Sn-2 Acyl Chain Research Articles

Membrane Insertion and Lipid-Protein Interactions of Bovine Seminal Plasma Protein PDC-109 Investigated by Spin-Label Electron Spin Resonance Spectroscopy

The interaction of the major acidic bovine seminal plasma protein, PDC-109, with dimyristoylphosphatidylcholine (DMPC) membranes has been investigated by spin-label electron spin resonance spectroscopy. Studies employing phosphatidylcholine spin labels, bearing the spin labels at different positions along the sn-2 acyl chain indicate that the protein penetrates into the hydrophobic interior of the membrane and interacts with the lipid acyl chains up to the 14th C atom. Binding of PDC-109 at high protein/lipid ratios (PDC-109:DMPC = 1:2, w/w) results in a considerable decrease in the chain segmental mobility of the lipid as seen by spin-label electron spin resonance spectroscopy. A further interesting new observation is that, at high concentrations, PDC-109 is capable of (partially) solubilizing DMPC bilayers. The selectivity of PDC-109 in its interaction with membrane lipids was investigated by using different spin-labeled phospholipid and steroid probes in the DMPC host membrane. These studies indicate that the protein exhibits highest selectivity for the choline phospholipids phosphatidylcholine and sphingomyelin under physiological conditions of pH and ionic strength. The selectivity for different lipids is in the following order: phosphatidylcholine ≈ sphingomyelin ≥ phosphatidic acid (pH 6.0) > phosphatidylglycerol ≈ phosphatidylserine ≈ androstanol > phosphatidylethanolamine ≥ N-acyl phosphatidylethanolamine ≫ cholestane. Thus, the lipids bearing the phosphocholine moiety in the headgroup are clearly the lipids most strongly recognized by PDC-109. However, these studies demonstrate that this protein also recognizes other lipids such as phosphatidylglycerol and the sterol androstanol, albeit with somewhat reduced affinity.

Spin-label electron paramagnetic resonance studies on the interaction of avidin with dimyristoyl-phosphatidylglycerol membranes

The interaction of avidin – a basic protein from hen egg-white – with dimyristoyl-phosphatidylglycerol membranes was investigated by spin-label electron paramagnetic resonance spectroscopy. Phosphatidylcholines, bearing the nitroxide spin label at different positions along the sn-2 acyl chain of the lipid were used to investigate the effect of protein binding on the lipid chain-melting phase transition and acyl chain dynamics. Binding of the protein at saturating levels results in abolition of the chain-melting phase transition of the lipid and accompanying perturbation of the lipid acyl chain mobility. In the fluid phase region, the outer hyperfine splitting increases for all phosphatidylcholine spin-label positional isomers, indicating that the chain mobility is decreased by binding avidin. However, there was no evidence for direct interaction of the protein with the lipid acyl chains, clearly indicating that the protein does not penetrate the hydrophobic interior of the membrane. Selectivity experiments with different spin-labelled lipid probes indicate that avidin exhibits a preference for negatively charged lipid species, although all spin-labelled lipid species indirectly sense the protein binding. The interaction with negatively charged lipids is relevant to the use of avidin in applications such as the ultrastructural localization of biotinylated lipids in histochemical studies.

The effect of ethanol on the phase transition temperature and the phase structure of monounsaturated phosphatidylcholines

