast and cytoplasm.

在高等植物中，细胞中积累的半胱氨酸量很少，而是带有半胱氨酸残基的谷胱甘肽（GSH）、三肽积累，为了回收GSH中的半胱氨酸，需要GSH的分解代谢。研究中，我们分析了高等植物中的γ-谷氨酰转移酶（γ-GT）和二肽酶（GT），首次从高等植物萝卜子叶中纯化出可溶型γ-GT。还阐明了编码可溶性γ-GT的cDNA。根据酶蛋白和cDNA的分析结果，γ-GT的转录物是单一的，因此翻译后的蛋白可能是直接的。然后，单个多肽被切割成大多肽和小多肽，它们是成熟γ-GT酶蛋白的组成部分，如在动物和大肠杆菌中所见。 γ-GT，我们证实了萝卜子叶细胞壁上存在结合的γ-GT。我们还证实了成熟叶和根中存在两种类型的γ-GT，因此我们得出结论，所有植物组织都具有两种类型。 γ-GT 的结合形式也被纯化至同质性。结合的γ-GT 的成熟形式由单一多肽组成，因此我们可以区分可溶性和束状γ-GT 两者。 γ-GT利用GSH作为良好的底物，并且其他性质大多相似，这是首次从萝卜子叶中纯化出均质性为约300,000的相同亚基的寡聚物(55,000)。是以半胱氨酰甘氨酸为底物的金属酶。这里获得的结果导致了一个新的假设：GSH 的分解代谢发生在质外体中。和细胞质。