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

(1)在缺乏GGT的大肠杆菌突变株中自克隆γ -谷氨酰丙转肽酶（GGT）基因（GGT），克隆菌株作为野生菌株低温依赖表达GGT。Western和Northern印迹结果表明，表达控制不在加工阶段，而在转录阶段。(2)将携带ggt的质粒转染到大肠杆菌中。转化物分泌GGT， GGT通过简单的两步法纯化。(3)研究了制备适合x射线分析的GGT晶体的条件，确定了获得大晶体的条件。(4) GGT中Arg 571向Ala的突变导致宿主细胞周围质间隙中无活性的前GGT的积累。(5)从酵母Issatchenkia orientalis中克隆了GILitathione S-transferase (GST) cDNA，并在大肠杆菌中进行了高表达。测定了GST CDNA的核苷酸序列，并推导了其氨基酸序列。GST的诱导形成在转录阶段受到控制。(6)发现谷胱甘肽缀合物在东方蓟细胞中通过羧肽酶样酶的作用被代谢为半胱氨酸缀合物。(7)将GST cDNA亚克隆到穿梭载体上，并将重组质粒导入酿酒酵母细胞。转化后的GST活性比宿主细胞高15倍。(8)通过细胞分离和免疫电镜分析，确定了GST在东方蓟马细胞表面的定位。

(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.