Previous studies from our laboratories have delineated the relationship between the acyl chain asymmetry of mixed-chain phosphatidylcholines, C(X):C(Y)PC, and the effect of ethanol concentration, [EtOH], on the main phase transition temperature, Tm, and the phase structure of the lipid bilayer composed of C(X):C(Y)PC using differential scanning calorimetry and X-ray diffraction techniques [Huang and McIntosh, Biophys. J. 72 (1997) 2702–2709]. In the present work, we have extended these studies to characterize the effect of [EtOH] on the Tm and the phase structure of the lipid bilayer composed of sn-1 saturated/sn-2 monounsaturated phosphatidylcholines with various positions of the cis double bond. Specifically, five positional isomers of 1-eicosanoyl-2-eicosenoyl-sn-glycero-3-phosphocholines, C(20):C(20:1Δn)PC with n=5, 8, 11, 13 and 17, were synthesized and studied. For C(20):C(20:1Δn)PC with n=5 and 8, results from the calorimetric experiments showed that in response to various concentrations of ethanol, the change in Tm of the lipid bilayer composed of monounsaturated lipids was characterized by a sigmoidal or biphasic profile in the plot of Tm versus [EtOH]. In contrast, a continuous depression of the Tm by ethanol was observed calorimetrically for C(20):C(20:1Δn)PC with n≥11. The X-ray diffraction experiments further demonstrated that C(20):C(20:1Δ5)PC and C(20):C(20:1Δ8)PC can undergo the ethanol-induced gel-to-fully interdigitated phase transition at T<Tm. Such a transition, however, was not observed for C(20):C(20:1Δ13)PC even at a very high ethanol concentration of 100 mg/ml. These distinct different effects of [EtOH] on the phase transition temperature and the phase structure can be attributed to various positions of the cis double bond in these monounsaturated phosphatidylcholines. And the different effects of ethanol can, in fact, be explained based on the molecular structures of these monounsaturated lipids packed in the gel-state bilayer as generated by molecular mechanics simulations. To the best of our knowledge, this is the first time that the ethanol-induced fully interdigitated bilayers are observed at T<Tm for unsaturated phospholipids with well defined double bond positions in their sn-2 acyl chains.

Dynamics of Membrane Penetration of the Fluorescent 7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl (NBD) Group Attached to an Acyl Chain of Phosphatidylcholine

Location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) covalently attached to a short (C6) or a long (C12) sn2 acyl chain of a phosphatidylcholine molecule was investigated by fluorescence and solid-state NMR spectroscopy. 2H NMR lipid chain order parameters indicate a perturbation of the phospholipid packing density in the presence of NBD. Specifically, a decrease of molecular order was found for acyl chain segments of the lower, more hydrophobic region. Molecular collision probabilities determined by 1H magic angle spinning nuclear Overhauser enhancement spectroscopy indicate a highly dynamic reorientation of the probe in the membrane due to thermal fluctuations. A broad distribution of the fluorophore in the lipid bilayer is observed with a preferential location in the upper acyl chain/glycerol region. The distribution of the NBD group in the membrane is quite similar for both the long- and the short-chain analog. However, a slight preference of the NBD group for the lipid-water interface is found for C12-NBD-PC in comparison with C6-NBD-PC. Indeed, as shown by dithionite fluorescence assay, the long-chain analog reacts more favorably with dithionite, indicating a better accessibility of the probe by dithionite present in the aqueous phase. Forces determining the location of the fluorophore in the lipid water interface are discussed.

Molecular Mechanics and Calorimetric Studies of Phosphatidylethanols

Phosphatidylethanols (PEths) are negatively charged diacyl phospholipids that are ubiquitously present in humans under the condition of alcohol intoxication. These lipids, derived in vivo from other naturally occurring phospholipids such as phosphatidylcholines (PC) via transphosphatidylation reaction as catalyzed by phospholipase D in the presence of ethanol, are well known to affect many biochemical properties of the cell membranes in humans. In this communication, we applied the combined approach of molecular mechanics (MM) simulations and high-sensitivity differential scanning calorimetry (DSC) to investigate the structure and phase transition behavior of PEth. We first determined the energy-minimized structures of tetrameric C(15):C(15)PEth arranged in two types of packing motif by the MM approach. An inwardly bent orientation of the lipid headgroup was observed; specifically, the methyl terminus of PEth's headgroup was juxtaposed intramolecularly to the C(2) atom of the sn-2 acyl chain. Clearly, this headgroup conformation was rather unique among all naturally occurring phospholipids. Subsequently, the phase transition behavior of the fully hydrated lipid bilayers prepared individually from 11 species of saturated C(X):C(Y)PEth with the same MW was studied by DSC, and the resulting Tm values were codified in terms of the normalized acyl chain asymmetry (ΔC/CL). A V-shaped Tm profile was observed in the plot of Tm versus ΔC/CL for each subclass of these lipids, suggesting two types of packing motif for C(X):C(Y)PEth at T <Tm. Moreover, it was observed that within each packing motif these Tm values were, on average, 2.0 ± 0.9°C smaller than the Tm values of the corresponding saturated PC. However, based on the unique headgroup conformation of PEth, we were able to predict that monounsaturated PEth with a cis double bond near the H2O/hydrocarbon interface would exhibit a higher Tm than the corresponding PC. Most interestingly, this prediction was indeed borne out by DSC results obtained with C(18):C(20:1Δ5)PC and C(18):C(20:1Δ5)PEth.

