Ebel H, Aulbert E, Merker H J
Biochim Biophys Acta. 1976 May 21;433(3):531-46. doi: 10.1016/0005-2736(76)90279-0.
A method was developed to isolate renal basolateral membranes from cortical kidney tubule cells of single rats. The isolated membrane fraction was characterized by the measurement of marker enzyme activities and by electron microscopy. 1. After centrifugation of crude plasma membranes on a discontinuous sucrose density gradient the basolateral membranes accumulated at a sucrose density of p= 1.14-1.15 g/ml. The yield was 147 mug membrane protein/g kidney wet weight. Protein recovery was 0.1%. 2. (Na+ + K+)-ATPase was enriched 22-fold from the homogenate. The recovery was 2.6%. The (Na+ + K+)/Mg2+-ATPase ratio was 4.1. 3. The contamination by brush borders was small. Alkaline phosphatase was 1.6-fold enriched and 0.2% was recovered. Aminopeptidase was 1-fold enriched with a recovery of 0.1%. The contamination by mitochondria, lysosomes and endoplasmic reticulum was negligible. 4. In electron micrographs the basolateral membranes showed a typical triple layered profile and were characterized by the presence of junctional complexes, gap junctions or tight junctions.
已开发出一种从单只大鼠的皮质肾小管细胞中分离肾基底外侧膜的方法。通过测量标记酶活性和电子显微镜对分离出的膜部分进行表征。1. 在不连续蔗糖密度梯度上对粗质膜进行离心后，基底外侧膜在蔗糖密度为p = 1.14 - 1.15 g/ml处积累。产量为147μg膜蛋白/克肾脏湿重。蛋白质回收率为0.1%。2. （Na + + K +）-ATP酶相对于匀浆富集了22倍。回收率为2.6%。（Na + + K +）/Mg2 + -ATP酶的比率为4.1。3. 刷状缘的污染很小。碱性磷酸酶富集了1.6倍，回收率为0.2%。氨肽酶富集了1倍，回收率为0.1%。线粒体、溶酶体和内质网的污染可忽略不计。4. 在电子显微镜照片中，基底外侧膜呈现典型的三层轮廓，并以存在连接复合体、缝隙连接或紧密连接为特征。
