Fundamental and applied research in microbial glutathione metabolizing enzymes and their genes

(1)将γ-谷氨酰塔兰肽酶（GGT）基因（ggt）自克隆到无GGT的大肠杆菌中。大肠杆菌突变株和该克隆菌株表现出低温依赖性的GGT表达的野生型菌株。Western和北方印迹分析结果表明，该基因的表达控制在转录水平，而不是加工水平。(2)将携带ggt基因的质粒转化大肠杆菌托拉菌株。杆菌通过简单的两步法纯化了γ-谷氨酰转肽酶。(3)研究了适合于X射线分析的GGT晶体的制备条件，并建立了获得大晶体的条件。(4)GGT中Arg 571突变为Ala导致无活性的pro-GGT在宿主细胞的周质空间中积累。(5)从东方伊萨酵母（Issatchenkia orientalis）中克隆了谷胱甘肽S-转移酶（GST）cDNA，并将其克隆到E.大肠杆菌中表达GST。测定了GST cDNA的核苷酸序列，并推导了其氨基酸序列。GST的诱导形成被阐明为在转录步骤中被控制。(6)谷胱甘肽结合物在I.东方细胞(7)将GST cDNA亚克隆到穿梭载体中，并将重组质粒转导到酿酒酵母细胞中。GST活性是宿主细胞的15倍。(8)GST在I.通过细胞分级分离和免疫电镜分析来指示orientalis细胞。

(1) gamma-Glutamyltaranspeptidase (GGT) gene (ggt) was self-cloned in GGT-less E. coli mutant and this cloned strain showed low-temperature dependent expression of GGT as a wild strain. The results from Western and Northern blotting indicated that the expression controlled not at the processing but at the transcriptional step.(2) The plasmid carrying ggt was transduced into a tola strain of E. coli. The transformant excreted GGT and the GGT was purified by a simple two-step method.(3) The conditions were investigated for the preparation of crystals of GGT suitable for the x-ray analysis and the conditions to obtain large crystals were established.(4) The mutation of Arg 571 to Ala in GGT caused accumulation of inactive pro-GGT in the periplasmic space of the host cell.(5) GILitathione S-transferase (GST) cDNA from a yeast Issatchenkia orientalis was cloned into an E. coli strain and this transformant highly expressed GST. The nucleotide sequence of GST CDNA was determined and the amino acid sequence was deduced. The inducible formation of GST was elucidated to be controlled at the transcriptional step.(6) The glutathione-conjugate was found to be metabolized to eysteine-conjugate by the action of carboxypeptidase-like enzyme in I. orientalis cells.(7) GST cDNA was subcloned to a shuttle-vector and the reconstituted plasmid was transduced into Saccharomyces cerevisiae cells. The transformant showed 15 times higher GST activity than the host cells.(8) The localization of GST at the surface of I. orientalis cells were indicated by cell-fractionation and immune-electromicroscope analysis.