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.     

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.     

Sterols have higher affinity for sphingomyelin than for phosphatidylcholine bilayers even at equal acyl-chain order

The interaction between cholesterol and phospholipids in bilayer membranes is important for the formation and maintenance of membrane structure and function. However, cholesterol does not interact favorably with all types of phospholipids and, for example, prefers more ordered sphingomyelins (SMs) over phosphatidylcholines (PCs). The reason for this preference is not clear. Here we have studied whether acyl-chain order could be responsible for the preferred sterol interaction with SMs. Acyl-chain order was deduced from diphenylhexatriene anisotropy and from the deuterium order parameter obtained by ²H-NMR on bilayers made from either 14:0/14:0_(d27)-PC, or 14:0_(d27)-SM. Sterol/phospholipid interaction was determined from sterol bilayer partitioning. Cholestatrienol (CTL) was used as a fluorescence probe for cholesterol, because its relative membrane partitioning is similar to cholesterol. When CTL was allowed to reach equilibrium partitioning between cyclodextrins and unilamellar vesicles made from either 14:0/14:0-PC or 14:0-SM, the molar-fraction partitioning coefficient (K_x) was approximately twofold higher for SM bilayers than for PC bilayers. This was even the case when the temperature in the SM samples was raised to achieve equal acyl-chain order, as determined from 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy and the deuterium order parameter. Although the K_x did increase with acyl-chain order, the higher K_x for SM bilayers was always evident. At equal acyl-chain order parameter (DPH anisotropy), the K_x was also higher for 14:0-SM bilayers than for bilayers made from either 14:0/15:0-PC or 15:0-/14:0-PC, suggesting that minor differences in chain length or molecular asymmetry are not responsible for the difference in K_x. We conclude that acyl-chain order affects the bilayer affinity of CTL (and thus cholesterol), but that it is not the cause for the preferred affinity of sterols for SMs over matched PCs. Instead, it is likely that the interfacial properties of SMs influence and stabilize interactions with sterols in bilayer membranes.     

Structure of polymerizable lipid bilayers. I--1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, a tubule-forming phosphatidylcholine

This report presents the first X-ray diffraction data on diacetylenic phospholipids. The tubule-forming polymerizable lipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC), was studied by low angle X-ray diffraction from partially dehydrated oriented multibilayers in both polymerized and unpolymerized form. Bilayers of this material were found to be highly ordered, yielding as many as 16 orders of lamellar diffraction, in both the polymerized and unpolymerized states. The unit cell dimension was very small for a lipid of this size. In addition to the features usually observed in the electron density profile structure of phospholipid bilayers, the electron-dense diacetylenic portions of the fatty acyl chain produced electron density maxima at two well-defined levels on each side of the bilayer approximately 15 A and 9 A from the bilayer midplane. A model molecular conformation deduced from the one-dimensional electron density map features all-trans acyl chains tilted at approximately 28 degrees from the bilayer normal that are interdigitated with chains of the opposing monolayer by approximately two carbons at the bilayer center. The linear diacetylene moieties on beta- and gamma-chains appear at different positions along the bilayer normal axis and are roughly parallel to the bilayer surface. This model is discussed in terms of a polymerization mechanism.     

Effect of phospholipid composition on an amphipathic peptide-mediated pore formation in bilayer vesicles

