Purification and characterization of the amiloride-sensitive sodium channel from A6 cultured cells and bovine renal papilla.
Purification and characterization of the amiloride-sensitive sodium channel from A6 cultured cells and bovine renal papilla.
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A6 培养细胞和牛肾乳头阿米洛利敏感钠通道的纯化和表征。
DOI：
10.1073/pnas.83.22.8525
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发表时间：
1986
影响因子：
11.1
通讯作者：
Sariban-Sohraby,S
中科院分区：
文献类型：
--
作者：
Benos,DJ;Saccomani,G;Brenner,BM;Sariban-Sohraby,S
从体外培养的A6蟾蜍肾细胞和牛肾乳头细胞中，溶解并纯化了高电阻上皮细胞的阿米洛利结合性Na+通道蛋白。通过富集[3 H]甲基溴阿米洛利特异性结合来评估纯化。3-[（3-胆酰胺丙基）二甲基铵]-1-丙磺酸（CHAPS）增溶质膜囊泡琼脂糖固定的小麦胚芽凝集素色谱法提供了130倍富集的阿米洛利结合组分相比，细胞匀浆。通过阿米洛利亲和层析或尺寸排阻HPLC实现进一步纯化。当将HPLC和阿米洛利亲和纯化的物质注入第二个较高分子量排阻HPLC柱时，仅发现Mr为800，000的单峰。只有700-kDa的组件显示特定的[3 H]甲基溴阿米洛利结合活性。达到的最终结合比活性为1300 pmol/mg蛋白质，对应于91%的蛋白质同质性。
The amiloride-binding Na+ channel protein of high electrical resistance epithelia was solubilized and purified from cultured A6 toad kidney cells and bovine renal papilla. Purification was assessed by enrichment in [3H]methylbromoamiloride specific binding. Chromatography of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilized plasma membrane vesicles on agarose-immobilized wheat-germ agglutinin provided a 130-fold enrichment of the amiloride-binding component compared to the cell homogenate. Further purification was achieved by either amiloride-affinity chromatography or size-exclusion HPLC. When the HPLC and amiloride affinity-purified material was injected into a second higher molecular weight exclusion HPLC column, only a single peak with Mr 800,000 was found. Further HPLC separation of the Mr 800,000 material at low ionic strength resolved two peaks with apparent Mrs 800,000 and 700,000. Only the 700-kDa component displayed specific [3H]methylbromoamiloride binding activity. The final binding specific activity achieved was 1300 pmol/mg of protein, corresponding to 91% homogeneity of the protein.