Sn-2 Acyl Research Articles

Miscibility and Phase Behavior of N-Acylethanolamine/Diacylphosphatidylethanolamine Binary Mixtures of Matched Acyl Chainlengths ( n = 14, 16)

The miscibility and phase behavior of hydrated binary mixtures of two N-acylethanolamines (NAEs), N-myristoylethanolamine (NMEA), and N-palmitoylethanolamine (NPEA), with the corresponding diacyl phosphatidylethanolamines (PEs), dimyristoylphosphatidylethanolamine (DMPE), and dipalmitoylphosphatidylethanolamine (DPPE), respectively, have been investigated by differential scanning calorimetry (DSC), spin-label electron spin resonance (ESR), and 31P-NMR spectroscopy. Temperature-composition phase diagrams for both NMEA/DMPE and NPEA/DPPE binary systems were established from high sensitivity DSC. The structures of the phases involved were determined by 31P-NMR spectroscopy. For both systems, complete miscibility in the fluid and gel phases is indicated by DSC and ESR, up to 35 mol % of NMEA in DMPE and 40 mol % of NPEA in DPPE. At higher contents of the NAEs, extensive solid-fluid phase separation and solid-solid immiscibility occur depending on the temperature. Characterization of the structures of the mixtures formed with 31P-NMR spectroscopy shows that up to 75 mol % of NAE, both DMPE and DPPE form lamellar structures in the gel phase as well as up to at least 65°C in the fluid phase. ESR spectra of phosphatidylcholine spin labeled at the C-5 position in the sn-2 acyl chain present at a probe concentration of 1 mol % exhibit strong spin-spin broadening in the low-temperature region for both systems, suggesting that the acyl chains pack very tightly and exclude the spin label. However, spectra recorded in the fluid phase do not exhibit any spin-spin broadening and indicate complete miscibility of the two components. The miscibility of NAE and diacyl PE of matched chainlengths is significantly less than that found earlier for NPEA and dipalmitoylphosphatidylcholine, an observation that is consistent with the notion that the NAEs are most likely stored as their precursor lipids ( N-acyl PEs) and are generated only when the system is subjected to membrane stress.

Conformation of an Endogenous Ligand in a Membrane Bilayer for the Macrophage Scavenger Receptor CD36

Phagocytic removal of aged or oxidatively damaged cells and macromolecules is an indispensable homeostatic function of the innate immune system. A structurally conserved family of oxidized phospholipids that serve as endogenous high-affinity ligands for the macrophage scavenger receptor CD36 (oxPC(CD36)) was recently identified. Enriched within atherosclerotic plaque and senescent cell membranes, oxPC(CD36) promote the uptake of oxidized lipoproteins and cell membranes by macrophages when present at only a few molecules per particle. How macrophages recognize oxPC(CD36) within cellular membranes and lipoprotein surfaces remains unknown. Herein, we deduce the conformation of oxPC(CD36) near the hydrophobic-hydrophilic interface within membrane bilayers by determining multiple critical internuclear distances using nuclear Overhauser enhancement spectroscopy. The molecular model reveals a unique conformation for oxPC(CD36) within bilayers whereby the distal end of the sn-2 acyl chain harboring the structurally conserved CD36 recognition motif protrudes into the aqueous phase. The remarkable conformation elucidated for oxPC(CD36) produces a surface accessible phagocytic "eat me signal" to facilitate senescent cell and oxidized lipoprotein recognition by the scavenger receptor CD36 as part of its immune surveillance function.

