Selectivity of lipid-protein interactions with trypsinized Na,K-ATPase studied by spin-label EPR

The selectivity of the lipid-protein interactions in trypsinised Na,K-ATPase membranes from Squalus acanthias has been determined by using EPR spectroscopy with different lipid probes spin-labelled on the 14-C atom of the fatty acid chain. From measurements at low ionic strength and different pH values, the pattern of selectivity is: (stearic acid)(-)> (phosphatidylserine)(-) > (stearic acid)(0) > (phosphatidylcholine)(+/-), where superscripts indicate the formal electrostatic charge on the lipid headgroup. This is in the same order as that determined with native Na,K-ATPase membranes [M. Esmann, D. Marsh, Biochemistry 24 (1985) 3572-3578]. The selectivity for phosphatidylserine is independent of pH, over the range pH 6.0-9.0, as found also for native membranes. For membranes trypsinised in the presence of Rb+ ions, and in the presence of Na+ (which allows more extensive proteolysis), the relative association constants, K-r, of all lipids are the same as for control membranes, with the exception of ionised (stearic a.cid)- that shows the highest specificity. Therefore, both the stoichiometry and the principal determinants of the specificity of lipid-protein interaction are preserved on extensive trypsinisation of Na,K-ATPase membranes. This has implications for the location and arrangement of those amino acid side chains that determine the lipid selectivity of the native Na,K-ATPase. (C) 1998 Elsevier Science B.V. All rights reserved.