The His gap motif in microbial lipases: A determinant of stereoselectivity toward triacylglycerols and analogs

Lipases preferably hydrolyze the sn-1 and sn-3 acyl chain of triacylglycerols and sn-2 substituted analogs. Molecular modeling studies of the stereopreference of microbial lipases from Rhizopus oryzae, Rhizomucor miehei, Candida rugosa, and lipase B from Candida antarctica toward the hydrolysis of triacylglycerols and analogs revealed that sterical interactions occurring between the sn-2 substituent and the His gap affect substrate geometry, which can be monitored by a single torsion angle. This torsion angle correlates to the experimentally determined stereopreference and is, therefore, suitable to predict stereopreference by molecular modeling. For a given microbial lipase, stereopreference can be estimated by measuring the distance between the side chains of the His gap residues: a narrow His gap cleft implies sn-3 stereopreference for all investigated substrates; a medium-sized His gap discriminates by flexibility of the substrates: flexible substrates are hydrolyzed in sn-1, while rigid substrates are hydrolyzed in sn-3. A wide open His gap implies sn-1 stereopreference for all substrates. This rule holds for all investigated microbial wild type lipases and mutants.

Effect of soybean oil and fish oil on individual molecular species of Atlantic salmon head kidney phospholipids determined by normal-phase liquid chromatography coupled to negative ion electrospray tandem mass spectrometry

The effect of soybean oil (SO) and fish oil (FO) on the relative molecular species distribution of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in Atlantic salmon head kidney was studied using normal-phase liquid chromatography coupled with negative ion electrospray tandem mass spectrometry. The conformation of identity of the phospholipid species was based on retention time, the mass of the [M−H] − ([M−15] − for PC) molecular ions and the carboxylate anion fragments in the product ion spectrum. The intensity ratio of sn-1/ sn-2 fragment ions increased with increasing number of double bonds in the sn-2 acyl chain but was not affected by increasing number of double bonds in the sn-1 acyl chain of the species examined. The relative distribution of the molecular species was determined by multiple reaction monitoring of the carboxylate anion fragment from the sn-1 position. A total of 68 different phospholipid species were determined in the head kidney and the largest amount was found in PE (22 species). Depending on the diet, the main species identified in the different phospholipid classes were; PC 16:0/18:1, PE 16:0/22:6, PI 18:0/20:4 and PS 16:0/22:6. The SO diet significantly increased the 18:2, 20:3 and most 20:4 containing species and significantly reduced the 14:0 and most 20:5 and 22:6 fatty acid containing species. The increase of the 20:4 and the decrease of the 20:5 and 22:6 containing species were dependent on the fatty acid combination of the species

Role of phosphatidylethanolamine in assembly and function of the factor IXa-factor VIIIa complex on membrane surfaces.

Phospholipid membranes play a significant role during the proteolytic activation of blood coagulation proteins. This investigation identifies a role for phosphatidylethanolamine (PE) during the activation of factor X by the tenase complex, an enzymatic complex composed of the serine protease, factor IXa, a protein cofactor, factor VIIIa, a phospholipid membrane, and Ca(2+). Phospholipid vesicles composed of PE, phosphatidylserine (PS), and phosphatidylcholine support factor Xa generation. The K(m) and k(cat) for factor X activation by the tenase complex are independent of PE in the presence of 20% PS. At lower PS concentrations, the presence of 20 or 35% PE lowers the K(m) and increases the k(cat) as compared to those in vesicles without PE. The effect of PE on the k(cat) of the tenase complex is mediated through factor VIIIa. PE also enhances factor Xa generation by facilitating tenase complex formation; PE lowers the K(d(app)) of factor IXa for both phospholipid/Ca(2+) and phospholipid/Ca(2+)/factor VIIIa complexes in the presence of suboptimal PS. In addition, the K(d)s of factor IXa and factor X were lower for phospholipid vesicles containing PE. N-Methyl-PE increased the k(cat) and decreased the K(d(app)), whereas N,N-dimethyl-PE had no effect on either parameter, indicating the importance of headgroup size. Lyso-PE had no effect on kinetic parameters, indicating the sn-2 acyl chain dependence of the PE effect. Together, these results demonstrate a role for PE in the assembly and activity of the tenase complex and further extend the understanding of the importance of PE-containing membranes in hemostasis.

