Harms V, Wright E M
J Membr Biol. 1980 Apr 15;53(2):119-28. doi: 10.1007/BF01870580.
Basal lateral membrane vesicles were isolated from rat intestinal epithelial cells. The sodium potassium triphosphatase (Na/K-ATPase) of these plasma membranes has been characterized by (1) the molecular weight of the phosphorylated intermediate, (2) the sensitivity of the phosphorylated intermediate to hydroxylamine, (3) its ouabain binding constants, and (4) its susceptibility to digestion by pronase. The phosphorylated intermediate was shown by SDS polyacrylamide gel electrophoresis to be a protein of 100,000 Daltons apparent mol wt. Its extensive hydrolysis in hydroxylamine demonstrated that it was an acyl phosphate. The isolated basal lateral membranes bound ouabain with a dissociation constant, Km (1.5 x 10(5) M), similar to the inhibitory constant KI (3 X 10(-5) M), measured for ouabain inhibition of the Na/K-ATPase activity. The association rate constant measured for ouabaiation rate constants reported for other tissues and species. The high dissociation rate constant 3.6 x 10(-2) sec-1, is consistent with the insensitivity of the rat to ouabain. Digestion of the intact cells by pronase yielded basal lateral membranes in which the Na/K-ATPase had been unaffected. The phosphorylated intermediate ran as a sharp band at 100,000 Daltons on electrophoresis, and the ouabain dissociation constant appeared to be unchanged. In these membranes, protein stains of polyacrylamide gels revealed digestion of the major high mol wt proteins including the major protein at 100,000 Daltons. This suggests that the Na/K-ATPase represents a minor component, less than 1%, of the basal lateral membrane protein. From these characteristics of the phosphorylated intermediate and the ouabain binding constants, we conclude that the Na/K-ATPase of the basal lateral membranes of rat intestinal epithelial cells is similar to that found in other tissues and species. Estimates of the number of pump sites and the turnover number predict rates of Na transport that are consistent with observed values.
从大鼠肠上皮细胞中分离出基底外侧膜囊泡。这些质膜的钠钾三磷酸酶（Na/K-ATP酶）已通过以下方面进行了表征：（1）磷酸化中间体的分子量；（2）磷酸化中间体对羟胺的敏感性；（3）其哇巴因结合常数；（4）其对链霉蛋白酶消化的敏感性。通过SDS聚丙烯酰胺凝胶电泳显示，磷酸化中间体是一种表观分子量为100,000道尔顿的蛋白质。它在羟胺中的广泛水解表明它是一种酰基磷酸。分离出的基底外侧膜以解离常数Km（1.5×10⁻⁵M）结合哇巴因，这与测量得到的哇巴因对Na/K-ATP酶活性抑制的抑制常数KI（3×10⁻⁵M）相似。测量得到的哇巴因结合速率常数与其他组织和物种报道的结合速率常数相似。高解离速率常数3.6×10⁻²秒⁻¹与大鼠对哇巴因不敏感一致。用链霉蛋白酶消化完整细胞产生的基底外侧膜中，Na/K-ATP酶未受影响。磷酸化中间体在电泳时在100,000道尔顿处呈现为一条清晰的条带，且哇巴因解离常数似乎未变。在这些膜中，聚丙烯酰胺凝胶的蛋白质染色显示主要的高分子量蛋白质包括100,000道尔顿的主要蛋白质被消化。这表明Na/K-ATP酶是基底外侧膜蛋白中的次要成分，占比不到1%。从磷酸化中间体的这些特征和哇巴因结合常数来看，我们得出结论，大鼠肠上皮细胞基底外侧膜的Na/K-ATP酶与其他组织和物种中的相似。对泵位点数量和周转数的估计预测的Na转运速率与观察值一致。