Structural Elements of Ligand Recognition Site in Secretory Phospholipase A2 and Structure-Based Design of Specific Inhibitors

Phospholipases A2 (phosphotide 2-acylhydrolases, PLA2s, EC 3.1.1.4) are widely distributed enzymes in the animal world. They catalyze the hydrolysis of the sn-2 acyl ester linkage of phospholipids, producing fatty acids and lysophospholipids. The mammalian type II secreted phospholipase A2 (PLA2-II) is one of the most extensively studied member of low molecular weight (13-18 kDa) PLA2s. PLA2-II contains 120-125 amino acid residues and seven disulphide bridges. The important features of overall structure of PLA2-II contain an N-terminal helix, H1 (residues: 2-12), an external loop (residues: 14-23), a calcium binding loop (Ca2+-loop, residues: 25-35), a second alpha-helix, H2 (residues: 40-55), a short two stranded anti-parallel beta-sheet referred to as beta-wing (residues: 75-84), a third alpha-helix, H3 (residues: 90-108) which is antiparallel to H2 and two single helical turns, SH4 (residues: 114-117) and SH5 (residues: 121-125). The three-dimensional structure of PLA2-II has defined a conserved active site within a hydrophobic channel lined by invariant hydrophobic residues. The active site residues His48, Asp49, Tyr52 and Asp99 are directly connected to the channel. An important water molecule that bridges His48 and Asp49 through hydrogen bonds is a part of catalytic network. Based on the structures of various complexes of group II PLA2, the ligand-recognition site has been divided into six subsites consisting of residues 2-10 (subsite 1), residues 17-23 (subsite 2), residues 28-32 (subsite 3), residues 48-52 (subsite 4), residues 68-70 (subsite 5) and residues 98-106 (subsite 6). It is observed that most of the currently available ligands saturate only part of the ligand-recognition site leaving a wide scope to improve the ligand complementarity. Naturally, the ligands that interact with the largest number of subsites would also correspond to the maximum affinity. Therefore, for the design of potent inhibitors of PLA2, the stereochemical knowledge of the binding site as well as their potential to interact with ligands must be known so as to make the structure-based ligand design successful.

Structural Biology of Recombinant Bovine Pancreatic Phospholipase A2 and its Inhibitor Complexes

The enzyme phospholipase A2 catalyzes the cleavage of the sn-2 acyl ester bond of phospholipids, leading to the production of free fatty acids and lysophospholipids, which leads to many inflammatory disorders. In view of its pharmaceutical interest, three phospholipase A2 + inhibitor (namely, (i) L-1-O-octyl-2-heptylphosphonyl-sn-glycero-3-phosphoethanolamine, Transition State Analogue, (ii) 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol, MJ33 and (iii) p-methoxybenzoic acid, anisic acid) complex structures have already been solved and analysed, using the data obtained from X-ray diffraction. These structures provide insight on the mode of binding of the inhibitor molecules at the active site of phospholipase A2. The knowledge of the active site geometry in these inhibitor bound structures, yield valuable information in the design of more useful therapeutic agents. This report reviews only the inhibitor bound recombinant bovine pancreatic phospholipase A2 structures solved using X-ray crystallography.

Phosphatidylcholine-derived bolaamphiphiles via click chemistry.

The copper-catalyzed azide alkyne cycloaddition is employed to modify phosphatidylcholine precursors with sn-2 acyl chains containing terminal alkyne or azide groups. Although the reactions are conducted as biphasic dispersions, the yields are essentially quantitative. Bolaamphiphiles are formed by simply clicking together two phosphatidylcholine alkyne precursors to a central bisazide scaffold. The chemistry introduces polar 1,4-triazole units into the lipophilic region of the bilayer membrane, and the bolaamphiphiles do not form stable vesicles. [structure: see text].

The Chemistry of Phospholipid Binding by the Saccharomyces cerevisiae Phosphatidylinositol Transfer Protein Sec14p as Determined by EPR Spectroscopy

The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C5 region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer.

Positional specificity of lysosomal phospholipase A2

Lysosomal phospholipase A(2) (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A(1) and phospholipase A(2) activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.