Spin-label electron spin resonance studies on the mode of anchoring and vertical location of the N-acyl chain in N-acylphosphatidylethanolamines.

Electron spin resonance (ESR) studies have been performed on N-myristoyl dimyristoylphosphatidylethanolamine (N-14-DMPE) membranes using both phosphatidylcholines spin-labeled at different positions in the sn-2 acyl chain and N-acyl phosphatidylethanolamines spin-labeled in the N-acyl chain to characterize the location and mobility of the N-acyl chain in the lipid membranes. Comparison of the positional dependences of the spectral data for the two series of spin-labeled lipids suggests that the N-acyl chain is positioned at approximately the same level as the sn-2 chain of the phosphatidylcholine spin-label. Further, similar conclusions are reached when the ESR spectra of the N-acyl PE spin-labels in dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylethanolamine (DMPE) host matrixes are compared with those of phosphatidylcholine spin-labels in these two lipids. Finally, the chain ordering effect of cholesterol has also been found to be similar for the N-acyl PE spin-label and PC spin-labels, when the host matrix is either DMPC and cholesterol or N-14-DMPE and cholesterol at a 6:4 mole ratio. In both cases, the gel-to-liquid crystalline phase transition is completely abolished but cholesterol perturbs the gel-phase mobility of N-14-DMPE more readily than that of DMPC. These results demonstrate that the long N-acyl chains are anchored firmly in the hydrophobic interior of the membrane, in an orientation that is parallel to that of the O-acyl chains, and are located at nearly the same vertical position as that of the sn-2 acyl chains in the lipid bilayer. There is a high degree of dynamic compatibility between the N-acyl chains and the O-acyl chains of the lipid bilayer core, although bilayers of N-acyl phosphatidylethanolamines possess a more hydrophobic interior than phosphatidylcholine bilayers. These results provide a structural basis for rationalizing the biological properties of NAPEs.

Comparative dynamics and location of chain spin-labelled sphingomyelin and phosphatidylcholine in dimyristoyl phosphatidylcholine membranes studied by EPR spectroscopy

The dynamics and environment of sphingomyelin spin-labelled at different positions in the N-acyl chain have been studied in dimyristoyl phosphatidylcholine bilayer membranes by using electron spin resonance spectroscopy. Comparison was made with phosphatidylcholine spin-labelled on the sn-2 acyl chain in the same host membrane. Spin-labelled sphingomyelin was found to mix well with the host phosphatidylcholine lipids in both gel and fluid phase membranes. At 1 mol%, mutual spin–spin interactions are no greater than for spin-labelled phosphatidylcholine. In the fluid membrane phase, the effective chain order parameters and polarity-sensitive isotropic hyperfine coupling constants of spin-labelled sphingomyelin display a similar dependence on the position of labelling to those of spin-labelled phosphatidylcholine. The values of both parameters are, however, generally larger for sphingomyelin than for phosphatidylcholine at equivalent positions of acyl chain labelling. This difference is attributed to the different chain linkage of sphingo- and glycero-lipids, combined with an offset of approximately one C-atom in transbilayer register between the respective N-acyl and O-acyl chains. In the gel phase, differences in chain configuration between sphingomyelin and phosphatidylcholine are indicated by differences in spin label spectral anisotropy between the two lipids, which appears to reverse towards the terminal methyl chain end.

