Abstract
A method is described for isolating plasma membrane vesicles from bovine tracheal epithelium. The procedure yields highly purified apical membranes which are enriched 19-fold in the marker enzyme, alkaline phosphatase. Contamination of this fraction by other organelles is minimal. Basolateral membranes isolated from the same preparation have a 4-fold enrichment of ( Na + + K + )- ATPase and a 2-fold reduction in alkaline phosphatase specific activity compared to the starting material. Assays of Na + uptake by the apical membrane vesicles demonstrate their suitability for transport studies. Transport of Na + into an intravesicular space was demonstrated by (1) a linear inverse correlation between Na + uptake and medium osmolarity; (2) complete release of accumulated Na + by treatment with detergent; and (3) a marked temperature-dependence of Na + uptake rate. Other features of Na + transport were (1) inhibition by amiloride; (2) insensitivity to furosemide; and (3) anion-dependence of uptake rate with the following selectivity: SCN − > Cl − > gluconate − .
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