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

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12660059/
• 关键词: Glutathine; Glutathione catabolism; Recycle of cysteine; γ-Glutamyltransferase; Dipeptidase; Cysteinylglycine; Radish cotyledons; γ-グルタミルトランスフェラーゼ; ジペプチダーゼ

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，半胱氨酸在细胞中积累的量很少，而具有半胱氨酸残基的三肽谷胱甘肽（GSH）则积累。对于GSH中半胱氨酸的再循环，需要GSH的分解代谢。本研究分析了高等植物中的γ-谷氨酰转移酶（γ-GT）和二肽酶（GT）。首次从萝卜子叶中分离纯化出可溶性γ-GT，并对其性质进行了研究。还克隆了可溶性γ-GT的cDNA。根据对酶蛋白和cDNA的分析结果，γ-GT的转录本是单一的，因此翻译后的蛋白质可能是单一的多肽。然后，单个多肽被切割成大的和小的多肽，它们是成熟γ-GT酶蛋白的组成部分，如在动物和E.杆菌除可溶性γ-GT外，我们还证实了萝卜子叶细胞壁结合γ-GT的存在。我们还证实了两种类型的γ-GT的存在，可溶性和结合，在成熟的叶片和根。因此，我们认为所有植物组织都含有两种类型的γ-GT，结合型γ-GT也得到了纯化。结合γ-GT的成熟形式由单一多肽组成。因此，可以将可溶性和结合型γ-GT区别开来。两种纯化的γ-GT都利用GSH作为良好的底物，其他性质基本相似。首次从萝卜子叶中分离纯化了三种DPs中的一种，并得到了纯度较高的DPs。Mw约为300，000，相同亚基的低聚物（55，000）。DP被认为是金属酶特异性半胱氨酰甘氨酸作为底物。本文的结果提出了一个新的假说，即GSH的催化作用既发生在质外体中，也发生在细胞质中。