Feit P W, Hoffmann E K, Schiødt M, Kristensen P, Jessen F, Dunham P B
Leo Pharmaceutical Products, Ballerup, Denmark.
J Membr Biol. 1988 Jul;103(2):135-47. doi: 10.1007/BF01870944.
Bumetanide-binding proteins were isolated from membranes of Ehrlich ascites tumor cells by affinity chromatography. An affinity column was constructed with the active moiety of bumetanide as a ligand using 4'-azidobumetanide, a photoactive analogue which inhibits Na/Cl cotransport in Ehrlich cells with high specificity. Covalent binding of the 4'-azidobumetanide with Sepharose was promoted by photolysis. Membranes isolated from Ehrlich cells were solubilized with n-octylglucoside. Solubilized proteins retarded by the affinity column were readily eluted by bumetanide. In reducing gels the major proteins eluted by bumetanide were approximately 76 kDa and 38-39 kDa. There were also two proteins of 32 to 35 kDa eluted in lesser amounts. No proteins retarded by the affinity column were eluted with extensive washing without bumetanide. Furthermore, bumetanide eluted no proteins from a "control" column lacking the specific ligand. Upon rechromatography with bumetanide in solution, bumetanide-eluted proteins were not retarded, but their purity was increased by the retardation of contaminating proteins. Bumetanide-binding protein purified in this manner were characterized further by electrophoresis in nonreducing, nondenaturing gels.
通过亲和层析从艾氏腹水癌细胞膜中分离出布美他尼结合蛋白。使用4'-叠氮布美他尼构建了以布美他尼的活性部分为配体的亲和柱，4'-叠氮布美他尼是一种光活性类似物，能高度特异性地抑制艾氏细胞中的Na/Cl共转运。通过光解促进4'-叠氮布美他尼与琼脂糖的共价结合。用正辛基葡糖苷溶解从艾氏细胞分离的膜。被亲和柱阻滞的溶解蛋白很容易被布美他尼洗脱。在还原凝胶中，被布美他尼洗脱的主要蛋白约为76 kDa和38 - 39 kDa。还有两种32至35 kDa的蛋白被少量洗脱。在没有布美他尼的情况下进行大量洗涤，亲和柱阻滞的蛋白没有被洗脱。此外，布美他尼从缺乏特异性配体的“对照”柱中没有洗脱任何蛋白。在用溶液中的布美他尼进行再层析时，被布美他尼洗脱的蛋白没有被阻滞，但通过污染蛋白的阻滞提高了它们的纯度。以这种方式纯化的布美他尼结合蛋白通过在非还原、非变性凝胶中的电泳进一步表征。
