Isolation of brush-border membrane from the rabbit descending colon epithelium. Partial characterization of a unique K+-activated ATPase

Abstract

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.