1-Cys Peroxiredoxin, a Bifunctional Enzyme with Glutathione Peroxidase and Phospholipase A2 Activities

This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional ("moonlighting") enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca(2+)-independent phospholipase A(2) (aiPLA(2)). The cDNA encoding aiPLA(2) was found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously reported E. coli construct which has a His-tag and 50 additional amino acids at the NH(2) terminus, did not exhibit aiPLA(2) activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn-2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H(2)O(2) at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA(2) activity is inhibited by the serine protease inhibitor, diethyl p-nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect on aiPLA(2) activity. Mutation of Ser(32) to Ala abolishes aiPLA(2) activity, yet the NSGPx activity remains unaffected; a Cys(47) to Ser mutant is devoid of peroxidase activity but aiPLA(2) activity remains intact. These results suggest that Ser(32) in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA(2), while Cys(47) in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of 1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as well as in protection against oxidative injury.

Raman spectroscopic study of polycrystalline mono- and polyunsaturated 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines: bilayer lipid clustering and acyl chain order and disorder characteristics.

Polycrystalline lipid samples of a series of mono- and polyunsaturated, double bond positional isomers of 1-eicosanoyl-d(39)-2-eicosenoyl-sn-glycero-3-phosphocholines [C(20-d(39)):C(20:1 Delta(j))PC, with j = 5, 8, 11, or 13; C(20-d(39)):C(20:2 Delta(11,14))PC; and C(20-d(39)):C(20:3 Delta(11, 14,17))PC] were investigated using vibrational Raman spectroscopy to assess the acyl chain packing order-disorder characteristics and putative bilayer cluster formation of the isotopically differentiated acyl chains. Perdeuteration of specifically the saturated sn-1 acyl chains for these bilayer systems enables each chain's intra- and intermolecular conformational and organizational properties to be evaluated separately. Various saturated chain methylene CD(2) and carbon-carbon (C&bond;C) stretching mode peak height intensity ratios and line width parameters for the polycrystalline samples demonstrate a high degree of sn-1 chain order that is unaffected by either the double bond placement or number of unsaturated bonds within the sn-2 chain. In contrast, the unsaturated sn-2 chain spectral signatures reflect increasing acyl chain conformational disorder as either the cis double bond is generally repositioned toward the chain terminus or the number of double bonds increases from one to three. The lipid bilayer chain packing differences observed between the sn-1 and sn-2 chains of this series of monounsaturated and polyunsaturated 20 carbon chain lipids suggest the existence of laterally distributed microdomains predicated on the formation of highly ordered, saturated sn-1 chain clusters.

Interactions of angiotensin II non-peptide AT 1 antagonist losartan with phospholipid membranes studied by combined use of differential scanning calorimetry and electron spin resonance spectroscopy

We used differential scanning calorimetry (DSC) and electron spin resonance (ESR) spectroscopy to investigate the interactions of Losartan, a potent, orally active Angiotensin II AT 1 receptor antagonist with phospholipid membranes. DSC results showed that Losartan sensitively affected the chain-melting behavior of dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes. ESR spectroscopy showed that phosphatidylcholines spin-labeled at the 5-position of the sn-2 acyl chain ( n-PCSL with n=5), incorporated either in DMPC or DPPC bilayers containing Losartan, were restricted in motion both in the gel and in the liquid–crystalline membrane phases, indicating a location of the antagonist close to the interfacial region of the phosphatidylcholine bilayer. At high drug concentrations (mole fraction ≥ x=0.60), the decrease in chain mobility registered by 5-PCSL in fluid-phase membranes is smaller than that found at lower concentrations, whereas that registered by 14-PCSL is further increased. This indicates a different mode of interaction with Losartan at high concentrations, possibly arising from a location deeper within the bilayer. Additionally, Losartan reduced the spin–spin broadening of 12-PCSL spin labels in the gel-phase of DMPC and DPPC bilayers. As a conclusion, our study has shown that Losartan interacts with phospholipid membranes by affecting both their thermotropic behavior and molecular mobility.