To better understand the influence of phospholipid acyl-chain composition on the formation of pores by cytotoxic amphipathic helices in biological membranes, the leakage of aqueous contents induced by the synthetic peptide GALA (WEAALAEALAE ALAEHLAEALAEALEALAA) from large unilamellar phospholipid vesicles of various compositions has been studied. Peptide-mediated leakage was examined at pH 5.0 from vesicles made of phosphatidylcholine (PC) and phosphatidylglycerol (PG) with the following acyl-chain compositions: 1-palmitoyl-2-oleoyl (PO), 1,2-dioleoyl (DO), 1, 2-dielaidoyl (DE), and 1,2-dipetroselinoyl (DPe). A mathematical model predicts and simulates the final extents of GALA-mediated leakage of 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and p-xylene-bis-pyridinium bromide (DPX) from 1-palmitoyl-2-oleoyl-phosphatidylcholine/1-palmitoyl-2-oleoyl-phospha tidylglycerol (POPC/POPG) and 1, 2-dielaidoyl-sn-glycero-3-phosphocholine/1, 2-dielaidoyl-phosphatidylglycerol (DEPC/DEPG) liposomes at pH 5.0 as a function of peptide concentration in the bilayer, by considering that GALA pores responsible for this leakage have a minimum size of 10 +/- 2 monomers and are formed by quasiirreversible aggregation of the peptide. With the phospholipid acyl-chain compositions tested, GALA-induced ANTS/DPX leakage follows the rank order POPC/POPG approximately DEPC/DEPG > DPePC/DPePG > DOPC/DOPG. Results from binding experiments reveal that this reduced leakage from DOPC/DOPG vesicles cannot be explained by a reduced binding affinity of the peptide to these membranes. As shown by monitoring the leakage of a fluorescent dextran, an increase in the minimum pore size also does not explain the reduction in ANTS/DPX leakage. The data suggest that surface-associated GALA monomers or aggregates are stabilized in bilayers composed of phospholipids containing a cis unsaturation per acyl chain (DO and DPe), while transbilayer peptide insertion is reduced. GALA-induced ANTS/DPX leakage is also decreased when the vesicles contain phosphatidylethanolamine (PE). This lends further support to the suggestion that factors stabilizing the surface state of the peptide reduce its insertion and subsequent pore formation in the bilayer.     

Phospholipid and detergent effects on (Ca2+ + Mg2+)ATPase purified from human erythrocytes

(Ca2+ + Mg2+)ATPase (EC 3.6.1.3) was solubilized from human erythrocyte membranes by detergent extraction with Triton N-101 (0.5 mg/mg membrane protein) and purified by calmodulin affinity chromatography. ATPase activity was assayed in mixtures of Triton N-101 and phospholipid, without reconstitution into bilayer vesicles. At low levels of phospholipid (5 micrograms/ml), the ATPase activity was highly sensitive to the detergent concentration, with maximal activity occurring at or near the critical micelle concentration of the detergent. With increased amounts of phospholipid (50 micrograms/ml), detergent concentrations greater than the critical micelle concentration were required for maximal activity. Detergent alone did not support ATPase activity. Sonicated phospholipid in the form of vesicles was equally ineffective. Activity seemed to be dependent on the presence of detergent/phospholipid mixed micelles. The acidic phospholipids, phosphatidylserine and phosphatidylinositol, as well as the commercial phospholipid preparation, Asolectin, gave activities five to eight times greater than the same amount of phosphatidylcholine. Mixtures of phosphatidylserine and phosphatidylcholine produced intermediate ATPase activities, with the maximal value dependent on the phosphatidylserine concentration. Addition of phosphatidylcholine to fixed concentrations of phosphatidylserine caused a rise in activity that was independent of the ratio of the two phospholipids or the total phospholipid concentration. Phosphatidylcholine may therefore be irreplaceable for some aspect of ATPase function. The number of phospholipid molecules present in mixed micelles at maximal ATPase activity was calculated to be near 50. This value implied that the hydrophobic surface of the ATPase molecule must be completely coated by a single layer of phospholipid molecules for maximum activity to occur.     

Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness.

Two mechanisms have been proposed to account for solute permeation of lipid bilayers. Partitioning into the hydrophobic phase of the bilayer, followed by diffusion, is accepted by many for the permeation of water and other small neutral solutes, but transient pores have also been proposed to account for both water and ionic solute permeation. These two mechanisms make distinctively different predictions about the permeability coefficient as a function of bilayer thickness. Whereas the solubility-diffusion mechanism predicts only a modest variation related to bilayer thickness, the pore model predicts an exponential relationship. To test these models, we measured the permeability of phospholipid bilayers to protons, potassium ions, water, urea, and glycerol. Bilayers were prepared as liposomes, and thickness was varied systematically by using unsaturated lipids with chain lengths ranging from 14 to 24 carbon atoms. The permeability coefficient of water and neutral polar solutes displayed a modest dependence on bilayer thickness, with an approximately linear fivefold decrease as the carbon number varied from 14 to 24 atoms. In contrast, the permeability to protons and potassium ions decreased sharply by two orders of magnitude between 14 and 18 carbon atoms, and leveled off, when the chain length was further extended to 24 carbon atoms. The results for water and the neutral permeating solutes are best explained by the solubility-diffusion mechanism. The results for protons and potassium ions in shorter-chain lipids are consistent with the transient pore model, but better fit the theoretical line predicted by the solubility-diffusion model at longer chain lengths.     

Cholesterol interactions with phospholipids in membranes.

Mammalian cell membranes are composed of a complex array of glycerophospholipids and sphingolipids that vary in head-group and acyl-chain composition. In a given cell type, membrane phospholipids may amount to more than a thousand molecular species. The complexity of phospholipid and sphingolipid structures is most likely a consequence of their diverse roles in membrane dynamics, protein regulation, signal transduction and secretion. This review is mainly focused on two of the major classes of membrane phospholipids in eukaryotic organisms, sphingomyelins and phosphatidylcholines. These phospholipid classes constitute more than 50% of membrane phospholipids. Cholesterol is most likely to associate with these lipids in the membranes of the cells. We discuss the synthesis and distribution in the cell of these lipids, how they are believed to interact with each other, and what cellular consequences such interactions may have. We also include a discussion about findings in the recent literature regarding cholesterol/phospholipid interactions in model membrane systems. Finally, we look at the recent trends in computer and molecular dynamics simulations regarding phospholipid and cholesterol/phospholipid behavior in bilayer membranes.     

Activation of (Na+ + K+)-dependent ATPase by lipid vesicles of negative phospholipids.

1. Kidney (Na+ + K+)-stimulated ATPase was depleted of phospholipids by extraction with lubrol and inserted in lipid structures of known composition. Both ouabain-sensitive ATPase and phosphatase reactions could be partially restored by lipid replacement. 2. Lipid vesicles of natural and synthetic negative phospholipids proved to be effective. The low activity of uncharged liposomes was increased when negative charges were included into the bilayer structure. 3. Reactivation by negative phospholipids was accompanied by spontaneous re-assembly of a stable lipid-protein complex. By contrast, the interaction of lipid deficient ATPase complex with uncharged lamellae was possible only after sonication of lipid-protein suspension. Reactivation did not ensue. 4. The ouabain-sensitive ATPase reactivated by synthetic dioleoylphosphatidylglycerol yielded curvilinear Arrhenius plots. The same pattern was seen with the original undepleted microsomal preparation. A discontinuity close to the temperature of fluid-order transition was found with dimyristoyl phosphatidylglycerol. 5. It is concluded that reassembly of lipid-deficient (Na+ + K+)-stimulated ATPase requires the addition of diacylphospholipids with fluid acyl-chains and negatively charged polar heads able to assemble in an expanded lamellar configuration.     

Intrinsic membrane targeting of the flagellar export ATPase FliI: interaction with acidic phospholipids and FliH

The specialised ATPase FliI is central to export of flagellar axial protein subunits during flagellum assembly. We establish the normal cellular location of FliI and its regulatory accessory protein FliH in motile Salmonella typhimurium, and ascertain the regions involved in FliH(2)/FliI heterotrimerisation. Both FliI and FliH localised to the cytoplasmic membrane in the presence and in the absence of proteins making up the flagellar export machinery and basal body. Membrane association was tight, and FliI and FliH interacted with Escherichia coli phospholipids in vitro, both separately and as the preformed FliH(2)/FliI complex, in the presence or in the absence of ATP. Yeast two-hybrid analysis and pull-down assays revealed that the C-terminal half of FliH (H105-235) directs FliH homodimerisation, and interacts with the N-terminal region of FliI (I1-155), which in turn has an intra-molecular interaction with the remainder of the protein (I156-456) containing the ATPase domain. The FliH105-235 interaction with FliI was sufficient to exert the FliH-mediated down-regulation of ATPase activity. The basal ATPase activity of isolated FliI was stimulated tenfold by bacterial (acidic) phospholipids, such that activity was 100-fold higher than when bound by FliH in the absence of phospholipids. The results indicate similarities between FliI and the well-characterised SecA ATPase that energises general protein secretion. They suggest that FliI and FliH are intrinsically targeted to the inner membrane before contacting the flagellar secretion machinery, with both FliH105-235 and membrane phospholipids interacting with FliI to couple ATP hydrolysis to flagellum assembly.     

