Isolation of a protein component of sodium-potassium transport adenosine triphosphatase containing ligand-protected sulfhydryl groups.

The sodiumand potassium-activated adenosine triphosphatase (Naf + 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[aH]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 sulfateprotein complexes by agarose titration 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 con&red to a single component. This component was coincident with the 98,000 molecular weight band.