Biaxial ordering of terminal diene groups in lipid membranes: an infrared linear dichroism study

The molecular order within the hydrophobic core of membranes of the diene lipid di-tetradecadienoylphosphatidylcholine was studied by means of infrared spectroscopy on multibilayer assemblies which orient macroscopically on the surface of an attenuated total reflection crystal. The relative humidity and temperature were used as variable parameters to demonstrate that there were profound differences in the melting transition of lipids possessing predominantly cis and trans diene groups. The cis isomer undergoes the phase transition at a vapor pressure which is increased by ∽0.15 GPa when compared with that of the trans isomer. The methylene wagging band progression gives no indication of differences between the acyl chain conformation of the cis and trans forms in the gel state. The frequencies of a number of absorption bands of the diene groups reveal that these moieties are predominantly in the s-trans conformation to accommodate a favorable packing within the bilayer. The linear dichroism of selected in-plane and out-of-plane vibrations of the diene groups gives indications of the biaxial ordering of these moieties. We present the basic equations for the quantitative analysis of IR dichroism data of lamellar structures in terms of transverse and longitudinal molecular order parameters. It turns out that the planes of the rigid diene groups orient preferentially in a perpendicular direction with respect to the bilayer surface and parallel to each other forming in this way a layer of well-aligned diene groups in the bilayer center. This finding is confirmed by the results of X-ray measurements. We suggest that the partial interdigitation of the diene groups of the sn-1 acyl chains promotes the formation of the inverse H II phase and/or enables the formation of covalent bonds between both the monolayers upon polymerization of diene lipids.

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.

Effects of Various Numbers and Positions of cisDouble Bonds in the sn-2 Acyl Chain of Phosphatidylethanolamine on the Chain-melting Temperature

In an attempt to investigate systematically the effects of various single and multiple cis carbon-carbon double bonds in the sn-2 acyl chains of natural phospholipids on membrane properties, we have de novo synthesized unsaturated C20 fatty acids comprised of single or multiple methylene-interrupted cis double bonds. Subsequently, 15 molecular species of phosphatidylethanolamine (PE) with sn-1 C20-saturated and sn-2 C20-unsaturated acyl chains were semi-synthesized by acylation of C20-lysophosphatidylcholine with unsaturated C20 fatty acids followed by phospholipase D-catalyzed base-exchange reaction in the presence of excess ethanolamine. The gel-to-liquid crystalline phase transitions of these 15 mixed-chain PE, in excess H2O, were investigated by high resolution differential scanning calorimetry. In addition, the energy-minimized structures of these sn-1 C20-saturated/sn-2 C20-unsaturated PE were simulated by molecular mechanics calculations. It is shown that the successive introduction of cis double bonds into the sn-2 acyl chain of C(20):C(20)PE can affect the gel-to-liquid crystalline phase transition temperature, Tm, of the lipid bilayer in some characteristic ways; moreover, the effect depends critically on the position of cis double bonds in the sn-2 acyl chain. Specifically, we have constructed a novel Tm diagram for the 15 species of unsaturated PE, from which the effects of the number and the position of cis double bonds on Tm can be examined simultaneously in a simple, direct, and unifying manner. Interestingly, the characteristic Tm profiles exhibited by different series of mixed-chain PE with increasing degree of unsaturation can be interpreted in terms of structural changes associated with acyl chain unsaturation.

Probing the role of C-1 ester group in Naja naja phospholipase A2-phospholipid interactions using butanetriol-containing phosphatidylcholine analogues.

To understand the role of the ester moiety of the sn-1 acyl chain in phospholipase A2-glycerophospholipid interactions, we introduced an additional methylene residue between the glycerol C1 and C2 carbon atoms of phosphatidylcholines, and then studied the kinetics of hydrolysis and the binding of such butanetriol-containing phospholipids with Naja naja phospholipase A2. Hydrolysis was monitored by using phospholipids containing a NBD-labelled sn-2 acyl chain and binding was ascertained by measuring the protein tryptophan fluorescence. The hydrolysis of butanetriol-containing phospholipids was invariably slower than that of the glycerol-containing phospholipids. In addition, the enzyme binding with the substrate was markedly decreased upon replacing the glycerol residue with the 1,3,4-butanetriol moiety in phosphatidylcholines. These results have been interpreted to suggest that the sn-1 ester group in glycerophospholipids could play an important role in phospholipase A2-phospholipid interactions.

Study of mechanisms involved in the collision-induced dissociation of carboxylate anions from glycerophospholipids using negative ion electrospray tandem quadrupole mass spectrometry.

