AJP - Cell Physiology, Vol 263, Issue 4 C888-C895, Copyright © 1992 by American Physiological Society
Immunolocalization of chloride-transporting membrane vesicles in tracheal epithelial cells
W. P. Dubinsky, C. L. Preston, M. A. Calenzo, G. J. White and E. R. Decker
Department of Physiology and Cell Biology, University of Texas Medical School, Houston 77225.
A membrane fraction eluted from a phenyl Sepharose column (MPS) 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 MPS 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 MPS 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.
