Isolation of the basal and lateral plasma membranes of rat kidney tubule cells.
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.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001485:Basement Membrane / Q000201:enzymology / Q000648:ultrastructure*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D007672:Kidney Cortex / Q000648:ultrastructure; D007684:Kidney Tubules / Q000201:enzymology / Q000648:ultrastructure*; D008297:Male; D008854:Microscopy, Electron; D051381:Rats
D000251:Adenosine Triphosphatases