A NOVEL ATPase OF ERYTHROYCTE MEMBRANES IN RELATION TO TRANSPORT OF GLUTATHIONE AND GLUTATHIONE CONJUGATES.

一种与谷胱甘肽和谷胱甘肽缀合物运输相关的新型红细胞膜ATP酶。

• 批准号: 63570127
• 负责人: KONDO Takahito
• 金额: $ 1.41万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63570127/
• 关键词: ERYTHROYCTES; MEMBRANE TRANSPORT; GLUTATHIONE; GLUTATHIONE S-CONJUGATE; ATPase; ATP use

We have purified a novel ATPase in human erythroyctes which is stimulated by oxidized form of glutathione(GSSG). Purification of the enzymes were performed to apparent homogeneity involving procedures of affinity chromatography on S-hexylglutathione-Sepharose 6B. Further characterization of the enzyme was performed using enzymatic, biochemical, physiological and immunological methods. The enzyme has two independent isozymes(type I and type II) which are indistinguishable by gel filtration, with an apparent molecular weight of 150,000. However they showed a different affinity for GSSG and ATP. We prepared polyclonal antibody using rabbits specific for each isozyme. These enzymes were immunologically cross reactive. The enzyme reconstituted into phospholipid vesicles showed both GSSG- stimulated ATPase activity and active GSSG transport activity. The GSSG-ATpase activity and the GSSG transport activity of type I isozyme were 25 nmol/mg protein/min, and 25 nmol pi released/mg protein/min, and those of type II isozyme were 248 nmol/mg protein/min and 133 nmol pi released/mg protein/min, respectively. These results indicate that these enzymes function in the active transport of GSSG and glutathione conjugates.

我们从人红细胞中纯化了一种新的ATP酶，它能被氧化型谷胱甘肽（GSSG）激活。酶的纯化进行到表观均一性，涉及在S-己基谷胱甘肽-Sepharose 6 B上的亲和层析程序。使用酶、生物化学、生理学和免疫学方法对酶进行进一步表征。该酶有两种独立的同工酶（I型和II型），通过凝胶过滤无法区分，表观分子量为150，000。然而，它们对GSSG和ATP表现出不同的亲和力。我们用兔制备了特异性的多克隆抗体。这些酶具有免疫交叉反应性。重组到磷脂囊泡中的酶显示出GSSG刺激的ATP酶活性和主动GSSG转运活性。Ⅰ型同工酶的GSSG-ATP酶活性和GSSG转运活性分别为25 nmol/mg蛋白/min和25 nmol pi释放/mg蛋白/min; Ⅱ型同工酶的GSSG-ATP酶活性和GSSG转运活性分别为248 nmol/mg蛋白/min和133 nmol pi释放/mg蛋白/min。这些结果表明，这些酶在GSSG和谷胱甘肽结合物的主动转运中起作用。

项目成果

近藤宇史: 蛋白質核酸酵素. 33. 1466-1473 (1988)

Ushi Kondo：蛋白质核酸酶 33. 1466-1473 (1988)

Takahito,Kondo: "Glutathione Centennial" Academic press, (1989)

近藤隆仁：“谷胱甘肽百年”学术出版社，（1989）