Binding of a nonionic detergent to membranes: flip-flop rate and location on the bilayer.
The kinetic aspects of amphiphile interaction with intact membranes (unilamellar and multilamellar liposomes, sarcoplasmic reticulum vesicles) were studied, with the nonionic detergent octa(ethylene glycol) dodecyl monoether (C12E8) as a prototype. C12E8 was bound to these membranes noncooperatively and with a maximum of 0.6-0.8 mol per mole of phospholipid, before the onset of solubilization. Binding was not affected by ultrasonication to expose internal binding sites on the inner leaflet. All detergent could be removed from the membranes by treatment with hydrophobic beads. Furthermore, bound detergent, also from the inside of multilayered liposomes, comprising 10-20 bilayers, was quickly released by dilution of the membranes, followed by gel filtration. The time course of these processes was investigated with a rapid-filtration apparatus, using glass fiber filters to deposit membrane material. Both detergent binding and removal could be described by a monoexponential process with a half-time of approximately 350 ms for all types of membranes. Binding of detergent enhanced the intrinsic fluorescence of sarcoplasmic reticulum vesicles. This occurred in less than 100 ms, probably as the result of direct interaction of C12E8 with Ca2+-ATPase at a few binding sites. The data show that flip-flop of C12E8 across lipid membranes is a rapid process that cannot account for incomplete detergent removal in reconstitution experiments [Ueno, M., Tanford, C., & Reynolds, J. A. (1984) Biochemistry 23, 3070-3076]. It is also suggested that other nonionized amphiphiles, including those with an anesthetic action, rapidly gain access to membrane proteins on the inside of the cell, even when used at low, clinical doses.
le Maire
M
M
Centre de Génétique Moléculaire, Laboratoire du Centre National de la Recherche Scientifique Associé à l'Université Pierre, Gif-sur-Yvette, France.
Møller
J V
JV
Champeil
P
P
D000818:Animals; D002850:Chromatography, Gel; D003902:Detergents; D007700:Kinetics; D008051:Lipid Bilayers; D008081:Liposomes; D009132:Muscles / Q000378:metabolism; D011092:Polyethylene Glycols; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013501:Surface-Active Agents
D003902:Detergents; D008051:Lipid Bilayers; D008081:Liposomes; D013501:Surface-Active Agents; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols