Studies on the Characterization of the Sodium-Potassium Transport Adenosine Triphosphatase X. PURIFICATION OF THE ENZYME FROM THE RECTAL GLAND OF SQUALUS ACANTHIAS

Abstract Membranes have been isolated from fresh rectal glands of the dogfish shark, Squalus acanthias, in which the specific activity of the (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) is as high as 400 µmoles of Pi per mg of protein per hour. Membranes isolated from frozen rectal glands have only one-fourth to one-third this specific activity. Solubilization of membranes from frozen glands with the nonionic detergent Lubrol WX activates the NaK ATPase 2-fold, but there is no activation of the enzyme with membranes from fresh glands. Purification of Lubrol extracts by zonal centrifugation and a novel ammonium sulfate fractionation gives an enzyme preparation with a specific activity of 1,500 µmoles of Pi per mg of protein per hour. The same specific activity and gel pattern is obtained with enzyme purified from membranes from either fresh or frozen glands. The loss of Lubrol activation of the enzyme from frozen glands occurs on zonal centrifugation, where free Lubrol is removed. The over-all yield of enzyme from the membrane stage is 70%. A mince of 10 rectal glands weighing about 10 to 15 g fresh weight yields about 20 to 30 mg of purified enzyme. The enzyme is completely stable at 0° for 10 days in either the membranous form or in the most highly purified form. It is stable on freezing at -70°. Disc gel electrophoresis shows a gradual elimination of proprotein bands on purification. At the final stage of purification, scanning of Coomassie blue-stained gels gives 72% of the protein as the 97,000 molecular weight catalytic subunit, 19% as the 55,000 molecular weight glycoprotein, and 8.5% as the protein running with the tracking dye. The catalytic subunit and the glycoprotein can be isolated in milligram quantities by solubilization and chromatography on Sephadex G-150. About 90% of the protein applied to the column can be recovered. Sephadex chromatography gives a protein composition of 66% for the catalytic subunit, 28% for the glycoprotein, and 5% for the protein running with the tracking dye. The catalytic subunit and the glycoprotein enrich more or less in parallel as purification proceeds. At the last stage of purification, both proteins are found in the form of vesicles, which bear a striking resemblance on negative staining to purified cytochrome oxidase. Electron-translucent rods and rings which are approximately 80 A in diameter are also formed with projections of about 35 to 55 A at regular intervals. These rods and rings resemble reconstituted oligomycin-sensitive mitochondrial ATPase. The projections appear to be more hydrophilic, and this, coupled with their size, suggests that they are the glycoprotein. Incubation of the enzyme with [γ-32P]ATP, magnesium, and sodium gives 4,080 pmoles of acyl phosphate per mg of protein, which is 2 to 3 times higher than the highest levels of phosphorylation previously reported, thus attesting to the high purity of the enzyme preparation (the level of phosphorylation is generally accepted as being proportional to the number of NaK ATPase molecules in the preparation). The turnover number of the preparation is 6,300 min-1, which is within the range found for most NaK ATPase preparations from different organs and species. Arguments are presented for and against the view that the glycoprotein is a subunit of the NaK ATPase. If the catalytic subunit of molecular weight of 97,000 and the glycoprotein of molecular weight of 55,000 are both integral components of the NaK ATPase, the enzyme is 90 to 95% pure. If the 97,000 molecular weight subunit is the only subunit in the enzyme, the present preparation is 66 to 72% pure.