PURIFICATION OF THE ALPHA-SUBUNIT AND BETA-SUBUNIT OF (NA,K)-ATPASE BY CONTINUOUS ELUTION ELECTROPHORESIS

Covalent structural information on membrane proteins is not easily acq uired since it is difficult to obtain pure membrane proteins in suffic ient 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)-AT Pase 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 c onstant power and continuous cooling conditions. Those fractions conta ining the beta subunit were combined and further purified by wheat ger m agglutinin affinity chromatography. Fractions containing the alpha s ubunit were combined and did not require further purification. The ide ntity and the degree of purity of the proteins obtained using this app roach was assessed utilizing SDS-PAGE, amino acid analysis and N-termi nal 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 cru de (Na,K)-ATPase preparation was estimated to be 28% and 81%, respecti vely.