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

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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.