Fluorescence quenching in model membranes. 2. Determination of local lipid environment of the calcium adenosinetriphosphatase from sarcoplasmic reticulum.
Fluorescence quenching by spin-labeled phospholipid is used to determine the affinities of different phospholipids species to an intrinsic membrane protein, the Ca2+-ATPase of sacroplasmic reticulum. The phospholipids in contact with the Ca2+ ATPase are examined in a reconstituted system in which the enzyme is incorporated into a model membrane of defined phospholipid composition. The local phospholipid environment of the protein is considered to be governed at each phospholipid binding site by an equilibrium: lipid A + (lipid B - protein) in equilibrium or formed from lipid B + (lipid A - protein). Phospholipid binding constants to the Ca2+ ATPase can be obtained from an analysis of fluorescence quenching data. The binding constants for a number of phospholipid species are nearly identical when the phospholipids are in the liquid-crystal state. However, temperature of Ca2+-induced phase separation of phospholipid induces striking changes in the composition of the phospholipids in contact with the Ca2+ATPase, relative to the overall composition of the membranes. The implications of these results with respect to the control of local phospholipids environment by intrinsic membrane proteins and the nature of the phospholipids binding sites on the proteins are discussed. General applicability of this type of fluorescence quenching study to the problem of lipid-protein interactions in membranes is considered, and this method is compared to other techniques.
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008433:Mathematics; D008563:Membrane Lipids / Q000502:physiology*; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000502:physiology*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013696:Temperature
D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010743:Phospholipids; D000252:Calcium-Transporting ATPases