|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1998 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1997 : ¥2,000,000 (Direct Cost : ¥2,000,000)
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.