Fluorescence quenching in model membranes. 3. Relationship between calcium adenosinetriphosphatase enzyme activity and the affinity of the protein for phosphatidylcholines with different acyl chain characteristics

The dependence of function and lipid binding affinity of an integral transport protein on the fatty acyl chain characteristics of a membrane-forming phospholipid have been determined. When a newly developed fluorescence quenching technique [London, E., & Feigenson, G. W. (1981) Biochemistry (first paper of three in this issue); London, E., & Feigenson, G. W. (1981) Biochemistry (preceding paper in this issue)] is used for examining lipid-protein interactions in membranes, the Ca2+ ATPase from rabbit sarcoplasmic reticulum is found to bind with equal affinity a large variety of phosphatidylcholines used to reconstitute the protein into enzymatically active vesicles, regardless of fatty acyl chain length or details of unsaturation. In parallel with the lipid binding studies, we have measured the sensitivity of the catalytic activity of the Ca2+ ATPase to the fatty acyl chain characteristics of the phosphatidylcholine membranes in which the enzyme was reconstituted. The enzyme appears to be sensitive only to the effective fatty acyl chain length, which determines the thickness of the bilayer in which the protein is inserted and displays little sensitivity to such details of unsaturation as degree, position, and isomeric type. Both ATP hydrolyzing and Ca2+ transporting activities of the enzyme were similarly affected by bilayer thickness, and maximum activity was observed in membranes of intermediate thickness. These observations are reconciled in a number of possible models for the manner in which this integral protein interacts with membranes of varying thickness. A freeze-thaw method was used to reconstitute the Ca2+ ATPase, and the vesicles so obtained have been characterized by gel permeation chromatography, density gradient centrifugation, and electron microscopy, (thin section). Convenient methods are described for (a) rapidly separating reconstituted Ca2+ ATPase from unincorporated protein simultaneously in a large number of small samples, giving good recovery of fractionated vesicles without significant dilution, and (b) measuring leakiness to Ca2+ of reconstituted vesicles. Additionally, the gel and liquid-crystal phase transition temperature and bilayer thickness have been determined respectively by differential thermal analysis and low-angle X-ray diffraction for some of the synthetic phosphatidylcholines, which range in chain length from 12 to 24 carbon atoms.     

The effect of bilayer thickness on the activity of (Na+ + K+)-ATPase

The activities of purified (Na+ + K+)-ATPase supported by a series of phosphatidylcholines with monounsaturated (cis-9) fatty acyl chains (di(n : 1) phosphatidylcholine) varying in length from n = 12 to n = 23 were determined by the lipid titration technique. The ATPase activity at 20 degrees C decreased from 2.9 to 0.1 mumol/min per mg protein as n was decreased from 16 to 12 and decreased from 2.9 to 1.0 mumol/min per mg protein as n was increased from 20 to 23. In further experiments, the di(n : 1) phosphatidylcholine-ATPase complexes were treated with increasing proportions of n-decane, which has been shown previously to increase the thickness of black lipid membranes. n-Decane caused a large increase (greater than 20-fold) in activity of the short-chain complexes (n = 12,13); for n = 14--18, the ATPase activity first increased and subsequently decreased as the proportion of decane was increased, and for n = 20 or 23 decane caused a progressive decrease in activity with increasing concentration. These effects confirm qualitatively that a major factor determining the activity in each bilayer is its thickness. This behaviour closely parallels that of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum [1] and suggests that a major class of trans-membrane transport proteins may have a similar dependence on bilayer thickness.     

Mechanism of action of "ruthenium red" compounds on Ca2+ ionophore from sarcoplasmic reticulum (Ca2+ + Mg2+)- adenosine triphosphatase and lipid bilayer.

Sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase was previously shown to have Ca2+-dependent and -selective ionophoric activity when tested in oxidized cholesterol lipid bilayer membranes (Shamoo, A. E., and MacLennan, D. H. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 3522). ruthenium red, a known inhibitor of (Ca2+ + Mg2+)-ATPase, is found to inhibit the Ca2+-ionophoric activity associated with (Ca2+ + Mg2+)-ATPase. Furthermore, ruthenium red alone acts as an anion-selective ionophore in lipid bilayers with the the following selectivity sequence for anions: l- greater than Cl-, Br- greater than F- greater than NO3-. The PCl-/PNa+ ratio was approximately 4/l. The presence of ruthenium red in excess of Ca2+ ionophore in lipid bilayer experiments converts the cation selectivity of the bilayer due to Ca2+ ionophore into anion selectivity.     

Effects of lipids and long-chain alkyl derivatives on the activity of (Ca2+-Mg2+)-ATPase

The ATPase activity of the (Ca2+-Mg2+)-ATPase reconstituted into bilayers of phosphatidylcholines depends on the fatty acyl chain length of the phospholipids. It is shown that the fluorescence response to Ca2+ of the ATPase modified with fluorescein isothiocyanate is also dependent on phospholipid structure and is interpreted in terms of a change in the equilibrium between two forms of the ATPase, E1 and E2. A kinetic scheme for the ATPase is presented in which ATPase activity is markedly dependent on the rate of the transition between two phosphorylated forms of the ATPase, E1'PCa2 and E2'PCa2, and it is postulated that changing the phospholipid structure changes this rate. The rate of dephosphorylation of the ATPase and the ATP dependence of the E1'PCa2-E2'PCa2 transition are also lipid dependent. Binding of oleyl alcohol causes large, lipid-dependent changes in ATPase activity, and these are interpreted in terms of changes in the rates of these same steps. Oleylamine, which has been shown to bind more strongly at annular sites than at nonannular sites, inhibits ATPase activity irrespective of lipid structure, whereas fatty acids, which bind less strongly at annular sites, only inhibit at high concentrations. Methyl oleate, which binds more strongly at nonannular sites than at annular sites, causes marked stimulation for the ATPase reconstituted with short-chain lipids.     

The behaviors of Ca(2+)-ATPase embedded in interdigitated bilayer.

We investigated the behavior of a membrane protein, Ca(2+)-ATPase, in interdigitated phospholipid bilayers. The results showed that Ca(2+)-ATPase does not cause significant alterations in the interdigitation of 16:0 LPC/DPPC (27.0 mol% LPC) vesicles when it is reconstituted with lipids. Intrinsic fluorescence, acrylodan fluorescent adducts, and CD spectra indicated that Ca(2+)-ATPase, when embedded in interdigitated bilayer structures, is more exposed to the hydrophilic environment and has a looser structure than when embedded in non-interdigitated bilayers. The interdigitation of acyl chains induces a rapid loss of enzyme activity. It is suggested that interdigitated bilayer structures may play an important role as negative regulatory factors in physiological functions.     

Calcium-dependent binding of annexin 12 to phospholipid bilayers: stoichiometry and implications

Annexins (ANXs) are a superfamily of proteins whose functional hallmark is Ca2+-dependent binding to anionic phospholipids. Their core domains are usually composed of a 4-fold repeat of a conserved amino acid sequence, with each repeat containing a type II Ca2+ binding site that is generally thought to mediate Ca2+-dependent binding to the membrane. We now report that ANX12 binding to phospholipid vesicles is highly cooperative with respect to Ca2+ concentration (Hill constant approximately 7), thereby suggesting that more than the four well-characterized type II Ca2+ binding sites are involved in phospholipid binding. Two independent approaches, a novel 45Ca2+ copelleting assay and isothermal titration calorimetry, indicate a stoichiometry of approximately 12 mol of Ca2+/mol of ANX12 for binding to phospholipid vesicles. On the basis of the "low-affinity" Ca2+-binding sites in a number of ANX X-ray crystal structures, we propose a model for ANX12 bilayer binding that involves three types of Ca2+ sites in each of the four repeats. In this model, there is a complementarity between the spacing of the ANX12 Ca2+ binding sites and the spacing of the phospholipid headgroups in bilayers. We tested the implications of the model by manipulating the physical state of vesicles composed of phospholipids with saturated acyl chains with temperature and measuring its influence on ANX12 binding. ANX12 bound to vesicles in a Ca2+-dependent manner when the vesicles were in the liquid crystal phase but not when the phospholipid was in the gel phase. Furthermore, ANX12 bound initially to fluid bilayers remained bound when cooled to 4 degrees C, a temperature that should induce the gel phase transition. Overall, these studies suggest that ANX12 is well suited to being a Ca2+ sensor for rapid all-or-none intercellular membrane-related events.     

