Change in oligomeric structure of solubilized Na+/K(+)-ATPase induced by octaethylene glycol dodecyl ether, phosphatidylserine and ATP.
Membrane-bound Na+/K(+)-ATPase purified from dog kidney was solubilized with octaethylene glycol dodecyl ether (C12E8), and the resultant solubilized enzyme was chromatographed on a TSKgel G4000SWXL or G3000SWXL column equilibrated with elution buffers containing various ligands affecting oligomerization of the enzyme. Weight-averaged molecular weight (Mw) values for the main protein components eluted were estimated by low-angle laser light-scattering photometry. With increasing concentration of C12E8 included in the elution buffer from 0.1 to 5 mg/ml, the Mw decreased from 230,000 to 153,000, indicating that C12E8 induced dissociation of the enzyme. In contrast, the Mw of the protein component increased up to 1.44.10(6) as the concentration of phosphatidylserine (PS) added to the elution buffer containing a fixed concentration of 0.3 mg/ml C12E8 was increased to 120 micrograms/ml. The association and/or aggregation were reversible by removal of the PS by rechromatography. Addition of PS to the elution buffer also allowed the solubilized enzyme to exhibit ATPase activity comparable to that of the membrane-bound enzyme during passage through the column. This was also the case with phosphatidylglycerol (PG) and phosphatidylinositol, but not with phosphatidylcholine or phosphatidylethanolamine. The specific refractive index increment (dn/dcp) of the solubilized enzyme was increased by addition of exogenous PG or PS, strongly suggesting that the phospholipid became bound to the enzyme, and that it induced association of the enzyme. The association induced by PS was inhibited by ATP and ADP, but not AMP. The concentrations for half-maximal inhibition were 0.44 mM for ATP and 0.88 mM for ADP. The PS-induced associated enzyme isolated by chromatography in the presence of 120 micrograms/ml PS was dissociated by ATP with K0.5 of 0.16 mM. The dissociating effect of C12E8, ATP and ADP and the associating effect of PS on the solubilized enzyme are consistent with the reports that C12E8 mimics the effect of regulatory ATP at the low-affinity site on the conformational transition from E2 to E1, and that phospholipids are essential for the reverse transition from E1 to E2. The results can be explained by assuming that the enzyme takes the form of a loosely associated diprotomer in the E1 state and a tightly associated one in the E2 state.