Isolation of the alpha and beta subunits of canine (Na+,K+)ATPase by using SDS-PAGE and lectin-Sepharose.
(Na+,K+)ATPase from dog kidney was solubilized and denatured by SDS treatment, then applied to a Con A- and WGA-Sepharose column. While the alpha subunit of the ATPase had no affinity for either of the lectin-Sepharoses, the beta subunit specifically bound to WGA-Sepharose and was eluted with N-acetylglucosamine. This property was utilized for the isolation of the alpha and beta subunits by using lectin-Sepharoses and SDS-polyacrylamide gel electrophoresis. The amino acid composition of the alpha subunit thus isolated was in reasonable agreement with the data reported by Kyte (Kyte, J. (1972) J. Biol. Chem. 247, 7642-7649). The amino acid and carbohydrate compositions of the beta subunit were, however, different from his data. The beta subunit contained little histidine (0.1 mol/100 mol amino acid) and a very large amount of carbohydrates (33%). The antibody raised against alpha or beta subunit reacted specifically with the corresponding subunit and with protease-fragmented alpha subunit and neuraminidase-treated beta subunit, respectively, but no cross-reactivity was observed between the two subunits. These results indicate that our alpha and beta subunits were highly purified.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D002847:Chromatography, Agarose; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007120:Immunochemistry; D007668:Kidney / Q000201:enzymology*; D037102:Lectins; D046911:Macromolecular Substances; D009439:Neuraminidase; D011817:Rabbits; D012794:Sialic Acids / Q000032:analysis; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification*
D000596:Amino Acids; D002241:Carbohydrates; D037102:Lectins; D046911:Macromolecular Substances; D012794:Sialic Acids; D009439:Neuraminidase; D000254:Sodium-Potassium-Exchanging ATPase