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.     

Influence of N-dodecyl-N,N-dimethylamine N-oxide on the activity of sarcoplasmic reticulum Ca(2+)-transporting ATPase reconstituted into diacylphosphatidylcholine vesicles: efects of bilayer physical parameters

Sarcoplasmic reticulum Ca-transporting ATPase (EC 3.6.1.38) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 degrees C, the specific activity of ATPase displays a maximum (31.5+/-0.8 IU/mg) for dioleoylphosphatidylcholine (diC18:1PC) and decreases progressively for both shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydration and lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstituted into dierucoylphosphatidylcholine (diC22:1PC), the zwitterionic surfactant N-dodecyl-N,N-dimethylamine N-oxide (C12NO) stimulates the ATPase activity from 14.2+/-0.6 to 32.5+/-0.8 IU/mg in the range of molar ratios C12NO:diC22:1PC=0/1.2. In dilauroylphosphatidylcholines (diC12:0PC) and diC18:1PC, the effect of C12NO is twofold-the ATPase activity is stimulated at low and inhibited at high C12NO concentrations. In diC18:1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO:diC18:1PC< or =1.3 in bilayers, where the bilayer thickness estimated by SANS decreases by 0.4+/-0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increases indicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N(+)-P- accompanied by a variation of the local membrane dipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO:diC18:1PC=1.3/2.5, where mixed tubular micelles are detected by SANS in C12NO+diC18:1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes in dipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca(2+)-ATPase activity.     

The role of phospholipid acyl chains in the activation of mitochondrial ATPase complex.

1. The role of length and unsaturation of phospholipid acyl chains in the activation of ATPase complex was studied with synthetic phosphatidylcholines and a phospholipid-dependent preparation obtained after cholate-extraction of submitochondrial particles (Kagawa, Y. and Racker, E. (1966) J. Biol. Chem. 241, 2467--2474). 2. Micelle-forming, short-chain phosphatidylcholines produced activation only at critical micellar concentration. The reactivated complex was cold-stable but the oligomycin sensitivity was low. 3. Bilayer-forming saturated phosphatidylcholines produced activation which was maximal at 9 carbon atoms in each chain but decreased sharply as the chain-length was increased and essentially disappeared at 14 carbon atoms. By contrast the oligomycin-sensitivity increased with the increase in chain length. 4. Activation of ATPase complex reappeared when bilayers were formed with long-chain unsaturated phosphatidylcholines. The activity was oligomycin sensitive. Significant inhibition of activity was observed also after incorporation of cholesterol into the bilayers. 5. By contrast the activation induced by negatively charged liposomes of diacylphosphatidylglycerol was independent on acyl-chain composition and occurred at very low amounts of phospholipid. 6. The discontinuity in the Arrhenius plot of activity of the ATPase complex reactivated with saturated phospholipids was found at temperatures close to the gel-to-liquid crystalline transition of the lipid showing that the activity of ATPase complex was sensitive to the physical state of membrane phospholipids. 7. It is concluded that (a) reactivation of ATPase complex by isoelectric phospholipids is an interfacial activation, the minimum requirement for the lipid effect being micelle formation. (b) In order to gain the properties of the native complex a stable lamellar phase is needed. Both activity and oligomycin sensitivity are regulated by the chain length and degree of unsaturation of phospholipid acyl chains.     

Organization of lipids in sarcoplasmic reticulum membrane and Ca2+-dependent ATPase activity.