A niosomal bilayer of sorbitan monostearate in complex with flavones: a molecular dynamics simulation study

Bilayers prepared from sorbitan fatty acid esters (Span) have been frequently used for delivery of drugs including flavonoids. We applied molecular dynamics simulation to characterize the structure of a sorbitan monostearate (Span 60) bilayer in complex with three representative flavones, a subclass of flavonoids. At a low concentration, unsubstituted flavone, the most hydrophobic member, was able to flip over and cross the bilayer with a large diffusion coefficient. At a high concentration, it was accumulated at the bilayer center resulting in a phase separation. The leaflets of the bilayer were pushed in the opposite directions increasing the membrane thickness. Order parameter of the stearate chain of Span 60 was not affected significantly by unsubstituted flavone. In contrast, chrysin with hydroxylated ring A was lined up with the acyl chains of Span 60 with its hydroxyl group facing the membrane surface. Neither flipping nor transbilayer movement were allowed. Diffusion coefficient was only 15–25% of that of unsubstituted flavone and order parameter decreased with the concentration of chrysin. Luteolin, the most hydroxylated member, interacted mainly with the headgroup of Span 60 and assumed many different orientations without crossing the bilayer. Unlike chrysin and unsubstituted flavone the bilayer integrity was disrupted at 50?mol% luteolin. These behaviors and structures of flavones in a Span 60 bilayer can be accounted for by their hydrophobicity and sites of hydroxylation.     

Phospholipid surface bilayers at the air-water interface. III. Relation between surface bilayer formation and lipid bilayer assembly in cell membranes.

Lipid bilayer assembly in cell membranes has been simulated with total lipid extracts from human red blood cells and from mesophilic and thermophilic bacteria grown at several temperatures. Aqueous dispersions of these natural lipid mixtures form surface bilayers, a single bimolecular lipid state, but only at the growth temperature of the source organism. Thus, a single isolated bilayer state forms spontaneously in vitro from lipids that are available in vivo at the growth temperature of the cell. Surface bilayers form at a specific temperature that is a function of hydrocarbon chain length and degree of fatty acid unsaturation of the phospholipids; this property is proposed as an essential element in the control of membrane lipid composition.     

Deciphering Membrane Insertion of the Diphtheria Toxin T Domain by Specular Neutron Reflectometry and Solid-State NMR Spectroscopy

Insertion and translocation of soluble proteins into and across biological membranes are involved in many physiological and pathological processes, but remain poorly understood. Here, we describe the pH-dependent membrane insertion of the diphtheria toxin T domain in lipid bilayers by specular neutron reflectometry and solid-state NMR spectroscopy. We gained unprecedented structural resolution using contrast-variation techniques that allow us to propose a sequential model of the membrane-insertion process at angstrom resolution along the perpendicular axis of the membrane. At pH 6, the native tertiary structure of the T domain unfolds, allowing its binding to the membrane. The membrane-bound state is characterized by a localization of the C-terminal hydrophobic helices within the outer third of the cis fatty acyl-chain region, and these helices are oriented predominantly parallel to the plane of the membrane. In contrast, the amphiphilic N-terminal helices remain in the buffer, above the polar headgroups due to repulsive electrostatic interactions. At pH 4, repulsive interactions vanish; the N-terminal helices penetrate the headgroup region and are oriented parallel to the plane of the membrane. The C-terminal helices penetrate deeper into the bilayer and occupy about two thirds of the acyl-chain region. These helices do not adopt a transmembrane orientation. Interestingly, the T domain induces disorder in the surrounding phospholipids and creates a continuum of water molecules spanning the membrane. We propose that this local destabilization permeabilizes the lipid bilayer and facilitates the translocation of the catalytic domain across the membrane.     

