Rapid procedure for obtaining tracheal apical membranes.
Apical membranes from cow tracheal epithelium were prepared in a two-step process. First, most of the unwanted membranes in the crude homogenate were aggregated with Mg and removed by a low-speed spin. The membranes remaining in the supernatant were pelleted by a high-speed spin, resuspended, and exposed to ouabain-affinity chromatography. This step removed approximately 50% of the protein, all the Na-K-adenosinetriphosphatase, but had no effect on total levels of alkaline phosphatase (a marker for apical membranes). The specific activity of the apical membrane marker, alkaline phosphatase, was 21 +/- 7-fold (mean +/- SD) greater in the apical membranes than in the homogenate. Markers for nuclei, mitochondria, and basolateral membranes were excluded compared with the homogenate. Similar results were obtained with primary cultures of cow tracheal epithelium. The vesicular nature of the membranes was demonstrated in isotope uptake studies that revealed an osmotically active space.
Shen
B Q
BQ
Cystic Fibrosis Research Center, University of California, San Francisco 94143.
Yang
C M
CM
Widdicombe
J H
JH
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D011232:Chemical Precipitation; D006652:Histological Techniques; D008274:Magnesium; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013048:Specimen Handling / Q000379:methods*; D014132:Trachea / Q000648:ultrastructure*
D000469:Alkaline Phosphatase; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium