Mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents. 1. Solubilization of large unilamellar liposomes (prepared by reverse-phase evaporation) by triton X-100, octyl glucoside, and sodium cholate.
The mechanisms governing the solubilization by Triton X-100, octyl glucoside, and sodium cholate of large unilamellar liposomes prepared by reverse-phase evaporation were investigated. The solubilization process is described by the three-stage model previously proposed for these detergents [Lichtenberg, D., Robson, R.J., & Dennis, E.A.(1983) Biochim. Biophys. Acta 737, 285-304]. In stage I, detergent monomers are incorporated into the phospholipid bilayers until they saturate the liposomes. At that point, i.e., stage II, mixed phospholipid-detergent micelles begin to form. By stage III, the lamellar to micellar transition is complete and all the phospholipids are present as mixed micelles. The turbidity of liposome preparations was systematically measured as a function of the amount of detergent added for a wide range of phospholipid concentrations (from 0.25 to 20 mM phospholipid). The results allowed a quantitative determination of RSat, the effective detergent to lipid molar ratios in the saturated liposomes, which were 0.64, 1.3, and 0.30 for Triton X-100, octyl glucoside, and sodium cholate, respectively. The corresponding ratios in the mixed micelles, RSol, were 2.5, 3.8, and 0.9 mol of detergent/mol of phospholipid. The monomer concentrations of the three detergents in the aqueous phase were also determined at the lamellar to micellar transitions (0.18, 17, and 2.8 mM, respectively). These transitions were also investigated by 31P NMR spectroscopy, and complete agreement was found with turbidity measurements. Freeze-fracture electron microscopy and permeability studies in the sublytic range of detergent concentrations indicated that during stage I of solubilization detergent partitioning between the aqueous phase and the lipid bilayer greatly affects the basic permeability of the liposomes without significantly changing the morphology of the preparations. A rough approximation of the partition coefficients was derived from the turbidity and permeability data (K = 3.5, 0.09, and 0.11 mM-1 for Triton X-100, octyl glucoside, and sodium cholate, respectively). It is concluded that when performed systematically, turbidity measurements constitute a very convenient and powerful technique for the quantitative study of the liposome solubilization process by detergents.
Paternostre
M T
MT
Département de Biologie, CEN Saclay, Gif-sur-Yvette, France.
Roux
M
M
Rigaud
J L
JL
D001436:Bacteriorhodopsins / Q000378:metabolism*; D019826:Cholic Acid; D002793:Cholic Acids; D003902:Detergents; D005452:Fluoresceins; D005614:Freeze Fracturing; D005960:Glucosides; D006027:Glycosides; D008081:Liposomes; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins; D008854:Microscopy, Electron; D008954:Models, Biological; D009391:Nephelometry and Turbidimetry; D017830:Octoxynol; D011092:Polyethylene Glycols; D011522:Protons; D013501:Surface-Active Agents
D002793:Cholic Acids; D003902:Detergents; D005452:Fluoresceins; D005960:Glucosides; D006027:Glycosides; D008081:Liposomes; D008565:Membrane Proteins; D011522:Protons; D013501:Surface-Active Agents; C018619:octyl-beta-D-glucoside; C024098:6-carboxyfluorescein; D011092:Polyethylene Glycols; D001436:Bacteriorhodopsins; D017830:Octoxynol; D019826:Cholic Acid