一种对芪敏感的氯离子通道的纯化以及氯离子传导性在磷脂囊泡中的重建。

Purification of a stilbene sensitive chloride channel and reconstitution of chloride conductivity into phospholipid vesicles.

作者信息

Blair H C, Schlesinger P H

机构信息

Department of Pathology, Jewish Hospital of St. Louis, Missouri.

出版信息

Biochem Biophys Res Commun. 1990 Sep 28;171(3):920-5. doi: 10.1016/0006-291x(90)90771-e.

DOI:10.1016/0006-291x(90)90771-e

PMID:1699531

Abstract

A protein conferring passive chloride permeability was isolated from a N-octylglucoside solubilized extract of partially purified H(+)-transporting osteoclast cell membranes. Purification was achieved by binding of solubilized protein to an amine-linked 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) Sepharose 4B column and elution with 50 mM KCl. A major protein, with MR = 60 kD on 10% SDS-PAGE, was obtained, which was further purified to homogeneity by HPLC gel filtration. This protein introduced 36Cl- permeability when reconstituted in phospholipid membranes by equilibrium dialysis. The Cl- transport recovered in reconstituted membranes retained sensitivity to DIDS confirming the identity of the isolated protein as a stilbene-sensitive chloride channel.

摘要

从部分纯化的H⁺转运破骨细胞膜的N - 辛基葡糖苷增溶提取物中分离出一种赋予被动氯离子通透性的蛋白质。通过将增溶的蛋白质与胺连接的4,4'-二异硫氰酸根合芪-2,2'-二磺酸盐（DIDS）琼脂糖4B柱结合并用50 mM KCl洗脱来实现纯化。在10% SDS - PAGE上得到一种主要蛋白质，其相对分子质量（MR）为60 kD，通过HPLC凝胶过滤进一步纯化至同质。当通过平衡透析在磷脂膜中重构时，这种蛋白质引入了³⁶Cl⁻通透性。重构膜中恢复的Cl⁻转运对DIDS仍保持敏感性，证实分离出的蛋白质是一种芪敏感的氯离子通道。