Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. VI. Large scale partial purification and properties of a lubrol-solubilized bovine brain enzyme.

The (sodium -Ipotassium)-activated adenosine triphosphatase (NaK ATPase) in Lubrol extracts of NaI-treated bovine brain microsomes has been purified 30 to 50 times over that in the microsomes by salt and isoelectric precipitation, zonal centrifugation, and a new ammonium sulfate fractionation procedure. The final step in the purification procedure renders the NaK ATPase insoluble. The partially purified enzyme has been examined by polyacrylamide gel electrophoresis after solubilization in either phenol-acetic acid-urea or sodium dodecyl sulfate-mercaptoethanol. The former system is unsatisfactory in that a considerable proportion of the protein, including the NaK ATPase, remains at the origin; however, all of the protein penetrates the gel with the latter system. The purification procedure removes the majority of proteins seen in the microsomal fraction. With the partially purified enzyme, one prominent band, two less prominent bands, and two to three rather minor bands are seen with the sodium dodecyl sulfate system. The most prominent band has been identified as the phosphorylated subunit of the NaK ATPase by labeling its glutamyl-y-phosphate residue with s2P by prior incubation of the partially purified enzyme with ATP-Y-~~P in the presence of magnesium and sodium. Its apparent molecular weight is 94,000. Scanning of the protein-stained gel indicates that 25 to 40% of the protein applied to the gel can be accounted for by this 94,000 molecular weight subunit. The protein, phospholipid, and carbohydrate content of the preparation remain remarkably constant as purif?cation proceeds, being approximately SO%, 25%, and 2 to 3%, respectively, of the dry weights of the preparations after correction for bound Lubrol. The protein-bound Lubrol at the final stage of purification is 17%. The cholesterol content falls with purification. Ninety-nine per cent of the ATPase in the partially purified