Membrane-surfactant interactions. The role of surfactant in mitochondrial complex III-phospholipid-Triton X-100 mixed micelles.
Complex III (ubiquinol-cytochrome c reductase) was purified from beef heart mitochondria in the form of protein-phospholipid-Triton X-100 mixed micelles (about 1:80:100 molar ratio). Detergent may be totally removed by sucrose density gradient centrifugation, and the resulting lipoprotein complexes retain full enzyme activity. In order to understand the role of surfactant in the mixed micelles, and the interaction of Triton X-100 with integral membrane proteins and phospholipid bilayers, both the protein-lipid-surfactant mixed micelles and the detergent-free lipoprotein system were examined from the point of view of particle size and ultrastructure, enzyme activity, tryptophan fluorescence quenching, 31P NMR, and Fourier transform infrared spectroscopy. The NMR and IR spectroscopic studies show that surfactant withdrawal induces a profound change in phospholipid architecture, from a micellar to a lamellar-like phase. However, electron microscopic observations fail to reveal the existence of lipid bilayers in the absence of detergent. We suggest that, under these conditions, the lipid:protein molar ratio (80:1) is too low to permit the formation of lipid bilayer planes, but the relative orientation and mobility of phospholipids with respect to proteins is similar to that of the lamellar phase. Protein conformational changes are also detected as a consequence of surfactant removal. Fourier transform infrared spectroscopy indicates an increase of peptide beta-structure in the absence of Triton X-100; changes in the amide II/amide I intensity ratio are also detected, although the precise meaning of these observations is unclear. Tryptophanyl fluorescence quenching by acrylamide shows that a significant fraction of the Trp residues sensing the quencher become less readily available to it in the absence of surfactant. The temperature dependence of enzyme activity (expressed in the form of Arrhenius plots) is also different in the presence and absence of detergent. The effects of surfactant removal do not appear to be readily reversible upon readdition of Triton X-100.
D020106:Acrylamide; D000178:Acrylamides; D000818:Animals; D002417:Cattle; D003102:Colloids; D014450:Electron Transport Complex III; D007425:Intracellular Membranes / Q000378:metabolism*; D007454:Iodides; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008854:Microscopy, Electron; D008929:Mitochondria, Heart / Q000032:analysis; D009097:Multienzyme Complexes; D017830:Octoxynol; D010743:Phospholipids; D011092:Polyethylene Glycols; D011808:Quinone Reductases; D012542:Scattering, Radiation; D013055:Spectrophotometry, Infrared; D013501:Surface-Active Agents / Q000378:metabolism*
D000178:Acrylamides; D003102:Colloids; D007454:Iodides; D008823:Micelles; D009097:Multienzyme Complexes; D010743:Phospholipids; D013501:Surface-Active Agents; D020106:Acrylamide; D011092:Polyethylene Glycols; D017830:Octoxynol; D011808:Quinone Reductases; D014450:Electron Transport Complex III