Low-Angle Neutron Scattering Analysis of Na/K—Atpase in Detergent Solution

Abstract

The Na/K—ATPase is a membrane-bound enzyme system found in all eucaryotic cells. This enzyme regulates Na and K concentrations and energy metabolism, as well as controlling the transport of other cellular constituents. The Na/K—ATPase has been isolated to about 90% purity in a membrane-bound form from kidney outer medulla by detergent extraction (Jorgenson et al., Biochim. Biophys. Acta 694:27). The pure enzyme consists of two major subunits: the catalytic subunit (a) having an estimated molecular weight of 90 to 120K, and another subunit of unknown function (β) with molecular weight estimated as 45 to 65K. The stoichiometry of the subunits has been the subject of some debate. However, the bulk of the evidence suggests that the ratio of subunits (α/β) is 1/1, and the enzyme exists as a dimer in vivo (α₂, β₂). Analysis of low-angle neutron scattering from a solution containing detergent-solublized membrane protein provides a direct method of determining molecular weight and radius of gyration of a membrane protein. In this study, a purified Na/K—ATPase preparation from guinea pig outer medulla was first solubilized with the nonionic detergent Brij 58 (a polyoxyethylene ether) at a weight ratio of 5 mg detergent/mg protein. The purified protein was further delipidated by ion-exchange chromatography on DEAE in the presence of 0.2% Brij 58. The ATPase-Brij complex preserved 30% of the original membrane-bound Na/K—ATPase activity at V_max. Contrast matching by varying the H₂O/D₂O ratio of the detergent-protein solution was used to eliminate the scattering contribution of the detergent. The scattering intensity at zero angle for a set of proteins of known molecular weight was derived experimentally; the molecular weight of the Na/K—ATPase was thus determined to be 385 to 421K by measuring the zero-angle scattering intensity from the protein-detergent complex at the contrast match-point of the detergent. The radius of gyration of the Na/K—ATPase was found to be 76.2 Å. The molecular weight and radius of gyration indicate that the Na/K—ATPase is in the dimer form (α₂, β₂) in this micellar preparation. (Work supported by DOE and partially by NIH and NSF.)