The collision-induced dissociation of the carboxylate anions from human blood phosphatdilycholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidic acid (PA) containing the C18:0 (sn-1) and C20:4 (sn-2) fatty acyl residues was studied using normal phase liquid chromatography coupled with negative ion electrospray tandem mass spectrometry. The product ion peak area ratio of C18:0 to C20:4 was calculated for each phospholipid species and was found to increase with increasing collision energy for all classes. For the phospholipids with a net neutral charge (PE, PC) there was a preferential loss of the sn-2 carboxylate anion (C20:4) at low collision energy, while at higher energy there was a preferential loss of the sn-1 carboxylate anion (C18:0). For the phospholipids with a net negative charge (PI, PA, PS) the intensity of the sn-1 carboxylate anion peak was equal to or higher than the sn-2 carboxylate anion peak at the energies measured. At a given collision energy the product ion peak area ratio decreased in the order PA > or = PS > PI. Studying PS and PE species at different collision energies, it was found that for both classes the increase in the abundance ratio with increasing collision energy was largely dependent on the chain length and degree of unsaturation of the sn-2 acyl chain.

Determinants of the selection of phosphatidylcholine molecular species for secretion into bile in the rat.

Certain phosphatidylcholine (PC) molecular species appear to be secreted into bile preferentially, but the mechanism for this selection remains obscure. We used multivariate analysis to examine the relationship between PC structure and the odds of secretion for individual PC species secreted into bile. PC was isolated from Folch extracts of bile and liver from rats, and individual molecular species of PC were quantified with reverse-phase high-performance liquid chromatography (HPLC). The odds of secretion for a given PC species were quantified as the ratio of its mole% in bile/mole% in liver. Regression analysis indicated that the odds of secretion were significantly related to length of both the sn-1 and sn-2 acyl chains (P < .0001 for both) and to relative hydrophobicity as determined by reverse-phase HPLC (P < .0001). In addition, the relationship between odds of secretion and sn-1 chain length was best described by a parabolic function. Considered together, these characteristics accounted for 88% of the observed differences in odds of secretion. This relationship between PC structure and odds of secretion was strikingly similar to the relationship between PC structure and affinity for bovine PC transfer protein. When multivariate models were used to predict both the odds of secretion and the affinity for PC transfer protein for a set of biologically plausible PC species, there was a linear relationship between the two. The likelihood of a given PC species being secreted into bile can be related to the structural characteristics of the acyl chains without having to postulate the existence of a special pool of PC destined for biliary secretion. Second, the structural characteristics that dictate selection of PC species for secretion into bile are similar to those that determine binding affinity for PC transfer protein, suggesting that the likelihood of a PC being secreted into bile is, in fact, closely related to its binding affinity for PC transfer protein (PC-TP).

Unsaturated phospholipid acyl chains are required to constitute membrane binding sites for factor VIII.

Membranes containing phosphatidyl-L-serine (PS) and phosphatidylethanolamine (PE) greatly enhance the function of the enzymatic cofactor factor VIII. The mechanisms of enhanced function involve condensation of enzyme (factor IXa), activated cofactor (factor VIIIa), and substrate (factor X) at a common location and, most dramatically, activation of the assembled enzyme-cofactor complex. We asked whether unsaturated phospholipid (PL) acyl chains are necessary to constitute factor VIII binding sites or to activate the factor VIIIa-factor IXa complex. We found that membranes composed of saturated, dimyristoyl phospholipids had 20-fold fewer factor VIII binding sites and that these sites supported less than 5% normal activity of the factor VIIIa-factor IXa complex. Thrombin-activated factor VIII bound to a similar number of membrane sites, and thrombin activation did not reduce the affinity for saturated membranes more than 2-fold so that the loss of functional activity is due to a requirement of the factor VIIIa-factor IXa complex for unsaturated acyl chains that exceeds the requirement for factor VIII binding alone. Replacement of dimyristoyl-PS, -PE, or -PC individually with the corresponding unsaturated phospholipid restored 75%, 60%, and 15%, respectively, of factor VIII binding sites but less than 10% of factor VIIIa-factor IXa activating activity. Lyso-PS did not support binding of factor VIII or function of the factor VIIIa-factor IXa complex even when PE and phosphatidylcholine contained unsaturated acyl chains. We conclude that the sn-2 acyl chain of PS and unsaturated phospholipid acyl chains are chemical requirements for constitution of fully functional factor VIII binding sites on phospholipid membranes.