Packing characteristics of highly unsaturated bilayer lipids: Raman spectroscopic studies of multilamellar phosphatidylcholine dispersions

The thermotropic properties and acyl chain packing characteristics of multilamellar dispersions of highly unsaturated lipids were examined by Raman spectroscopy. Bilayer assemblies were composed of POPC (1-palmitoyl-2-oleoylphosphatidylcholine), PAPC (1-palmitoyl-2-arachidonylphosphatidylcholine), and PDPC (1-palmitoyl-2-docosahexaenoylphosphatidylcholine), lipid systems possessing saturated sn-1 chains and unsaturated sn-2 chains with one, four, and six double bonds, respectively. Raman spectra were recorded in the acyl chain 2800-3100-cm-1 carbon-hydrogen (C-H) stretching and 1100-1200-cm-1 carbon-carbon (C-C) stretching mode regions, spectral intervals reflecting both the inter- and intrachain order/disorder properties of the various lipid dispersions. In order to obtain C-H stretching mode spectra relevant solely to the sn-1 chains of PAPC and PDPC, liquid-phase spectra of arachidonic and docosahexaenoic acid, respectively, were subtracted from the observed phospholipid spectra. The unsaturated sn-2 chains of PAPC and PDPC undergo minimal conformational reorganizations as the bilayers pass from the gel to liquid-crystalline phases. Phase transition temperatures, Tm, derived from statistically fitting the temperature-dependent Raman spectral data are approximately -2.5, -22.5, and -3 degrees C for POPC, PAPC, and PDPC, respectively. As the degree of unsaturation increases from POPC to PAPC and PDPC, the cooperativity of the phase transition, as measured by its breadth, decreases. Estimates of the transition widths from the temperature profiles are approximately 15 degrees C for PAPC and 20 degrees C for PDPC. The behavior of various Raman spectral parameters for the lipid gel phase reflects the formation of lateral microdomains, or clusters, whose packing properties maximize the van der Waals interactions between sn-1 chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acids 149 and 294 of human lecithin:cholesterol acyltransferase affect fatty acyl specificity

We identified two regions of human LCAT (hLCAT) that when mutated separately to the corresponding rat sequence (E149A and Y292H/W294F) and transiently expressed in COS-1 cells increased phospholipase A2 (PLA2) activity by 5.5- and 2.8-fold, respectively, and increased cholesteryl ester (CE) formation by 2.9- and 1.4-fold, respectively, relative to hLCAT using substrate particles containing 1-16:0,2-20:4-sn-glycero-3-phosphocholine (PAPC). In contrast, both activities with 1-16:0,2-18:1-sn-glycero-3-phosphocholine (POPC) substrate were similar among the three LCAT proteins. The triple mutant (E149A/Y292H/W294F) had increased PLA2 activity with PAPC similar to that observed with the E149A mutation alone; however, unlike E149A, the triple mutant demonstrated a 50% decrease in activity with POPC for both PLA2 activity and CE formation, suggesting an interaction between the two regions of LCAT. Additional mutagenesis studies demonstrated that W294F, but not Y292H, increased PLA2 activity by 3-fold with PAPC without affecting activity with POPC. The E149A/W294F double mutation mimicked the LCAT activity phenotype of the triple mutant (more activity with PAPC, less with POPC). In conclusion, separate mutation of two amino acids in hLCAT to the corresponding rat sequence increases activity with PAPC, whereas the combined mutations increase PAPC and decrease POPC activity, suggesting that these amino acids participate in the LCAT PC binding site and affect fatty acyl specificity.     

Inhibition of lecithin:cholesterol acyltransferase activity by synthetic phosphatidylcholine species containing eicosapentaenoic acid or docosahexaenoic acid in the sn-2 position.

Phospholipids isolated from the plasma of monkeys fed a diet enriched in fish oil were poor substrates for cholesteryl ester (CE) synthesis by the lecithin:cholesterol acyltransferase (LCAT) reaction relative to those from animals fed a lard containing diet when the phospholipids were used for the preparation of recombinant particles by cholate dialysis (Parks, J. S., B. C. Bullock, and L. L. Rudel. 1989. J. Biol. Chem. 264: 2545-2551). The purpose of the present study was to directly test the influence of eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3) in the sn-2 position of phosphatidylcholine (PC) on the activity of LCAT. PC species containing 1-palmitoyl-2-oleoyl PC (POPC), 1-palmitoyl-2-linoleoyl PC (PLPC), 1-palmitoyl-2-arachidonoyl PC (PAPC), 1-palmitoyl-2-eicosapentaenoyl PC (PEPC), or 1-palmitoyl-2-docosahexaenoyl PC (PDPC) were purchased or synthesized and made into recombinant particles of uniform size and composition with [14C]cholesterol and apoA-I using the cholate dialysis procedure. The recombinant particles (PC:cholesterol:apoA-I molar ratio = 42:1.9:1) exhibited the following order of reactivity towards purified human LCAT in vitro: POPC greater than PLPC greater than PEPC = PAPC greater than PDPC. The apparent Vmax/Km for recombinant particles containing PEPC and PDPC was 17% and 7% that of particles containing POPC, respectively. There was a linear decrease in CE formation when the percentage of PEPC or PDPC was increased from 0 to 100% relative to POPC in recombinant particles with a constant PC:cholesterol:apoA-I molar ratio, suggesting that the PEPC and PDPC were competitive inhibitors of the LCAT reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transbilayer diffusion of phospholipids: dependence on headgroup structure and acyl chain length

The kinetics and thermodynamics of the transmembrane movement (flip-flop) of fluorescent analogs of phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were investigated to determine the contributions of headgroup composition and acyl chain length to phospholipid flip-flop. The phospholipid derivatives containing n-octanoic, n-decanoic or n-dodecanoic acid in the sn-1 position and 9-(1-pyrenyl)nonanoic acid in the sn-2 position were incorporated at 3 mol% into sonicated single-bilayer vesicles of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC). The kinetics of diffusion of the pyrene-labeled phospholipids from the outer and inner monolayers of the host vesicles to a large pool of POPC acceptor vesicles were monitored by the time-dependent decrease of pyrene excimer fluorescence. The observed kinetics of transfer were biexponential, with a fast component due to the spontaneous transfer of pyrenyl phospholipids in the outer monolayer of labeled vesicles and a slower component due to diffusion of pyrenyl phospholipid from the inner monolayer of the same vesicles. Intervesicular transfer rates decreased approx. 8-fold for every two carbons added to the first acyl chain. Correspondingly, the free energy of activation for transfer increased approx. 1.3 kcal/mol. With the exception of PE, the intervesicular transfer rates for the different headgroups within a homologous series were nearly the same, with the PC derivative being the fastest. Transfer rates for the PE derivatives were 5-to 7-fold slower than the rates observed for PC. Phospholipid flip-flop, in contrast, was strongly dependent on headgroup composition with a smaller dependence on acyl chain length. At pH 7.4, flip-flop rates increased in the order PC less than PG less than PA less than PE, where the rates for PE were at least 10-times greater than those of the homologous PC derivative. Activation energies for flip-flop were large, and ranged from 38 kcal/mol for the longest acyl chain derivative of PC to 25 kcal/mol for the PE derivatives. Titration of the PA headgroup at pH 4.0 produced an approx. 500-fold increase in the flip-flop rate of PA, while the activation energy decreased 10 kcal/mol. Increasing acyl chain length reduced phospholipid flip-flop rates, with the greatest change observed for the PC analogs, which exhibited an approx. 2-fold decrease in flip-flop rate for every two methylene carbons added to the acyl chain at the sn-1 position.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

Evidence that phospholipids play a key role in pre-beta apoA-I formation and high-density lipoprotein remodeling

The initial plasma acceptor of unesterified cholesterol and phospholipids from peripheral cells has been identified as pre-beta migrating, lipid-free, or lipid-poor apolipoprotein (apo) A-I (pre-beta apoA-I). Pre-beta apoA-I is formed when plasma factors, such as cholesteryl ester transfer protein (CETP), remodel high-density lipoproteins (HDL). The aim of this study is to determine how phospholipids influence pre-beta apoA-I formation during the CETP-mediated remodeling of HDL. Reconstituted HDL (rHDL) containing either 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonyl phosphatidylcholine (PAPC), or 1-palmitoyl-2-docosahexanoyl phosphatidylcholine (PDPC) as the only phospholipid were prepared. The rHDL were comparable in size and core lipid/protein molar ratio and contained only cholesteryl esters in their core and apoA-I as the sole apolipoprotein. The (POPC)rHDL, (PLPC)rHDL, (PAPC)rHDL, and (PDPC)rHDL were respectively incubated for 0-24 h with CETP and microemulsions containing triolein and either POPC, PLPC, PAPC, or PDPC. The rate at which the rHDL were depleted of core lipids and remodeled to small particles varied widely with (POPC)rHDL < (PLPC)rHDL < (PDPC)rHDL approximately (PAPC)rHDL. Pre-beta apoA-I was not formed in the (POPC)rHDL incubations. Pre-beta apoA-I was apparent by 24 h in the (PLPC)rHDL incubations and by 12 h in the (PAPC)rHDL and (PDPC)rHDL incubations. The enhanced formation of pre-beta apoA-I in the (PAPC)rHDL and (PDPC)rHDL incubations reflected the increased core lipid depletion of the particles combined with the destabilization and progressive exclusion of apoA-I from the particle surface. In conclusion, these results show that phospholipids play a key role in the CETP-mediated remodeling of rHDL and pre-beta apoA-I formation.     

Loss of stereospecificity of phospholipases C and D upon introduction of a 2-alkyl group into rac-1,2-diacylglycero-3-phosphocholine

rac-1-[1-14C]Lauroyl-2-oleylglycero-3-phospho[methyl-3H]choline and rac-1-lauroyl-2-[1-14C]oleoylglycero-3-phospho[methyl-3H]choline along with rac-1-palmitoyl-2-oleylglycero-3-phosphocholine and sn-1-palmitoyl-2-oleylglycero-3-phosphocholine were synthesized and subjected to hydrolysis with phospholipase C (EC 3.1.4.3) from Clostridium perfringens and phospholipase D (EC 3.1.4.4) from cabbage. Kinetics of hydrolysis of the radioactive substrates were determined by measuring the 3H radioactivity retained in the aqueous phase due to free choline and phosphocholine and the 3H and 14C radioactivity recovered in the organic phase due to the released diacylglycerols and phosphatidic acids and the residual phosphatidylcholines. The rate of hydrolysis of the unlabelled substrates by phospholipase C was determined by thin-layer chromatography and gas-liquid chromatography of the methanolysis products. The relative initial rates of hydrolysis of sn-1,2,- and sn-2,3-enantiomers were 100-200:1 for phospholipase C and 40-50:1 for phospholipase D using rac-1-lauroyl-2-oleoylglycero-3-phosphocholine as the substrate. The substitution of the 2-acyl group by an alkyl group resulted in a loss of stereospecificity, which was partial for phospholipase C (relative rates equal to 8-13:1) and total for phospholipase D. There was a parallel dramatic decrease (500-1000-fold) in the initial rate of hydrolysis with phospholipase C but the activity of phospholipase D was only moderately reduced (18-fold). These findings are consistent with the earlier observed loss of the stereospecificity of lipoprotein lipase following introduction of a 2-alkyl group into triacylycerols, and point to a general unsuitability of 2-alkyl-linked acylglycerols as substrates for the assay of the stereospecificity of lipases, as well as for the isolation of enantiomeric 2-alkylacylglycerols by means of stereospecific lipases.     

Acyl chain dynamics of phosphatidylethanolamines containing oleic acid and dihydrosterculic acid: 2H NMR relaxation studies

The dynamical behavior of the acyl chains of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, and 1-palmitoyl-2-dihydrosterculoyl-sn-glycero-3-phosphoethanolamine has been investigated by using 2H T1 and T2 relaxation times. Lipids were labeled at the 5-,9-,10-, and 16-positions of the sn-2 acyl chain. The profile of deuterium spin-lattice relaxation rate (T1(-1) vs. chain position is characterized in all systems by a marked discontinuity at the positions of the carbon-carbon double bond and the cyclopropane ring; the deuterons at these positions have relaxation rates which are greater than at any other labeled position of the sn-2 chain. For both types of sn-2 acyl chain, assuming a single-exponential correlation time and that the motion is within the rapid regime, the phosphatidylcholine lipid systems are less mobile than their phosphatidylethanolamine analogues. Systems containing an oleoyl chain are more dynamic than their analogues containing a dihydrosterculoyl chain. The rates of motion of the sn-2 acyl chains of phosphatidylethanolamine in a bilayer structure are slower than those of the lipid in an inverted hexagonal structure. In the hexagonal phase, the motional rates of a dihydrosterculoyl chain are slower than those of the corresponding positions of an oleoyl chain.     

Barotropic and thermotropic bilayer phase behavior of positional isomers of unsaturated mixed-chain phosphatidylcholines

The bilayer phase transitions of six kinds of mixed-chain phosphatidylcholines (PCs) with an unsaturated acyl chain in the sn-1 or sn-2 position, 1-oleoyl-2-stearoyl- (OSPC), 1-stearoyl-2-oleoyl- (SOPC), 1-oleoyl-2-palmitoyl- (OPPC), 1-palmitoyl-2-oleoyl- (POPC), 1-oleoyl-2-myristoyl- (OMPC) and 1-myristoyl-2-oleoyl-sn-glycero-3-phosphocholine (MOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light transmittance measurements. Bilayer membranes of SOPC, POPC and MOPC with an unsaturated acyl chain in the sn-2 position exhibited only one phase transition, which was identified as the main transition between the lamellar gel (L_β) and liquid crystalline (L_α) phases. On the other hand, the bilayer membranes of OSPC, OPPC and OMPC with an unsaturated acyl chain in the sn-1 position exhibited not only the main transition but also a transition from the lamellar crystal (L_c) to the L_β (or L_α) phase. The stability of their gel phases was markedly affected by pressure and chain length of the saturated acyl chain in the sn-2 position. Considering the effective chain lengths of unsaturated mixed-chain PCs, the difference in the effective chain length between the sn-1 and sn-2 acyl chains was proven to be closely related to the temperature difference of the main transition. That is, a mismatch of the effective chain length promotes a temperature difference of the main transition between the positional isomers. Anomalously small volume changes of the L_c/L_α transition for the OPPC and OMPC bilayers were found despite their large enthalpy changes. This behavior is attributable to the existence of a cis double bond and to significant inequivalence between the sn-1 and sn-2 acyl chains, which brings about a small volume change for chain melting due to loose chain packing, corresponding to a large partial molar volume, even in the L_c phase. Further, the bilayer behavior of unsaturated mixed-chain PCs containing an unsaturated acyl chain in the sn-1 or sn-2 position was well explained by the chemical-potential diagram of a lipid in each phase.     

The acylation of lipophilic alcohols by lysosomal phospholipase A2

A novel lysosomal phospholipase A(2) (LPLA2) with specificity toward phosphatidylethanolamine and phosphatidylcholine was previously purified and cloned. LPLA2 transfers sn-1 or sn-2 acyl groups of phospholipids to the C1 hydroxyl of the short-chain ceramide N-acetylsphingosine (NAS) under acidic conditions. The common features of lipophilic alcohols serving as acceptor molecules in the transacylase reaction were examined. 1-O-Hexadecyl-2-acetyl-sn-glycerol (HAG) was acylated by LPLA2 similar to NAS. HAG competed with NAS and inhibited NAS acylation. The transacylation of 1-O-hexadecyl-glycerol (HG), 1-O-palmityl-2-O-methyl-sn-glycerol (PMG), and monoacylglycerols was also investigated. HG, PMG, 1- or 3-palmitoyl-sn-glycerol, and 2-palmitoylglycerol were converted to 1,3-alkylacylglycerol, 1,2-dialkyl-3-acylglycerol, 1,3-diacylglycerol, and 1,2- or 2,3-diacylglycerol, respectively. HG and monoacylglycerol inhibited the acylation of NAS by the enzyme with IC(50) values of 35 and 45 microM, respectively. Additionally, the enzyme acylated glycerol to produce 1- or 3-acyl-sn-glycerol but not 2-acylglycerol. Therefore, the preferred acceptor molecules for LPLA2 are primary alcohols with one long carbon chain and one small nonpolar residue linked to the C2 position of ethanol. The enzyme acylated other natural lipophilic alcohols, including anandamide and oleoylethanolamide. Thus, LPLA2 may function to remodel acyl groups and modulate the biological and pharmacological activities of some lipophilic alcohols.     

Conversion of Bacillus thermocatenulatus lipase into an efficient phospholipase with increased activity towards long-chain fatty acyl substrates by directed evolution and rational design.

