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

我们在人红细胞中纯化了一种新的ATPase，它是由氧化形式的谷胱甘肽(GSSG)刺激的。采用S-己基谷胱甘肽-Sepharose6B亲和层析的方法对酶进行纯化，以获得明显的均一性。用酶学、生化、生理学和免疫学方法对该酶进行了进一步的鉴定。该酶有两个独立的同工酶(类型I和类型II)，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。我们用兔制备了针对每种同工酶的多克隆抗体。这些酶在免疫上有交叉反应。重组成磷脂囊泡的酶既有GSSG刺激的ATPase活性，又有GSSG转运活性。I型同工酶的GSSG-ATPase活性和GSSG转运活性分别为25nmoL/mg蛋白/min和25nmolpi释放/mg蛋白/min，II型同工酶的GSSG-ATPase活性和GSSG转运活性分别为248nmol/mg蛋白/min和133nmolpi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发挥作用。

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.