Purification of the alpha and beta subunits of (Na,K)-ATPase by continuous elution electrophoresis.

Abstract

Covalent structural information on membrane proteins is not easily acquired since it is difficult to obtain pure membrane proteins in sufficient quantities. We have therefore examined the Bio-Rad 491 prep cell continuous elution electrophoresis apparatus as a method for providing the quantities of purified alpha and beta subunits from (Na,K)-ATPase required for these studies. Twenty-four milligrams of crude (Na,K)-ATPase preparation was applied to the prep cell which consisted of a 7% Laemmli separating gel 4.5 cm in length. The prep cell was run under constant power and continuous cooling conditions. Those fractions containing the beta subunit were combined and further purified by wheat germ agglutinin affinity chromatography. Fractions containing the alpha subunit were combined and did not require further purification. The identity and the degree of purity of the proteins obtained using this approach was assessed utilizing SDS-PAGE, amino acid analysis and N-terminal sequencing. This simple and fast method provides approximately 1.8 milligrams of each purified subunit from 24 milligrams of relatively crude microsomes. Recovery of the alpha and beta subunits from the crude (Na,K)-ATPase preparation was estimated to be 28% and 81%, respectively.