The thermoalkalophilic lipase from Bacillus thermocatenulatus BTL2 exhibits a low phospholipase activity (lecithin/tributyrin ratio 0.03). A single round of random mutagenesis of the BTL2 gene followed by screening of 6000 transformants on egg-yolk plates identified three variants with 10-12-fold increased phospholipase activities, corresponding to lecithin/tributyrin ratios of 0.16-0.36. All variants were specific for the sn-1 acyl ester bond of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Mutations occurred predominantly in the N-terminal part of BTL2 with regions surrounding the predicted helix alpha(4) and lid as hotspots. Two mutations, L184P located in the predicted helix alpha(4) and H15P found in the highly conserved oxy-anion hole motif among hydrolases, were identified to account for increased phospholipase activity. Two of the three variants showed reduced activities towards medium- and long-chain fatty acyl methyl esters compared to the wild-type enzyme. Substitution of Leu353 with Ser, which is located adjacent to the active site histidine and is important for phospholipase activity in the Staphylococcus hyicus lipase, increased the absolute phospholipase activities of the variants, but not of BTL2, approximately 2-fold. The engineered best variant displayed a lecithin/tributyrin ratio of 0.52, corresponding to a 17-fold increase compared to the wild-type enzyme. Moreover, this variant exhibited a 1.5-4-fold higher activity towards long-chain fatty acyl methyl ester (C18:1, C18:2, C18 and C20) compared to BTL2. A second round of mutagenesis and screening on lecithin-plates yielded no new variants with further increased phospholipase/lipase activity ratios, but instead one variant with a 5-fold increased expression rate and two variants with a 3-fold reduced activity towards triolein were obtained.     

Altered positional specificity of human plasma lecithin-cholesterol acyltransferase in the presence of sn-2 arachidonoyl phosphatidyl cholines. Mechanism of formation of saturated cholesteryl esters.

The positional specificity of purified human lecithin-cholesterol acyltransferase (LCAT) was studied by analyzing the labeled cholesteryl ester (CE) species formed in the presence of proteoliposome substrates containing mixed chain phosphatidylcholine (PC) species, labeled cholesterol and apoprotein A-I. Whereas over 90% of the acyl groups used for CE synthesis were derived from the sn-2 position of most of the naturally occurring PC substrates, about 75% of the CE species formed in the presence of sn-1-myristoyl 2-arachidonoyl PC, sn-1-palmitoyl-2-arachidonoyl (PAPC) and sn-1-palmitoyl 2-docosahexaenoyl PC were derived from the sn-1-position. On the other hand, rat LCAT utilized mostly sn-2-acyl group from either PAPC or from sn-1-palmitoyl 2-linoleoyl PC. The positional specificity of the human enzyme was not affected by the alteration in the matrix fluidity, type of the apoprotein activator used, or by the free cholesterol/PC ratio in the substrate. These results show that the positional specificity of human plasma LCAT is altered in the presence of sn-2-arachidonoyl PC, or sn-2-docosahexaenoyl PC, probably due to steric restrictions at the active site, and this may account for the formation of disproportionately high concentrations of saturated CE, and low concentrations of long-chain polyunsaturated CE in human plasma, relative to the composition of sn-2-acyl groups in plasma PC.     

Endothelial lipase releases saturated and unsaturated fatty acids of high density lipoprotein phosphatidylcholine

We assessed the ability of endothelial lipase (EL) to hydrolyze the sn-1 and sn-2 fatty acids (FAs) from HDL phosphatidylcholine. For this purpose, reconstituted discoidal HDLs (rHDLs) that contained free cholesterol, apolipoprotein A-I, and either 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or 1-palmitoyl-2-arachidonylphosphatidylcholine were incubated with EL- and control (LacZ)-conditioned media. Gas chromatography analysis of the reaction mixtures revealed that both the sn-1 (16:0) and sn-2 (18:1, 18:2, and 20:4) FAs were liberated by EL. The higher rate of sn-1 FA cleavage compared with sn-2 FA release generated corresponding sn-2 acyl lyso-species as determined by MS analysis. EL failed to release sn-2 FA from rHDLs containing 1-O-1'-hexadecenyl-2-arachidonoylphosphatidylcholine, whose sn-1 position contained a nonhydrolyzable alkyl ether linkage. The lack of phospholipase A(2) activity of EL and its ability to liberate [(14)C]FA from [(14)C]lysophosphatidylcholine (lyso-PC) led us to conclude that EL-mediated deacylation of phosphatidylcholine (PC) is initiated at the sn-1 position, followed by the release of the remaining FA from the lyso-PC intermediate. Thin-layer chromatography analysis of cellular lipids obtained from EL-overexpressing cells revealed a pronounced accumulation of [(14)C]phospholipid and [(14)C]triglyceride upon incubation with 1-palmitoyl-2-[1-(14)C]linoleoyl-PC-labeled HDL(3), indicating the ability of EL to supply cells with unsaturated FAs.     