Diversity of the human erythrocyte membrane sialic acids in relation with blood groups

The aim of the present study was to detect defective structural properties in bilayers of mitochondrial phospholipids after oxidative stress of isolated mitochondria in vitro, reportedly during respiration state IV. The structural behaviour of extracted phospholipids was studied by electron paramagnetic resonance (EPR) spectrometry in oriented phospholipid bilayers spin-labelled with 5-doxyl-lecithin, by detecting of the degree of EPR spectral anisotropy loss, indicative of the phospholipid bilayer packing order. Bilayers of phospholipids from untreated mitochondria showed the highest spectral anisotropy, hence highly ordered structure, while chemically oxidised phospholipid yielded almost completely disordered supported phospholipid bilayers. Samples from mitochondria after respiration state IV showed bilayer disorder increasing with oxidation time, while inclusion of the antioxidant resveratrol in the respiration medium almost completely prevented bilayer disordering. On the other hand, β-n-doxylstearoyl-lecithin spin-labelled mitochondria showed unchanged order parameter S at C positions 5, 12 and 16 after respiration state IV, confirming the insensitivity of this parameter to phospholipid oxidative stress. It is concluded that reactive oxygen species attack to the membrane affects lipid packing order more than fluidity, and that EPR anisotropy loss reveals oxidative damage to the bilayer better than the order parameter.     

Interaction of the pore-forming domain of colicin A with phospholipid vesicles

The interaction of the 20-kDa pore-forming domain of colicin A with phospholipid vesicles was investigated by gel permeation chromatography, analytical centrifugation, and electron microscopy. Under the experimental conditions of this study, this peptide was found to interact only with vesicles containing negatively charged phospholipids. It forms a well-defined disklike complex with phosphatidylglycerols with a preference for those containing 12-14 atoms of carbon in their fatty acid chain. This complex has a diameter of 120 A and is about one bilayer thick. It contains nine molecules of peptide and is formed both at acidic pH (pH 5.0) and at neutral pH (pH 7.2).     

Non-bilayer lipids stimulate the activity of the reconstituted bacterial protein translocase

To determine the phospholipid requirement of the preprotein translocase in vitro, the Escherichia coli SecYEG complex was purified in a delipidated form using the detergent dodecyl maltoside. SecYEG was reconstituted into liposomes composed of defined synthetic phospholipids, and proteoliposomes were analyzed for their preprotein translocation and SecA translocation ATPase activity. The activity strictly required the presence of anionic phospholipids, whereas the non-bilayer lipid phosphatidylethanolamine was found stimulatory. The latter effect could also be induced by dioleoylglycerol, a lipid that adopts a non-bilayer conformation. Phosphatidylethanolamine derivatives that prefer the bilayer state were unable to stimulate translocation. In the absence of SecG, activity was reduced, but the phospholipid requirement was unaltered. Remarkably, non-bilayer lipids were found essential for the activity of the Bacillus subtilis SecYEG complex. Optimal activity required a mixture of anionic and non-bilayer lipids at concentrations that correspond to concentrations found in the natural membrane.     

Modulation of Na,K-ATPase and Na-ATPase activity by phospholipids and cholesterol. I. Steady-state kinetics

The effects of phospholipid acyl chain length (n(c)), degree of acyl chain saturation, and cholesterol on Na,K-ATPase reconstituted into liposomes of defined lipid composition are described. The optimal acyl chain length of monounsaturated phosphatidylcholine in the absence of cholesterol was found to be 22 but decreased to 18 in the presence of 40 mol % cholesterol. This indicates that the hydrophobic matching of the lipid bilayer and the transmembrane hydrophobic core of the membrane protein is a crucial parameter in supporting optimal Na,K-ATPase activity. In addition, the increased bilayer order induced by both cholesterol and saturated phospholipids could be important for the conformational mobility of the Na,K-ATPase changing the distribution of conformations. Lipid fluidity was important for several parameters of reconstitution, e.g., the amount of protein inserted and the orientation in the liposomes. The temperature dependence of the Na,K-ATPase as well of the Na-ATPase reactions depends both on phospholipid acyl chain length and on cholesterol. Cholesterol increased significantly both the enthalpy of activation and entropy of activation for Na,K-ATPase activity and Na-ATPase activity of Na,K-ATPase reconstituted with monounsaturated phospholipids. In the presence of cholesterol the free energy of activation was minimum at a lipid acyl chain length of 18, the same that supported maximum turnover. In the case of ATPase reconstituted without cholesterol, the minimum free energy of activation and the maximum turnover both shifted to longer acyl chain lengths of about 22.     

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.     

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values <6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.