Isolation and functional reconstitution of a 38-kDa chloride channel protein from bovine tracheal membranes.

摘要: Abstract Secretion of chloride ions via apically located anion-selective channels in epithelia regulates fluid formation and cytosolic Cl- homeostasis. In order to understand the biochemical basis channel function, we attempted isolate this transporter from bovine tracheal apical membranes. Initially, peripheral polypeptides were removed enriched vesicles by washing with alkaline buffer (pH 10.8) containing 2 mM CHAPS. The resulting pellet contained 50-60% original protein displayed 2-fold enhanced activity compared untreated vesicles. was treated Triton X-100, solubilized proteins separated on cationic exchanger CM-cellufine. Washing resin a pH 8.0-8.3 eluted fraction activity. This less than 5% total protein. A subsequent separation performed using anionic AM-cellufine. highest found fractions 80-120 KCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed major 38,000-Da band. band electroeluted under nondenaturing nonreducing conditions reconstituted into phosphatidylcholine liposomes. KCl-loaded purified 38-kDa transported up 5 nmol 125I-/mg protein/5 min. value 15-fold higher uptake measured membrane 4-fold CM-cellufine-enriched fraction. observed 125I- 90% inhibited 100 microM 4,4-bis(isothiocyano)-2,2'-stilbenedisulfonate or 10 valinomycin. summary, have developed protocol for isolation 38 kDa mediating potential-dependent 4,4-bis(isothiocyano)-2,2'-stilbenedisulfonate-sensitive