Evidence that hepatic lipase and endothelial lipase have different substrate specificities for high-density lipoprotein phospholipids

Hepatic lipase (HL) and endothelial lipase (EL) are both members of the triglyceride lipase gene family. HL hydrolyzes phospholipids and triglycerides in triglyceride-rich lipoproteins and high-density lipoproteins (HDL). EL hydrolyzes HDL phospholipids and has low triglyceride lipase activity. The aim of this study was to determine if HL and EL hydrolyze different HDL phospholipids and whether HDL phospholipid composition regulates the interaction of EL and HL with the particle surface. Spherical, reconstituted HDL (rHDL) containing either 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-linoleoylphosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonylphosphatidylcholine (PAPC), or 1-palmitoyl-2-docosahexanoylphosphatidylcholine (PDPC) as the only phospholipid, apolipoprotein A-I as the only apolipoprotein, and either cholesteryl esters (CE) only or mixtures of CE and triolein (TO) in their core were prepared. The rHDL were similar in size and had comparable core lipid/apoA-I molar ratios. The CE-containing rHDL were used to determine the kinetics of HL- and EL-mediated phospholipid hydrolysis. For HL the V(max) of phospholipid hydrolysis for (POPC)rHDL > (PLPC)rHDL approximately (PDPC)rHDL > (PAPC)rHDL, while the K(m)(app) for (POPC)rHDL > (PDPC)rHDL > (PLPC)rHDL > (PAPC)rHDL. For EL the V(max) for (PDPC)rHDL > (PAPC)rHDL > (PLPC)rHDL approximately (POPC)rHDL, while the K(m)(app) for (PAPC)rHDL approximately (PLPC)rHDL > (POPC)rHDL > (PDPC)rHDL. The kinetics of EL- and HL-mediated TO hydrolysis was determined using rHDL that contained TO in their core. For HL the V(max) of TO hydrolysis for (PLPC)rHDL > (POPC)rHDL > (PAPC)rHDL > (PDPC)rHDL, while the K(m)(app) for (PLPC)rHDL > (POPC)rHDL approximately (PAPC)rHDL > (PDPC)rHDL. For EL the V(max) and K(m)(app) for (PAPC)rHDL > (PDPC)rHDL > (PLPC)rHDL > (POPC)rHDL. These results establish that EL and HL have different substrate specificities for rHDL phospholipids and that their interactions with the rHDL surface are regulated by phospholipids.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.     

Solubilization and properties of Ca2+-dependent human platelet phospholipase A2

Using a sonicated dispersion of radiolabeled 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine as substrate, we found that phospholipase A2 activity of human platelets was enhanced 2.4-fold by albumin (1 mg/ml). The enzyme was recovered predominantly in the cytosolic fraction of platelets with less than a third of its activity being associated with the membrane fraction. In the presence of 24 mM n-octyl-beta-D-glucopyranoside (octylglucoside) phospholipase A2 was effectively (more than 90%) extracted from platelet lysates without solubilization of platelet membranes. Ion exchange chromatography of the soluble enzyme yielded a phospholipase A2 of unchanged total activity and great stability. This phospholipase A2 was active only in the presence of divalent cations (Ca2+ greater than Sr2+ greater than Mg2+ = 0), required albumin for optimal activity and exhibited exclusive positional specificity for the acyl ester bond at the 2-position of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. Indomethacin (500 microM), mepacrine (500 microM) and N-ethylmaleimide (4 mM) inhibited the phospholipase A2 by 69, 62 and 19%, respectively. The results are discussed in the light of previous findings on human platelet phospholipase A2.     

Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows that PAF receptor antagonists block the action of both PAF and these PAF-like lipids.     

Identification and characterization of kink motifs in 1-palmitoyl-2-oleoyl- phosphatidylcholines: a molecular mechanics study.