The organization of lipids in sarcoplasmic reticulum membrane was studied with a variety of stearic spin labels and a phosphatidylcholine spin label. The ESR spectra of the spin-labeled membranes consisted of two components, one due to labels in lipid bilayer structure and the other due to more immobilized labels. The relative intensity of the immobilized component increased when the lipid content of the membrane was decreased by treatment with phospholipase A [EC 3.1.1.4] and subsequent washing with bovine serum albumin. Membrane containing 30% of the intact phospholipid, i.e.0.15 mg of phospholipid per mg of protein, showed a spectrum consisting only of the immobilized component (the overall splitting ranged from 58.5 G to 60.5 G). The immobilized component was ascribed to lipids complexed with protein. The fraction of lipids in the two different organizations was determined from the ESR spectrum. The activity of the Ca2+-Mg2+ dependent ATPase [ATP phosphohydrolase, EC 3.6.1.3] was found to increase almost linearly with the lipid bilayer content in the membrane, whereas phosphoenzyme formation was almost independent of the bilayer content. This indicated that the bilayer structure is necessary for the ATPase to attain its full transport activity.     

Effects of lipid fatty acyl chain structure on the activity of the (Ca2+ + Mg2+)-ATPase

The (Ca2+ + Mg2+)-ATPase purified from rabbit muscle sarcoplasmic reticulum has been reconstituted into a series of phosphatidylcholines in the liquid crystalline phase. For phosphatidylcholines containing monounsaturated fatty acyl chains, optimal activity is observed for a chain length of C18, with longer or shorter chains supporting lower activities. Phospholipids with methyl-branched chain saturated fatty acids support somewhat lower activities than the corresponding phospholipids with mono-unsaturated fatty acids. Mixed chain phospholipids support ATPase activities comparable to those shown by an unmixed chain phospholipid with the same average chain length. However, the response of the ATPase reconstituted with mixed chain phospholipids to the addition of oleyl alcohol is dominated by the longest fatty acyl chain. Based on their ability to displace brominated phospholipids, relative binding constants to the ATPase of a series of phosphatidylcholines have been determined. Binding to the ATPase is virtually unaffected by fatty acyl chain length or the presence of methyl branches.     

The effect of Mg2+ on hepatic microsomal Ca2+ and Sr2+ transport

The ATP-dependent uptake of Ca2+ by rat liver microsomal fraction is dependent upon Mg2+. Studies of the Mg2+ requirement of the underlying microsomal Ca2+-ATPase have been hampered by the presence of a large basal Mg2+-ATPase activity. We have examined the effect of various Mg2+ concentrations on Mg2+-ATPase activity, Ca2+ uptake, Ca2+-ATPase activity and microsomal phosphoprotein formation. Both Mg2+-ATPase activity and Ca2+ uptake were markedly stimulated by increasing Mg2+ concentration. However, the Ca2+-ATPase activity, measured concomitantly with Ca2+ uptake, was apparently unaffected by changes in the Mg2+ concentration. In order to examine the apparent paradox of Mg2+ stimulation of Ca2+ uptake but not of Ca2+-ATPase activity, we examined the formation of the Ca2+-ATPase phosphoenzyme intermediate and formation of a Mg2+-dependent phosphoprotein, which we have proposed to be an attribute of the Mg2+-ATPase activity. We found that Ca2+ apparently inhibited formation of the Mg2+-dependent phosphoprotein both in the absence and presence of exogenous Mg2+. This suggests that Ca2+ may inhibit (at least partially) the Mg2+-ATPase activity. However, inclusion of the Ca2+ inhibition of Mg2+-ATPase activity in the calculation of Ca2+-ATPase activity reveals that this effect is insufficient to totally account for the stimulation of Ca2+ uptake by Mg2+. This suggests that Mg2+, in addition to stimulation of Ca2+-ATPase activity, may have a direct stimulatory effect on Ca2+ uptake in an as yet undefined fashion. In an effort to further examine the effect of Mg2+ on the microsomal Ca2+ transport system of rat liver, the interaction of this system with Sr2+ was examined. Sr2+ was sequestered into an A23187-releasable space in an ATP-dependent manner by rat liver microsomal fraction. The uptake of Sr2+ was similar to that of Ca2+ in terms of both rate and extent. A Sr2+-dependent ATPase activity was associated with the Sr2+ uptake. Sr2+ promoted formation of a phosphoprotein which was hydroxylamine-labile and base-labile. This phosphoprotein was indistinguishable from the Ca2+-dependent ATPase phosphoenzyme intermediate. Sr2+ uptake was markedly stimulated by exogenous Mg2+, but the Sr2+-dependent ATPase activity was unaffected by increasing Mg2+ concentrations. Sr2+ uptake and Sr2+-dependent ATPase activity were concomitantly inhibited by sodium vanadate. In contrast to Ca2+, Sr2+ had no effect on Mg2+-dependent phosphoprotein formation. Taken together, these data indicate that Mg2+ stimulated Ca2+ and Sr2+ transport by increasing the Ca2+ (Sr2+)/ATP ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphorylated intermediates of two hepatic microsomal ATPases.

The hepatic microsomal Ca2+- and Mg2+-dependent ATPase phosphoenzyme intermediates were distinguished by using the chelators EGTA and CDTA (trans-cyclohexane-1,2-diamine-NNN'N'-tetra-acetic acid). The Ca2+-ATPase intermediate is a hydroxylamine-labile base-labile 125 000-Mr phosphoprotein. The Mg2+-ATPase intermediate is a hydroxylamine-stable base-stable 30 000-Mr phosphoprotein. This enzyme intermediate probably reflects the large basal ATPase activity of hepatic microsomal fraction. It is dependent on Mg2+, since formation of the phosphoenzyme is abolished in the presence of CDTA. Under these conditions, the basal ATPase activity is dramatically decreased. These data demonstrate two separate and distinct enzymes which are responsible for the two ATPase activities of hepatic microsomal fraction. Furthermore, these data indicate that more meaningful data about the microsomal Ca2+-ATPase might be obtained if the free ion concentrations are controlled with CDTA.     

Fluorescence and kinetic studies of the interactions of pyrethroids with the (Ca2(+) + Mg2+)-ATPase

The fluorescence quenching properties of a series of brominated and iodinated pyrethroids have been used to study the binding of pyrethroids to the (Ca2(+) + Mg2+)-ATPase purified from skeletal muscle sarcoplasmic reticulum. It is suggested that binding at the lipid/protein interface of the ATPase is weak but that binding can occur at other (non-annular sites) on the ATPase. Pyrethroids containing either a brominated fatty acyl or iodinated alcohol moiety quench the tryptophan fluorescence of the ATPase, suggesting that the pyrethroids bound to the ATPase adopt a folded conformation with both the acid and alcohol moieties in contact with hydrophobic regions of the ATPase. Whereas effects of the pyrethroids on the activity of the ATPase in bilayers of dioleoylphosphatidylcholine are small, large increases are observed in the activity of the ATPase reconstituted into bilayers of the short-chain phospholipid, dimyristoleoylphosphatidylcholine (DMPC). The rate of phosphorylation of DMPC-ATPase by ATP is slow, but is increased on addition of pyrethroid. The level of phosphorylation of the ATPase by Pi is reduced on reconstitution into bilayers of DMPC, and this is also increased by addition of pyrethroid.     

Inhibition of gastric (H+ + K+)-ATPase by unsaturated long-chain fatty acids

Arachidonic acid and unsaturated C18 fatty acids at concentrations near 10(-5) M markedly inhibited (H+ + K+)-ATPase in hog or rat gastric membranes. Arachidonic acid was a more potent inhibitor than unsaturated C18 fatty acids, but the involvement of the metabolites of arachidonic acid cascade was ruled out. Linolenic acid inhibited the formation of phosphoenzyme and the K+ -dependent p-nitrophenylphosphatase activity of the hog ATPase. Treatment with fatty acid-free bovine serum albumin abolished only the inhibitory effect of the fatty acid on the phosphatase activity without restoring the overall ATPase action. These data suggest the existence of at least two groups of hydrophobic binding sites in the gastric ATPase for unsaturated long-chain fatty acids which affect differentially the catalytic reactions of the ATPase. (H+ + K+)-ATPase in rat gastric membranes was found more susceptible to the fatty acid inhibition and also more unstable than the ATPase in hog gastric membranes. The presence of a millimolar level of lanthanum chloride or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid stabilized the rat ATPase probably via the inhibition of Ca2+ -dependent phospholipases in the gastric membranes.     

Studies on the purified, lipid-reconstituted (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes. Dependence of enzyme activity on lipid headgroup and hydrocarbon chain structure

The purified (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes was successfully reconstituted with a number of different phospho- and glycolipids, and the ability of these lipids to support the function of this enzyme was evaluated by their ability to increase the specific activity of the purified enzyme and by their ability to restore its lipid-phase state-dependent properties which were lost during purification. The incorporation of this ATPase into liposomes composed of the endogenous membrane lipids of the organism, or of zwitterionic phospholipids such as phosphatidylcholine or phosphatidylethanolamine, results in a full reconstitution of its activity and its lipid-phase state-dependent properties. In contrast, anionic phospholipids alone, or in combination with zwitterionic phospholipids at concentrations higher than 10 mol % of the anionic phospholipid, cause an irreversible inhibition of this ATPase. However, when combined with neutral glycolipids, larger amounts of anionic phospholipid can be tolerated without enzyme inhibition. Phosphatidylcholines with acyl chains of 14-24 linear carbon atoms and varying degrees of branching and unsaturation successfully reconstitute the enzyme, in marked contrast to the shorter chain homologues, which were ineffective. Our results indicate that the full expression of the activity of the A. laidlawii B ATPase requires a host lipid bilayer membrane of low to moderate negative surface charge which is predominantly liquid-crystalline and of a minimal bilayer thickness. Once such requirements are met, the enzyme exhibits considerable flexibility regarding the nature of the lipids which can effectively support its function. In particular, the activity of the A. laidlawii B ATPase is not very sensitive to lipid "fluidity" in the liquid-crystalline state.     

Stages of the bilayer-micelle transition in the system phosphatidylcholine-C12E8 as studied by deuterium- and phosphorous-NMR, light scattering, and calorimetry.

The perturbation of phospholipid bilayer membranes by a nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), was investigated by 2H- and 31P-NMR, static and dynamic light scattering, and differential scanning calorimetry. Preequilibrated mixtures of the saturated phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), and 1,2-dilauroyl-sn-glycero-3-phosphorylcholine (DLPC) with the detergent were studied over a broad temperature range including the temperature of the main thermotropic phase transition of the pure phospholipids. Above this temperature, at a phospholipid/detergent molar ratio 2:1, the membranes were oriented in the magnetic field. Cooling of the mixtures below the thermotropic phase transition temperatures of the pure phospholipids led to micelle formation. In mixtures of DPPC and DMPC with C12E8, a narrow calorimetric signal at the onset temperature of the solubilization suggested that micelle formation was related to the disorder-order transition in the phospholipid acyl chains. The particle size changed from 150 nm to approximately 7 nm over the temperature range of the bilayer-micelle transition. The spontaneous orientation of the membranes at high temperatures enabled the direct determination of segmental order parameters from the deuterium spectra. The order parameter profiles of the phospholipid acyl chains could be attributed to slow fluctuations of the whole membrane and to detergent-induced local perturbations of the bilayer order. The packing constraints in the mixed bilayers that eventually lead to bilayer solubilization were reflected by the order parameters of the interfacial phospholipid acyl chain segments and of the phospholipid headgroup. These results are interpreted in terms of the changing average shape of the component molecules. Considering the decreasing cross sectional areas in the acyl chain region and the increasing hydration of the detergent headgroups, the bilayer-micelle transition is the result of an imbalance in the chain and headgroup repulsion. A neutral or pivotal plane can be defined on the basis of the temperature dependence of the interfacial quadrupolar splittings.     

