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.

Preferential interaction of trans-membrane alpha-helices whose hydrophobic length matches the hydrophobic thickness of the lipid bilayer could be a mechanism of retention in the Golgi apparatus. We have used fluorescence methods to study the interaction of peptides Ac-K2-G-Lm-W-Ln-K2-A-amide (Pm+n) with bilayers of phosphatidylcholines with chain lengths between C14 and C24. The peptide P22 (m = 10, n = 12) incorporates into all bilayers, but P16 (m = 7, n = 9) does not incorporate into bilayers when the fatty acyl chain length is C24 and only partly incorporates into bilayers where the chain length is C22. The strongest binding is seen when the hydrophobic length of the peptide matches the calculated hydrophobic thickness of the bilayer. It is suggested that a too-thin bilayer can match to a too-long peptide both by stretching of the lipid and by tilting of the peptide. However, a too-thick bilayer can only match a too-thin peptide by compression of the lipid, which becomes energetically unfavorable when the difference between the bilayer thickness and the peptide length exceeds about 10 A. The presence of cholesterol in the bilayer leads to a marked reduction in the incorporation of P16 into bilayers where the chain length is C18. Hydrophobic mismatch could explain retention of proteins with short trans-membrane alpha-helical domains in the Golgi, the effect following largely from the low concentration of cholesterol in the Golgi membrane compared to that in the plasma membrane.

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)

A spin-labeled phospholipid is used to study lipid-protein interactions in the (Ca2+,Mg2+)-ATPase of sarcoplasmic reticulum from muscle. A novel null method is used to decompose composite electron spin resonance spectra into two components, characteristic of immobilized and mobile environments. Calculations based on a random mixing model suggest that protein-protein interactions will be relatively rare in these systems and that the immobilized lipid does not represent lipid trapped between proteins but rather represents annular phospholipid at the lipid-protein interface of the adenosinetriphosphatase. The apparent decrease in the amount of immobilized lipid with increasing temperature is shown to be consistent with lipid exchange between bulk and annulus, characterized by an exchange time of 10(-7) s at 37 degrees C. A minimum number of annular phospholipid sites of 32 and 22 are calculated at 0 and 37 degrees C, respectively.

The report of the crystal structure of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum in its Ca(2+)-bound form [Toyoshima, Nakasako and Ogawa (2000) Nature (London) 405, 647-655] provides an opportunity to interpret much kinetic and mutagenic data on the ATPase in structural terms. There are no large channels leading from the cytoplasmic surface to the pair of high-affinity Ca(2+) binding sites within the transmembrane region. One possible access pathway involves the charged residues in transmembrane alpha-helix M1, with a Ca(2+) ion passing through the first site to reach the second site. The Ca(2+)-ATPase also contains a pair of binding sites for Ca(2+) that are exposed to the lumen. In the four-site model for transport, phosphorylation of the ATPase leads to transfer of the two bound Ca(2+) ions from the cytoplasmic to the lumenal pair of sites. In the alternating four-site model for transport, phosphorylation leads to release of the bound Ca(2+) ions directly from the cytoplasmic pair of sites, linked to closure of the pair of lumenal binding sites. The lumenal pair of sites could involve a cluster of conserved acidic residues in the loop between M1 and M2. Since there is no obvious pathway from the high-affinity sites to the lumenal surface of the membrane, transport of Ca(2+) ions must involve a significant change in the packing of the transmembrane alpha-helices. The link between the phosphorylation domain and the pair of high-affinity Ca(2+) binding sites is probably provided by two small helices, P1 and P2, in the phosphorylation domain, which contact the loop between transmembrane alpha-helices M6 and M7.