IMMUNOLOCALIZATION OF CHLORIDE-TRANSPORTING MEMBRANE-VESICLES IN TRACHEAL EPITHELIAL-CELLS

A membrane fraction eluted from a phenyl Sepharose column (M(PS)) was isolated from renal cortex and bovine tracheal epithelia that is enriched in a single type of Cl- channel [C. L. Preston, M. A. Calenzo, and W. P. Dubinsky. Am. J. Physiol. 263 (Cell Physiol. 32): C879-C887, 1992]. A 200-kDa membrane protein that copurifies with and appears to be specific to this fraction was purified and used to raise antisera for immunological characterization of these membranes. The antisera reacted in immunoblots with a 200-kDa protein in homogenates of bovine trachea, kidney, pancreas, lung, and intestine. There was also cross-reactivity with a 200-kDa protein in immunoblots in rat stomach, pancreas, and lung. There was no cross-reaction with rat skeletal muscle, cardiac muscle, or aorta. Thus this protein appears to be preferentially enriched in epithelial tissues. Examination of each major fraction during the purification of M(PS) membranes from trachea shows no enrichment of the 200-kDa protein in plasma, mitochondrial, or nuclear membrane fractions. The only significant enrichment was observed in M(PS) that is purified by hydrophobic chromatography. In frozen sections, antisera and monospecific immunoaffinity-purified antibodies localize the protein primarily to the apical domain of tracheal columnar epithelial cells with small punctate structures throughout the cytoplasmic compartment.