Li X Q, Hegazy M G, Mahdi F, Jezek P, Lane R D, Garlid K D
Department of Pharmacology, Medical College of Ohio, Toledo 43699.
J Biol Chem. 1990 Sep 5;265(25):15316-22.
We describe purification of three different states of the 82-kDa K+/H+ antiporter from rat liver mitochondria. The denatured 82-kDa protein, identified by its selective labeling with [14C]dicyclohexylcarbodiimide (DCCD), was purified by preparative two-dimensional gel electrophoresis. This purified product was used to raise and immunopurify monospecific polyclonal antibodies. Western blot analysis showed that the [14C] DCCD-labeled 82-kDa protein is not a DCCD-crosslinked product. The native, [14C]DCCD-labeled, 82-kDa protein was purified by (NH4)2SO4 fractionation and column chromatography, using 14C labeling and gel electrophoresis to track the protein. The native, non-DCCD-labeled 82-kDa protein was purified by similar procedures, using immunopurified antibodies to track the protein. DCCD binding had no effect on chromatographic behavior of the antiporter protein. This protocol resulted in purification of the 82-kDa protein to apparent homogeneity. The purified, native 82-kDa protein was reconstituted into proteoliposomes and assayed for K+ transport with the new fluorescent probe, PBFI. K+ transport was electroneutral and was inhibited by DCCD, Mg2+, and timolol. The turnover number for K+ transport was about 1000 s-1, very similar to the value previously estimated in intact mitochondria.
我们描述了从大鼠肝线粒体中纯化82-kDa K+/H+反向转运体三种不同状态的方法。通过制备性二维凝胶电泳纯化经[14C]二环己基碳二亚胺（DCCD）选择性标记鉴定的变性82-kDa蛋白。该纯化产物用于制备和免疫纯化单特异性多克隆抗体。蛋白质免疫印迹分析表明，[14C] DCCD标记的82-kDa蛋白不是DCCD交联产物。通过硫酸铵分级分离和柱色谱法纯化天然的、经[14C]DCCD标记的82-kDa蛋白，使用14C标记和凝胶电泳追踪该蛋白。通过类似的方法纯化天然的、未用DCCD标记的82-kDa蛋白，使用免疫纯化抗体追踪该蛋白。DCCD结合对反向转运体蛋白的色谱行为没有影响。该方案导致82-kDa蛋白纯化至明显的均一性。将纯化的天然82-kDa蛋白重建到蛋白脂质体中，并用新型荧光探针PBFI测定K+转运。K+转运是电中性的，并受到DCCD、Mg2+和噻吗洛尔的抑制。K+转运的周转数约为1000 s-1，与先前在完整线粒体中估计的值非常相似。
