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

在高等植物中，半胱氨酸在细胞中积累的量很少，而谷胱甘肽（GSH）、三肽和半胱氨酸残基反而积累起来。谷胱甘肽中半胱氨酸的再循环需要谷胱甘肽的分解代谢。本研究对高等植物中γ-谷氨酰转移酶（γ-GT）和二肽酶（GT）进行了分析。在高等植物中首次从萝卜子叶中纯化出可溶性γ-GT，并对其性质进行了澄清。同时克隆了可溶性γ-GT的cDNA。从酶蛋白和cDNA的分析结果来看，γ-GT的转录物是一个单一的，因此翻译后的蛋白质可能是一个单一的多肽。然后将单个多肽切割成大多肽和小多肽，这些多肽是动物和大肠杆菌中成熟的γ-GT酶蛋白的组成部分。除了可溶性γ-GT外，我们还证实了γ-GT与萝卜子叶细胞壁的结合。我们还证实了γ-GT在成熟叶片和根中存在可溶性和结合型两种类型。因此，我们认为所有植物组织都有两种类型的γ-GT。γ-GT的结合形式也被纯化到均匀性。成熟的结合γ-GT由一个多肽组成。从而可以区分出可溶性γ-GT和粘附性γ-GT。两种纯化的γ-GT均以谷胱甘肽为底物，其他性质基本相似。3个DPs中有1个为首次从萝卜子叶中分离得到。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酸甘氨酸作为底物。本研究结果提出了一种新的假说，即谷胱甘肽的分解代谢发生在细胞质和外质体中。

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.