Isolation of a Protein Component of Sodium-Potassium Transport Adenosine Triphosphatase Containing Ligand-protected Sulfhydryl Groups

The sodium- and potassium-activated adenosine triphosphatase (Na+ + K+)-ATPase was partially purified from a microsomal fraction of whole rabbit brain, homogenized in a deoxycholate buffer. Suspension and separation by flotation of the resulting lipoproteins in a linear gradient of potassium iodide formed the basis of a rapid preparative technique, whereby the enzyme-containing lipoproteins were harvested as a lipoid pellicle, following centrifugation in 1.5 m potassium iodide. After treatment of the resulting preparation with sodium dodecyl sulfate, disc gel electrophoresis revealed seven bands, the most prominent of which had a mobility corresponding to a molecular weight of 98,000. The enzyme protein was labeled in parallel incubations with N-ethyl[14C]maleimide and N-ethyl[3H]maleimide in the absence and presence, respectively, of protecting ligands after which it was solubilized in sodium dodecyl sulfate. The initial separation of the resulting sodium dodecyl sulfate-protein complexes by agarose filtration revealed the presence of ligand-protected sulfhydryl groups on a protein of high molecular weight. The sodium dodecyl sulfate-protein complexes were subsequently resolved by preparative disc gel electrophoresis, and the protected sulfhydryl groups were found to be confined to a single component. This component was coincident with the 98,000 molecular weight band.