Effects of phosphatidylcholine fatty acyl chain length on calcium binding and other functions of the calcium-magnesium-ATPase

Abstract

The ATPase activity of the (Ca(2+)-Mg2+)-ATPase purified from skeletal muscle sarcoplasmic reticulum and reconstituted into phosphatidylcholine bilayers of defined composition depends on the fatty acyl chain length of the surrounding phospholipid. The stoichiometry of Ca2+ binding to the ATPase is also sensitive to fatty acyl chain length, changing from the normal two Ca2+ ions bound per ATPase molecule to one Ca2+ ion bound for the ATPase reconstituted with phosphatidylcholines of chain lengths C12, C14, or C24. For the ATPase reconstituted with mixture of phosphatidylcholines where one phosphatidylcholine supports a Ca2+ binding stoichiometry of two and the other a stoichiometry of one, a highly cooperative change in binding stoichiometry with change in phospholipid composition is observed, suggesting that the effects of phospholipids follow from binding to a large number of sites at the lipid-protein interface of the ATPase. For the ATPase reconstituted with either 1-myristoyl-2-oleoylphosphatidylcholine or 1-oleoyl-2-myristoylphosphatidylcholine, the stoichiometry of Ca2+ binding is the normal two per ATPase molecule. Effects of short-chain phosphatidylcholines on Ca2+ binding stoichiometry and on ATPase activity can be reversed by addition of androstenol, oleic acid, methyl oleate, or oleyl alcohol but these molecules have no effect on the ATPase reconstituted with dinervonylphosphatidylcholine (C24:1). For the ATPase reconstituted with phosphatidylcholines with chain lengths between C16 and C22, release of the two bound Ca2+ ions is sequential, with release of the second Ca2+ being inhibited by high concentrations of Ca2+ in the bathing medium.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…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].…”

Mutual adaptation of a membrane protein and its lipid bilayer during conformational changes

“…On the basis of a vast amount of experimental data on SERCA, much is known about the structure and function of this transport protein. Nevertheless, some structural aspects remain relatively unexplored, such as the interplay of SERCA with its immediate lipid environment as well as the effect of the general physical properties of the lipid bilayer 5,13,14 . For an analysis of these effects, we took advantage of SERCA crystals grown from purified SR membranes, which are solubilized by octaethyleneglycol dodecyl monoether (C 12 E 8 ) detergent in a manner that retains the functional properties of the ATPase [15][16][17] .…”

Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

“…In the simulations, an internal volume of 174 µl\mg protein was assumed with a random orientation of the ATPase across the membrane, in agreement with experimental data ( Table 1). The proportion of active ATPase in the SR preparations can be determined by measuring the maximal level of phosphorylation observed with ATP in the presence of high concentrations of Ca# + ; maximal levels of phosphorylation have been shown to vary between preparations, but are typically about 3n2 nmol\mg protein [42], corresponding to 35 % of the ATPase being active. With these parameters, the simulations match the experimental data with slippage rates of 250 s −" , 65 s −" and 0 in the absence of anionic phospholipid and in the presence of 10 mol % DOPA and cardiolipin respectively, and with a slippage rate of 45 s −" in the presence of 5 mol % cardiolipin (Table 3).…”