Modulation of the Ca2+,Mg2+-ATPase Activity of Synaptosomal Plasma Membrane by the Local Anesthetics Dibucaine and Lidocaine

It has been previously shown that local anesthetics inhibit the total Ca2+, Mg2(+)-ATPase activity of synaptosomal plasma membranes. We have carried out kinetic studies to quantify the effects of these drugs on the different Ca2(+)-dependent and Mg2(+)-dependent ATPase activities of these membranes. As a result we have found that this inhibition is not altered by washing the membranes with EDTA or EGTA. We have also found that the Ca2(+)-dependent ATPase activity is not significantly inhibited in the concentration range of these local anesthetics and under the experimental conditions used in this study. The inhibition of the Mg2(+)-dependent ATPase activities of these membranes was found to be of a noncompetitive type with respect to the substrate ATP-Mg2+, did not significantly shift the Ca2+ dependence of the Ca2+, Mg2(+)-ATPase activity, and occurred in a concentration range of local anesthetics that does not significantly alter the order parameter (fluidity) of these membranes. Modulation of this activity by the changes of the membrane potential that are associated with the adsorption of local anesthetics on the synaptosomal plasma membrane is unlikely, on the basis of the weak effect of membrane potential changes on the Ca2+,Mg2(+)-ATPase activity. It is suggested that the local anesthetics lidocaine and dibucaine inhibit the Ca2+, Mg2(+)-ATPase of the synaptosomal plasma membrane by disruption of the lipid annulus.     

Reconstitution and photolabeling of the purified (Na+ + Mg2+)-ATPase from the plasma membrane of Acholeplasma laidlawii B with phospholipids containing a photosensitive fatty acyl group

The purified membrane (Na+ + Mg2+)-ATPase of Acholeplasma laidlawii B was reconstituted into vesicles composed of phospholipids containing a photoactivatable aryl nitrene-generating fatty acyl group. The reconstitution with phospholipid resulted in an enhancement of ATPase activity and a reduction in the sensitivity of the enzyme to radiation inactivation. The incorporation of the enzyme into the lipid vesicles results in a broadening of the gel-to-liquid-crystalline phase transition of the photolabeled phospholipid and the appearance of two partially resolved endotherms in the calorimetric traces. The temperatures and the total enthalpy of these overlapping transitions are higher than in the absence of incorporated enzyme. After photolysis of the lipid-reconstituted ATPase and separation of the polypeptide subunits by sodium dodecyl sulfate (SDS) gel electrophoresis, a significant labeling of the alpha-subunit of the enzyme was demonstrated. These results indicate that at least the alpha-subunit of this ATPase must penetrate into or traverse the phospholipid bilayer.     

Influence of chain length and unsaturation on sphingomyelin bilayers

Sphingomyelins (SMs) are among the most common phospholipid components of plasma membranes, usually constituting a mixture of several molecular species with various fatty acyl chain moieties. In this work, we utilize atomistic molecular dynamics simulations to study the differences in structural and dynamical properties of bilayers comprised of the most common natural SM species. Keeping the sphingosine moiety unchanged, we vary the amide bonded acyl chain from 16 to 24 carbons in length and examine the effect of unsaturation by comparing lipids with saturated and monounsaturated chains. As for structural properties, we find a slight decrease in average area per lipid and a clear linear increase in bilayer thickness with increasing acyl chain length both in saturated and unsaturated systems. Increasing the acyl chain length is found to further the interdigitation across the bilayer center. This is related to the dynamics of SM molecules, as the lateral diffusion rates decrease slightly for an increasing acyl chain length. Interdigitation also plays a role in interleaflet friction, which is stronger for unsaturated chains. The effect of the cis double bond is most significant on the local order parameters and rotation rates of the chains, though unsaturation shows global effects on overall lipid packing and dynamics as well. Regarding hydrogen bonding or properties related to the lipid/water interface region, no significant effects were observed due to varying chain length or unsaturation. The significance of the findings presented is discussed.