Purification and characterization of (Na+ + K+)-ATPase III. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate

Analysis of the protein composition by sodium dodecylsulphate gel electrophoresis and determination of the capacities for binding of ATP and ouabain and for sodium-dependent phosphorylation show that the procedures lead to a true purification of the enzyme.

Abstract

1. A procedure is presented that allows preparation of (Na+ + K+)-ATPase with specific activities of 32–37 units · mg−1protein (units refer to μmoles ATP hydrolysed per min) by incubation of a microsomal fraction with sodium dodecylsulphate and ATP followed by a single zonal centrifugation. 2. In a technically simple version of this procedure preparations with specific activities of 20–26 units · mg−1 protein are obtained in quantities of 12–20 mg protein by a single centrifugation, in an angle rotor, of microsomal fractions from the outer medulla of rabbit, pig or sheep kidneys. 3. Binding of ATP and ADP partially protects (Na+ + K+)-ATPase against inactivation by sodium dodceylsulphate. In the incubation with sodium dodecylsulphate and ATP, 80% of the protein in the microsomal fraction is solubilized whereas the (Na+ + K+)-ATPase remains bound to membrane fragments. Tracer analysis shows that sodium dodecylsulphate forms less than 0.5% of the mass of the purified preparations. 4. Analysis of the protein composition by sodium dodecylsulphate gel electrophoresis and determination of the capacities for binding of ATP and ouabain and for sodium-dependent phosphorylation (Jørgensen, P. L. (1974) Biochim. Biophys. Acta 356, 53–67 show that the procedures lead to a true purification of the enzyme.