Photoaffinity labelling of a 150 kDa (Na + K + Cl)-cotransport protein from duck red cells with an analog of bumetanide.
Photoaffinity labelling of a 150 kDa (Na + K + Cl)-cotransport protein from duck red cells with an analog of bumetanide.
复制标题
使用布美他尼类似物对来自鸭红细胞的 150 kDa (Na K Cl) 共转运蛋白进行光亲和标记。
DOI：
10.1016/0005-2736(88)90054-5
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Forbush3rd,B
中科院分区：
文献类型：
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作者：
Haas,M;Forbush3rd,B
我们使用放射性标记的布美他尼的二苯甲酮类似物4-[3 H]苯甲酰基-5-氨磺酰基-3-（3-噻吩氧基）苯甲酸（[3 H] BSTBA）对鸭红细胞的质膜进行光标记。与布美他尼一样，BSTBA是一种袢利尿剂和（Na+ K+ Cl）共转运的有效抑制剂，（[3 H] BSTBA）与完整鸭红细胞的结合亲和力与[3 H]布美他尼相似（K1 2 ≤ 0.1 gmM）。我们用[3 H] BSTBA孵育鸭红细胞，然后裂解细胞并将血影暴露于紫外线下。重影和光解在0° C下进行以防止（[3 H] BSTBA）解离。然后对鬼进行超声处理以去除细胞核并在SDS-聚丙烯酰胺凝胶上运行。H2 O2消化的凝胶切片分析显示（[3 H] BSTBA）被掺入到约100 μ g/ml的蛋白质中。150 kDa。这与我们从狗肾膜中获得的蛋白质的分子量相同，该蛋白质被（[3 H] BSTBA）以与（Na+ K+ Cl）共转运蛋白的标记高度一致的方式光标记（哈斯和Forbush（1987）Am. 253，C243-C252）。几条证据有力地表明，来自鸭红细胞膜的150 kDa蛋白质是鸭红细胞膜的组成部分。（Na+ K+ Cl）-共转运系统：（1）在含[3 H] BSTBA的初始培养液中加入μM未标记的布美他尼，可阻断[3 H] BSTBA对该蛋白的光标记;（2）[3 H]的光掺入当初始孵育培养基是高渗的或含有去甲肾上腺素时，BSTBA进入150 kDa蛋白质的量显著增加，同样刺激两者的条件鸭红细胞Na+ K+ Cl共转运和饱和[3 H]布美他尼结合（3）该蛋白的光标记对[~ 3 H] BSTBA浓度有饱和依赖性，K值为1 2（0.06 μM）与[3 H] BSTBA和[3 H]布美他尼与鸭红细胞的可逆饱和结合相似;和（4）[3 H] BSTBA光掺入到150 kDa蛋白质中，类似于饱和[3 H]布美他尼结合到完整细胞，需要在含有放射性标记利尿剂的培养基中同时存在Na+、K+和Cl−。
We have used a radiolabelled, benzophenone analog of bumetanide, 4-[3 H] benzoyl-5-sulfamoyl-3-(3-thenyloxy) benzoic acid ([3 H] BSTBA) to photolabel plasma membranes from duck red blood cells. BSTBA, like bumetanide, is a loop diuretic and a potent inhibitor of (Na+ K+ Cl) cotransport, and ([3 H] BSTBA) binds to intact duck red cells with a high affinity similar to that of [3 H] bumetanide (K 1 2≌ 0.1 gmM). We incubated duck red cells with ([3 H] BSTBA), then lysed the cells and exposed the ghosts to ultraviolet light. The ghosting and photolysis was done at 0° C to prevent dissociation of the ([3 H] BSTBA). The ghosts were then sonicated to remove the nuclei and run on SDS-polyacrylamide gels. Analysis of H 2 O 2-digested gel slices revealed ([3 H] BSTBA) to be incorporated into a protein of approx. 150 kDa. This is the same molecular weight we obtain for a protein from dog kidney membranes which is photolabelled by ([3 H] BSTBA) in a manner highly consistent with labelling of the (Na+ K+ Cl) cotransporter (Haas and Forbush (1987) Am. J. Physiol. 253, C243-C252). Several lines of evidence strongly suggest that the 150 kDa protein from duck red cell membranes is an integral component of the (Na+ K+ Cl)-cotransport system in these cells:(1) Photolabelling of this protein by [3 H] BSTBA is blocked when μM unlabelled bumetanide is included in the initial incubation medium with [3 H] BSTBA;(2) Photoincorporation of [3 H] BSTBA into the 150 kDa protein is markedly increased when the initial incubation medium is hypertonic or contains norepinephrine, conditions which similarly stimulate both (Na+ K+ Cl) cotransport and saturable [3 H] bumetanide binding in duck red cells;(3) The photolabelling of this protein shows a saturable dependence on [3 H] BSTBA concentration, with a K 1 2 (0.06 μM) similar to that for the reversible, saturable binding of [3 H] BSTBA and [3 H] bumetanide to duck red cells; and (4)[3 H] BSTBA photoincorporation into the 150 kDa protein, like saturable [3 H] bumetanide binding to intact cells, requires the simultaneous presence of Na+, K+, and Cl− in the medium containing the radiolabelled diuretic.