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, Kr, of all lipids are the same as for control membranes, with the exception of ionised (stearic acid)- 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.
Arora
A
A
Max-Planck-Institut für biophysikalische Chemie, Abt. Spektroskopie, D-37070 Göttingen, Germany.
Esmann
M
M
Marsh
D
D
D000818:Animals; D004284:Dogfish; D004578:Electron Spin Resonance Spectroscopy; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013113:Spin Labels; D014357:Trypsin / Q000378:metabolism*
D008565:Membrane Proteins; D013113:Spin Labels; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase