Isolation of transporting plasma membrane vesicles from bovine tracheal epithelium.
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- greater than Cl- greater than gluconate-.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D004848:Epithelium / Q000201:enzymology / Q000648:ultrastructure*; D007700:Kinetics; D009092:Mucous Membrane / Q000201:enzymology / Q000648:ultrastructure; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014132:Trachea / Q000201:enzymology
D012964:Sodium; D000469:Alkaline Phosphatase; D000254:Sodium-Potassium-Exchanging ATPase