QUANTITATIVE-ANALYSIS OF LIPID-LIPID AND LIPID-PROTEIN INTERACTIONS IN MEMBRANES BY USE OF PYRENE-LABELED PHOSPHOINOSITIDES

The lateral and rotational dynamics of phosphoinositides and their int eractions with proteins were characterized using pyrene-labeled lipid analogues. In these systems, the collision frequency of pyrene-labeled lipids was studied by monitoring the monomeric pyrene fluorescence yi eld as a function of their mole fraction in the membranes. From this d ependence, the lateral diffusion coefficient and a repulsion factor be tween two pyrene phosphoinositides could be estimated by applying an e xtended form of the Milling Crowd model [Eisinger, J., Flores, J., and Petersen, W. P. (1986) Biophys, J. 49, 987-1001]. The repulsion appea red to be highly dependent on the amount of negative charge of the lip id headgroups. From experiments with dioleoylphosphatidylcholine vesic les containing band 3 protein, the fraction of lipid molecules bound t o this protein and the minimum number of sites possessing affinity for phosphatidylinositol-4-phosphate could be approximately estimated. Th e results of this study indicate that phosphoinositides are located pr eferentially adjacent to band 3. Intramolecular excimer formation of d ipyrene-labeled phosphatidylcholine, phosphatidylinositol, and phospha tidylinositol-4-phosphate yielded information about the acyl chain dyn amics of Lipids surrounding the protein and of lipids in the bulk memb rane. Time-resolved measurements of the pyrene fluorescence anisotropy showed that in membranes of resealed erythrocyte ghost cells the rota tional freedom of pyrene-labeled phosphatidylinositol-4,5-bisphosphate is smaller than that of pyrene-labeled phosphatidylcholine. In contra st, no significant differences could be detected when these pyrene lip ids were dispersed in dioleoylphosphatidylcholine membranes, It is pro posed that the nonrandom distribution of the phosphoinositides induced by Lipid-lipid repulsion and protein-lipid attraction will have a pro found effect on the phospholipase C-catalyzed hydrolysis of the phosph oinositides into second-messenger molecules.