Lipid selectivity of the calcium and magnesium ion dependent adenosine triphosphatase, studied with fluorescence quenching by a brominated phospholipid

Abstract

1,2-Bis(9,10-dibromooleoyl)phosphatidylcholine (BRPC) has been prepared from dioleoylphosphatidylcholine (DOPC). It is shown that the gel to liquid-crystalline phase transition for BRPC occurs below ca. 5 degrees C and that the motional properties of bilayers of BRPC and DOPC as detected by spin-labeled fatty acids are similar. The ATPase activities of the (Ca2+-Mg2+)-ATPase from rabbit muscle sarcoplasmic reticulum reconstituted with BRPC and DOPC are similar. The brominated lipid quenches the fluorescence of the ATPase and can be used to determine selectivity of lipid binding to the ATPase. We show that there is little selectivity on the basis of fatty acyl chain length. Binding constants for phosphatidylcholines and phosphatidylserines are similar in the absence of calcium, although that for phosphatidylserine decreases in the presence of calcium. Phosphatidylethanolamines binds less strongly than phosphatidylcholines, although the difference is small. The largest difference in binding constants is seen between phosphatidylcholines in the gel and liquid-crystalline phases, with a distribution coefficient of 30 in favor of the liquid-crystalline phase. It is shown that the distribution of the ATPase in mixtures of dipalmitoylphosphatidylcholine and BRPC can be understood in terms of the phase diagram for this mixture of lipids. Activities of the ATPase in the presence of mixtures of lipids can be explained in terms of the relative binding constants obtained from the fluorescence experiments.

Preservation of the native structure and function of Ca2+-ATPase from sarcoplasmic reticulum: Solubilization and reconstitution by new short-chain phospholipid detergent 1,2-diheptanoyl-sn-phosphatidylcholine

“…Light SR vesicles were prepared from the fast-twitch skeletal muscle of New Zealand White female rabbits by using a combination of the protocols of Fernandez et al [8] and East and Lee [3]. The SR preparation was frozen rapidly in liquid nitrogen and stored at 80 mC until use.…”

“…Ca# + -ATPase couples the transport of 2 mol of Ca# + across the SR membrane with the hydrolysis of 1 mol of ATP ; a conformational change accompanies the reaction cycle [1][2][3]. To elucidate fully the structure and function of Ca# + -ATPase, and particularly the role of lipid-protein interactions in the conformational changes that accompany ion translocation, it is necessary to isolate the protein and separate it from other membrane constituents.…”

“…To elucidate fully the structure and function of Ca# + -ATPase, and particularly the role of lipid-protein interactions in the conformational changes that accompany ion translocation, it is necessary to isolate the protein and separate it from other membrane constituents. The most successful approach so far involves the use of detergents for solubilization [3][4][5][6][7][8][9][10] and for reconstitution into defined lipids [11][12][13][14][15][16][17][18][19][20].…”

“…Ionic detergents such as deoxycholate [3] and cholate [4] and nonionic detergents such as Triton X-100 [8], C "# E * [7] and poly(oxyethylene)8-lauryl ether (C "# E ) ) [9,21] have been employed for the solubilization and purification of Ca# + -ATPase from SR. These procedures have produced varied results in terms of active enzyme yield, specific activity and formation of phosphorylated enzyme.…”

Photo-Crosslinking Analysis of Preferential Interactions between a Transmembrane Peptide and Matching Lipids

“…Previously, this has been probed mainly by fluorescence and ESR 1 methods using labeled lipids (5)(6)(7)(8)(9)(10)(11). In this study, we explore a more direct method to investigate the immediate surroundings of membrane proteins.…”