As a cis carbon-carbon double bond (delta) is introduced into the middle of an isolated all-trans hydrocarbon chain, it can be shown by molecular graphics that this delta-bond makes a bend of 130 degrees in the chain axis, thus producing a boomerang-like conformation. Such a bent structure, indeed, has been detected experimentally for oleic acid by x-ray crystallography (Abrahamson and Ryderstedt-Nahringbaur, 1962). Membrane diacyl phospholipids are largely mixed-chain lipids containing a saturated sn-1 acyl chain and an unsaturated sn-2 acyl chain. 1-Palmitoyl-2-oleoyl-phosphatidylcholine (POPC), the most abundant phospholipid in animal cell membranes, is a typical example in which the sn-2 acyl chain is the acyl chain of an oleic acid. However, this sn-2 acyl chain of POPC is unlikely to adopt a boomerang-like configuration in the gel-state lipid bilayer due to the steric hindrance imposed by neighboring chains. Instead, it has been suggested that the oleate chain in POPC is kinked in the shape of a crankshaft in the gel-state bilayer (Huang, 1977; Lagaly et al., 1977), because POPC with such a kinked sn-2 acyl chain, which is denoted here as the secondary structural element or motif, can pack efficiently against other neighboring phospholipids. In this communication, 16 different types of secondary structural elements or motifs are derived for POPC at T < Tm based on a single protocol guided by two-dimensional steric contour maps and computer-based molecular graphics. After subjecting these derived molecular species to energy minimization using the molecular mechanics method, the number of the secondary structural motifs is reduced to 13 as a result of conformational degeneracy. The structure and steric energy of each of the energy-minimized lipid rotomers are presented in this communication. Furthermore, these rotomers packed in small clusters are also simulated to mimic the lipid bilayer structure of 1-palmitoyl-2-oleoyl-phosphatidylcholines at T < Tm.     

Phospholipid composition of reconstituted high density lipoproteins influences their ability to inhibit endothelial cell adhesion molecule expression

The ability of different phosphatidylcholine (PC) species to inhibit cytokine-induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) was investigated. PC species containing palmitoyl- in the sn-1 position and palmitoyl- (DPPC), arachidonyl- (PAPC), linoleoyl- (PLPC) or oleoyl- (POPC) in the sn-2 position were compared. These PC species were studied as components of reconstituted high density lipoproteins (rHDL) (containing apolipoprotein A-I [apoA-I] as the sole protein) or as small unilamellar vesicles (SUVs). The rHDL containing PLPC and PAPC inhibited VCAM-1 expression in activated HUVECs by 95 and 70%, respectively, at an apoA-I concentration of 16 micrometer. At this concentration of apoA-I, POPC rHDL inhibited by only 16% and DPPC rHDL did not inhibit at all. These differences could not be explained by differential binding of the rHDL to HUVECs. The same hierarchy of inhibitory activity was observed when these PC species were presented to the cells as SUVs but only when the SUVs also contained an antioxidant. It was concluded that rHDL PC is responsible for their inhibitory activity and that this varies widely with different PC species.     

Acyl chain length affects ceramide action on sterol/sphingomyelin-rich domains

The effects of ceramides with varying saturated N-linked acyl chains (C2-C14) on cholesterol displacement from sphingomyelin-rich domains and on the stability of ordered domains were studied. The bilayers examined were made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), D-erythro-N-palmitoyl-sphingomyelin (PSM), D-erythro-N-acyl-sphingosine, and cholesterol (60:15:15:10 mol%, respectively). Cholestatrienol (CTL) or D-erythro-N-trans-parinoyl-sphingomyelin (tParSM) were used as reporter molecules (at 1 mol%) for the ordered domains, and 1-palmitoyl-2-stearoyl-(7-doxyl)-sn-glycero-3-phosphocholine (7SLPC) as a fluorescence quencher (30 mol%, replacing POPC) in the liquid-disordered phase. The results indicate that the ceramide had to have an N-linked acyl chain with at least 8 methylene units in order for it to displace cholesterol from the sphingomyelin-rich domains at the concentration used. The melting of the sphingomyelin-rich domain shifted to higher temperatures (compared to the ceramide-free control) with C2, C12 and longer chain ceramides, whereas C4-C10 ceramides led to domain melting at lower temperatures than control. This study shows that short-chain ceramides do not have the same effects on sterol- and sphingomyelin-rich domains as naturally occurring longer-chain ceramides have.