Phospholipids at the lipid--protein interface of membrane proteins are in dynamic equilibrium with fluid bilayer. In order to express the number of binding sites (N) and the relative binding constants (K) in terms of measurable quantities, the equilibrium is formulated as an exchange reaction between lipid molecules competing for hydrophobic sites on the protein surface. Experimental data are reported on two integral membrane proteins, cytochrome oxidase and (Na,-K)-ATPase, reconstituted into defined phospholipids. Electron spin resonance measurements on reconstituted preparations of beef heart cytochrome oxidase in 1,2-dioleoyl-sn-3-phosphatidylcholine containing small quantities of the spin-labeled phospholipid 1-palmitoyl-2-(14-proxylstearoyl)-sn-3-phosphatidylcholine (PC*) gave a linear plot of bilayer/bound PC* vs. the lipid/protein ratio as predicted by the theory, with K congruent to 1 and N = 40 (normalized to heme aa3). This demonstrates that the spin-label moiety attached to the hydrocarbon chain does not significantly perturb the binding equilibria. In the second experimental system, (Na,K)-ATPase purified from rectal glands of Squalus acanthias was reconstituted with defined phosphatidylcholines as the lipid solvent and spin-labeled phospholipids with choline or serine head groups (PC*, PS*) as the solute. The (Na,K)-ATPase has a larger number of lipid binding or contact sites (N = 60-65 per alpha 2 beta 2 dimer) and exhibits a detectably larger average binding constant for the negatively charged phosphatidylserine than for the corresponding phosphatidylcholine. These results show that a multiple equilibria, noninteracting site binding treatment can account for the behavior of lipids exchanging between the protein surface and the lipid bilayer. Selective sites among a background of nonselective sites are experimentally detectable as a change in the measured relative binding constant.

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Lipid interactions with the integral membrane protein NaK-ATPase (ATP phosphohydrolase, EC 3.6.1.3) purified from the electric organ of Electrophorus electricus were studied by spin labeling. A protein-associated component (boundary layer) in equilibrium with the fluid bilayer is clearly evident in the electron spin resonance spectra. The influence of charge on this equilibrium was determined by varying the head group of the lipid while maintaining the chain length and the position of the label constant. The lipid spin labels were 14-proxylstearylmethyl phosphate and the corresponding dimethylphosphate, alcohol, and quaternary amine. By using a pairwise spectral analysis, as well as a conventional spectral analysis, the binding affinity was found to decrease in the order of negative > neutral > positive charges. The fraction bound decreased from about 0.57 for the negatively charged phosphate to 0.25 for the positively charged quaternary amine. The amount of each boun lipid was nearly constant over the temperature range investigated (5-35 'C) High salt concentrations reversibly abolished the selectivity between the labels, confirming the role of charge in the binding equilibria. Na,K-ATPase (ATP phosphohydrolase, EC 3.6.1.3) is a transmembranous protein involved in the active transport of Na+ and K+ in animal cells (1). All active Na,K-ATPase preparations contain phospholipids, and delipidation by detergents or phospholipase digestion inactivates the enzyme. The activity often can be restored partially by adding lipids (for review, see ref.2). Anionic lipids, including several detergents (3-8), reactivate lipid-depleted Na,K-ATPase the most efficiently. Cationic lipids have not been observed to reactivate the enzyme (6-8); conflicting results have been obtained with neutral phospholipids (2).These enzyme activity measurements provide important information on the effects of lipids at the functional level. However, there is little information available on the corresponding structure and organization of protein and lipids at the molecular level. The activation by negatively charged lipids could be caused indirectly by altering the properties of the bilayer or directly by selective interactions with the protein. In order to test for direct interactions, we have examined membranous preparations of purified NaK-ATPase from the electric eel by using lipid spin labels with polar head groups of different charges. Electron spin resonance (ESR) provides a means of distinguishing between protein-associated lipid label and the fluid bilayer component (9). Our results show that there is a definite preference for the negatively charged lipid at the lipid-protein interface. The temperature dependence and the origin of this charge preference are examined. MATERIALS AND METHODSSample Preparation. Na,K-ATPase was purified from the electric organ of the electric eel Electrophorus electricus (10) and had a specific activity of 25 ,mol of Pi/min per mg of protein. Of the protein staining with Coomassie blue on sodium dodecyl...