Immunopurification and structural analysis of a putative epithelial Cl- channel protein isolated from bovine trachea.
We have purified to homogeneity a 38-kDa protein (called p38) from bovine tracheal epithelium. This protein, when reconstituted into liposomes, mediates stilbene disulfonate-sensitive 125I- conductive uptake. On nonreduced or partially reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this protein associates into a doublet of 62-64 kDa. In some experiments a multimer of 141 kDa was also observed. Rabbit polyclonal anti-P38 antibodies have been produced and used to immunopurify the native transporter. Upon reconstitution of the immunoaffinity-purified protein into liposomes, a 260-fold enhancement of 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate and valinomycin-sensitive 125I- uptake was observed as compared to proteoliposomes containing unseparated material. On Western blots of total solubilized tracheal membrane proteins or semipurified fractions, the antibody recognized the 62-64-kDa doublet much better than the original 38-kDa antigen. Similar protein bands were detected in T84 and CFPAC cells as well. However, if apical membrane proteins were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, the antibody recognized major bands at 140 and approximately 240 kDa. Upon partial reduction, immunolabeling of these proteins diminished with the concomitant appearance of the 62-64-kDa doublet. Upon complete reduction, the appearance of 32- and 38-kDa proteins was evident with the disappearance of the 62-64-kDa doublet. We hypothesize that the native Cl-channel is a heteromer containing at least four subunits connected by S-S bridges.
Ran
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Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
Benos
D J
DJ
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011233:Precipitin Tests; D014132:Trachea / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D018118:Chloride Channels; D002712:Chlorides; D007473:Ion Channels; D008565:Membrane Proteins