Open AccessPurification and characterization of the amiloride-sensitive sodium channel from A6 cultured cells and bovine renal papilla.
Author(s) -
Dale J. Benos,
G. Saccomani,
Barry M. Brenner,
Sarah SaribanSohraby
Publication year - 1986
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.83.22.8525
Subject(s) - amiloride , chromatography , toad , chemistry , wheat germ agglutinin , high performance liquid chromatography , affinity chromatography , size exclusion chromatography , sodium , biochemistry , chaps , membrane , biology , lectin , enzyme , ecology , organic chemistry
The amiloride-binding Na+ channel protein of high electrical resistance epithelia was solubilized and purified from cultured A6 toad kidney cells and bovine renal papilla. Purification was assessed by enrichment in [3H]methylbromoamiloride specific binding. Chromatography of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-solubilized plasma membrane vesicles on agarose-immobilized wheat-germ agglutinin provided a 130-fold enrichment of the amiloride-binding component compared to the cell homogenate. Further purification was achieved by either amiloride-affinity chromatography or size-exclusion HPLC. When the HPLC and amiloride affinity-purified material was injected into a second higher molecular weight exclusion HPLC column, only a single peak with Mr 800,000 was found. Further HPLC separation of the Mr 800,000 material at low ionic strength resolved two peaks with apparent Mrs 800,000 and 700,000. Only the 700-kDa component displayed specific [3H]methylbromoamiloride binding activity. The final binding specific activity achieved was 1300 pmol/mg of protein, corresponding to 91% homogeneity of the protein.