Analysis of transport system for glutathione and glutathione conjugates as a factor of cell defense and drug metabolism.

分析谷胱甘肽和谷胱甘肽缀合物的转运系统作为细胞防御和药物代谢的因素。

• 批准号: 02671030
• 负责人: KONDO Takahito
• 金额: $ 0.32万
• 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: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02671030/
• 关键词: Erythrocyte; Membrane Transport; Glutathione; Glutathione S-Conjugate; ATPase; グルタチオン抱合体

We have purified and characterized a novel ATPase in human erytlirocyLes which is stimulated by glutathione S-conjugate. Erythrocyte plasma membranes were prepared and solubilized into Triton-X 100, and applied on an affinity chromatography of S-hexylglutathione-Sepharose 6B. An protein fraction eluted by approximately 1.5 mM S-hexylglutathione was found to show ATPase activity stimulated by S-(2, 4-dinitrophenyl)glutathione(GS-DNP). An apparte Km of the enzyme was 38 mu H for GS-DNP, and 140 mu H for ATP. This enzyme protein reconstituted into phospholipid vesicles showed both GS-DUP-stimulated mg^<2+> -ATPase and tile GS-DNP transport activity. T he enzyme activity was stimulated by other glutatlilone S-co-niugates, and not inhibited by vanadate nor EGTA, suggesting that this is different from Na^+, K^+ -ATPase or Ca^<2+> -ATPase. Immunological study using a specific antisera against this enzyme showed that this enzyme presents in membranes of platelets, leukocytes, hepatocytes, as well as erythrocytes. These results suggest that GS-DNP-stimulated ATPase functions in the active transport of glutathione S-conjugates and cell defense system against oxidative stress.

我们纯化并鉴定了人红细胞中一种新的ATP酶，它能被谷胱甘肽S-结合物激活。制备红细胞质膜并溶解于Triton-X 100中，并应用于S-己基谷胱甘肽-Sepharose 6 B亲和层析。发现由约1.5mM S-己基谷胱甘肽洗脱的蛋白质级分显示由S-（2，4-二硝基苯基）谷胱甘肽（GS-DNP）刺激的ATP酶活性。该酶对GS-DNP的Km值为38 μ H，对ATP的Km值为140 μ H。这种重组到磷脂囊泡中的酶蛋白显示出GS-DUP刺激的mg^<2+> -ATP酶和GS-DNP转运活性。该酶活性可被其他谷氨酰胺S-共刺激物激活，而不被钒酸盐或EGTA抑制，表明该酶不同于Na^+，K^+ -ATP酶或Ca^<2+> -ATP酶。使用针对该酶的特异性抗血清的免疫学研究表明，该酶存在于血小板、白细胞、肝细胞以及红细胞的膜中。这些结果表明GS-DNP刺激的ATP酶在谷胱甘肽S-结合物的主动转运和细胞防御氧化应激系统中起作用。

项目成果

T.Kondo,: "A simple enzymeーlinked immunoassay of human corbonic anbydrase 1 for the study of developing erythroid cells." Clin.Chim.Acta,. 191. 169-174 (1990)

T.Kondo，：“用于研究发育红系细胞的人碳酸酐酶 1 的简单酶联免疫测定。” 191. 169-174 (1990)

T.Kondo: "Active Transprt of glutathiore S-conjugate in Human Erythrocytes"

T.Kondo：“谷胱甘肽 S-缀合物在人红细胞中的活性转运”

S.Tagami: "Effect of Insulin on the Impaired Antioxidant Actiuities in Aortic Endothelical Cells from Diaberic Rabbits"

S.Tagami：“胰岛素对糖尿病兔主动脉内皮细胞抗氧化活性受损的影响”

S. Tagami: "Effect of Insulin on the lmpaired Antioxidant Actiuities in Aortic Endothelial Cells from Diabetic Rabbits" Metabolism.

S. Tagami：“胰岛素对糖尿病兔主动脉内皮细胞受损抗氧化活性的影响”代谢。

T.Kondo: "Active Transport of Glutathione Sーconjugate Highly Responses to Heat Shock in K562 Erythroid cells." Proc.Natl.Acad.Sci.USA.

T.Kondo：“谷胱甘肽 S 结合物的主动转运对 K562 红细胞中的热休克有高度反应。”Proc.Natl.Acad.Sci.USA。