Studies on the Characterization of the Sodium-Potassium Transport Adenosine Triphosphatase

The (sodium + potassium)-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-γ-phosphate residue with 32P by prior incubation of the partially purified enzyme with ATP-γ-32P 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 purification proceeds, being approximately 50%, 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 enzyme is ouabain-sensitive. Ouabain-sensitive, potassium-activated p-nitrophenylphosphatase activity parallels NaK ATPase. All detectable cholinesterase in the microsomes is removed by purification. With the method of purification reported here, as much as 1.2 kg of bovine brain cortex can be worked up weekly giving 30 to 50 mg of final enzyme with a specific activity of 450 to 750 µmoles of ATP split per mg of protein per hour. Based on the gel scans and on calculations from the picomoles of phosphorylated subunit and molecular weight of the native enzyme, the best preparations are judged to be approximately half pure. The partially purified enzyme is quite stable on storage at 0°, retaining full activity for two months. Electron microscopy of the enzyme preparation after zonal centrifugation shows rods of about 60 A in diameter, spherical protein, and aggregated protein. The ammonium sulfate fraction shows chains of protein averaging 60 A in diameter, which are believed to be end-to-end aggregates of the rods seen in the zonal fraction. Many chains form ringlike structures. Sheets of aggregated protein are also seen. Resolubilization of the ammonium sulfate enzyme with Lubrol to protein ratios of 20:1 shows predominantly protein subunits averaging 60 A in diameter and some aggregates of subunits.