Isolation of brush-border membrane from the rabbit descending colon epithelium. Partial characterization of a unique K+-activated ATPase.
Isolation of brush-border membrane from the rabbit descending colon epithelium. Partial characterization of a unique K+-activated ATPase.
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从兔降结肠上皮中分离刷状缘膜。
DOI：
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发表时间：
1981
影响因子：
4.8
通讯作者：
D. Goodman
中科院分区：
文献类型：
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作者：
M. Gustin;D. Goodman
以前只在完整的组织中研究过离子穿过结肠顶膜的运动机制。为了直接研究这些机制，我们进行了家兔降结肠顶端刷状缘膜的分离和表征。纯化方案包括在EDTA中解离粘膜层后初始分离单个上皮细胞，高pH（8.3），细胞的低离子强度均质化，差速离心和顶膜与细胞核的分离，以及在7.5%Percoll梯度上的丝状物质。在最终的膜组分中观察到碱性磷酸酶（顶端膜酶标记物）比活性超过初始匀浆值的20倍富集。该组分还含有K+激活的pH 7.8的最佳ATP酶（在匀浆上纯化20倍）具有以下性质：（2 X 10（-4）M）; 2）耐高离子强度（1 M Tris）溶解; 3）Na+的竞争性抑制（K1 = 14 mM），不受Na+激活; 4）受原钒酸盐抑制（K1 = 40 nM），但寡霉素（20微克/ml蛋白质）或哇巴因（10（-3）M）无作用;和5）K+敏感性磷酸化中间体。这些特征表明，这种膜结合ATP酶不同于其他已知的ATP酶，包括基底外侧膜的Na+ + K+ -ATP酶-Na+泵。
The mechanisms of ion movement across the apical membrane of the colon have previously been investigated only in intact tissue. To investigate these mechanisms directly, we have undertaken the isolation and characterization of the apical brush-border membrane of the rabbit descending colon. The purification protocol consists of an initial isolation of single epithelial cells after dissociation of the mucosal layer in EDTA, a high pH (8.3), low ionic strength homogenization of the cells, and differential centrifugation and separation of apical membrane from nuclei, and filamentous material on a 7.5% Percoll gradient. A 20-fold enrichment in alkaline phosphatase (an apical membrane enzyme marker) specific activity over the initial homogenate value is observed in the final membrane fraction. This fraction also contains a K+-activated, pH 7.8, optimum ATPase (20 times purified over homogenate) with the following properties: 1) low Kact (2 X 10(-4) M) for K+; 2) resistance to high ionic strength (1 M Tris) solubilization; 3) competitive inhibition by Na+ (K1 = 14 mM), no activation by Na+; 4) inhibition by orthovanadate (K1 = 40 nM), but no effect of oligomycin (20 micrograms/ml of protein) or ouabain (10(-3) M); and 5) a K+-sensitive phosphorylated intermediate. These characteristics suggest that this membrane-bound ATPase is distinct from other known ATPases including the Na+ + K+ - ATPase-Na+ pump of the basolateral membrane.