Purification and characteristics of (Na +,K +)-ATPase from canine kidney by zonal centrifugation in sucrose density gradient

Abstract

Microsomes were prepared from the outer medulla of canine kidney. Partially purified preparation of (Na +,K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) was obtained by solubilization of microsomes with sodium deoxycholate, and by precipitation with dilution. The deoxycholate-enzyme thus obtained was further purified by incubation with sodium dodecyl sulphate in the presence of ATP followed by a single zonal centrifugation in a sucrose density gradient by the method of Jørgensen [(1974) Biochim. Biophys. Acta 356, 36–52]. The (Na +,K +)-ATPase was purified to a specific activity of 1600–1800 μmol P i · h −1 · mg −1 protein. The yield was 20 mg per single centrifugation with a zonal rotor. Electron microscopy showed that the sectioned pellet of the purified enzyme contained exclusively membranous fragments in contrast with membranous vesicles of starting microsomes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that almost all proteins were accounted for by two polypeptides with molecular weights of 105 000 and 58 000, and that the mass ratio of the large to the small polypeptide was 82: 18.