Competition between cholesterol and phosphatidylcholine for the hydrophobic surface of sarcoplasmic reticulum Ca2+-ATPase.
A multiple equilibrium binding model is used to examine phospholipid and cholesterol binding with the transmembranous protein Ca2+-ATPase (calcium pump). The protein was reconstituted in egg phosphatidylcholine bilayers by lipid substitution of rabbit muscle sarcoplasmic reticulum. Electron spin resonance spectra of a phosphatidylcholine spin-label and a recently developed cholesterol spin-label show two major spectral contributions, a motionally restricted component consistent with interactions between the label and the protein surface and another component characteristic of motion of the label in a fluid lipid bilayer. The number of lipid binding (or contact) sites at the hydrophobic surface of the protein is calculated to be N = 22 +/- 2. Experiments with intact sarcoplasmic reticulum membranes give approximately the same value for N. The relative binding constants are Kav approximately 1 for the phosphatidylcholine label and Kav approximately 0.65 for the cholesterol spin-label. Thus, cholesterol does contact the surface of the protein, but with a somewhat lower probability than phosphatidylcholine. This is confirmed by competition experiments where unlabeled cholesterol and the phospholipid spin-label are both present in the bilayer. Evidently the flexible acyl chains of the phospholipid molecules accommodate more readily to the irregular surface of the protein than does the rigid steroid structure of cholesterol.
D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002645:Chickens; D002784:Cholesterol / Q000378:metabolism*; D004530:Egg Yolk; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D008051:Lipid Bilayers; D008958:Models, Molecular; D008968:Molecular Conformation; D009132:Muscles / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D013113:Spin Labels
D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D013113:Spin Labels; D002784:Cholesterol; D000252:Calcium-Transporting ATPases