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

我们纯化并鉴定了一种在人红细胞环中受s -谷胱甘肽偶联物刺激的新型三磷酸腺苷酶。制备红细胞膜，并将其溶解于triton - x100中，应用于s -己基谷胱甘肽- sepharose 6B亲和层析。用约1.5 mM S-己基谷胱甘肽洗脱的蛋白质片段显示S-（2,4 -二硝基苯）谷胱甘肽（GS-DNP）刺激的atp酶活性。GS-DNP酶的表观Km为38 μ H， ATP酶的表观Km为140 μ H。该酶蛋白重组成磷脂囊泡，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胱甘肽s -缀合物刺激，而不受钒酸盐和EGTA的抑制，这表明它不同于Na^+， K^+ - atp酶或Ca^<2+> - atp酶。使用特异性抗血清对这种酶进行免疫学研究表明，这种酶存在于血小板、白细胞、肝细胞和红细胞的膜中。这些结果表明，gs - dnp刺激的atp酶在主动转运谷胱甘肽s缀合物和细胞防御氧化应激系统中起作用。

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.