Endocannabinoid Biosynthesis Proceeding through Glycerophospho-N-acyl Ethanolamine and a Role for α/β-Hydrolase 4 in This Pathway

N-Acyl ethanolamines (NAEs) are a large class of signaling lipids implicated in diverse physiological processes, including nociception, cognition, anxiety, appetite, and inflammation. It has been proposed that NAEs are biosynthesized from their corresponding N-acyl phosphatidylethanolamines (NAPEs) in a single enzymatic step catalyzed by a phospholipase D (NAPE-PLD). The recent generation of NAPE-PLD(-/-) mice has revealed that these animals possess lower brain levels of saturated NAEs but essentially unchanged concentrations of polyunsaturated NAEs, including the endogenous cannabinoid anandamide. These findings suggest the existence of additional enzymatic routes for the production of NAEs in vivo. Here, we report evidence for an alternative pathway for NAE biosynthesis that proceeds through the serine hydrolase-catalyzed double-deacylation of NAPE to generate glycerophospho-NAE, followed by the phosphodiesterase-mediated cleavage of this intermediate to liberate NAE. Furthermore, we describe the functional proteomic isolation and identification of a heretofore uncharacterized enzyme alpha/beta-hydrolase 4 (Abh4) as a lysophospholipase/phospholipase B that selectively hydrolyzes NAPEs and lysoNAPEs. Abh4 accepts lysoNAPEs bearing both saturated and polyunsaturated N-acyl chains as substrates and displays a distribution that closely mirrors lysoNAPE-lipase activity in mouse tissues. These results support the existence of an NAPE-PLD-independent route for NAE biosynthesis and suggest that Abh4 plays a role in this metabolic pathway by acting as a (lyso)NAPE-selective lipase.

Phospholipid Hydroxyalkenals, a Subset of Recently Discovered Endogenous CD36 Ligands, Spontaneously Generate Novel Furan-containing Phospholipids Lacking CD36 Binding Activityin Vivo

We recently identified a novel family of oxidized choline glycerophospholipid (oxPC) molecular species enriched in atheroma that serve as endogenous ligands for the scavenger receptor CD36 (oxPC(CD36)), facilitating macrophage cholesterol accumulation and foam cell formation (Podrez, E. A., Poliakov, E., Shen, Z., et al. (2002) J. Biol. Chem. 277, 38517-38523). A high affinity CD36 recognition motif was defined within oxPC(CD36), an oxidatively truncated sn-2 acyl group with a terminal gamma-hydroxy (or oxo)-alpha,beta-unsaturated carbonyl. The fate of these species once formed in vivo is unknown. Here we show that a subset of oxPC(CD36), a phosphatidylcholine molecular species possessing sn-2 esterified fatty acyl hydroxyalkenal groups, can undergo a slow intramolecular cyclization and dehydration reaction to form novel oxPC species possessing a sn-2 acyl group that incorporates a terminal furyl moiety (oxPC-furan). Using high performance liquid chromatography with on-line tandem mass spectrometry in combination with unambiguous organic synthesis, we confirm that oxPC-furans, ultimately derived from phospholipids with sn-2 esterified docosahexaenoic, arachidonic, or linoleic acids, are formed during exposure of model membranes and isolated lipoproteins to physiological oxidant systems. In vivo generation of oxPC-furans at sites of enhanced oxidant stress is also demonstrated, such as within brain tissues following cerebral ischemia. Cell binding studies reveal that in contrast to their oxPC(CD36) precursors, oxPC-furans lack CD36 binding activity. Taken together, the present studies identify oxPC-furans as a novel family of oxidized phospholipids that are formed in vivo from phospholipid hydroxyalkenals but that lack CD36 binding activity.

Absence of clustering of phosphatidylinositol-(4,5)-bisphosphate in fluid phosphatidylcholine

Phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] plays a key role in the modulation of actin polymerization and vesicle trafficking. These processes seem to depend on the enrichment of PI(4,5)P(2) in plasma membrane domains. Here, we show that PI(4,5)P(2) does not form domains when in a fluid phosphatidylcholine matrix in the pH range of 4.8-8.4. This finding is at variance with the spontaneous segregation of PI(4,5)P(2) to domains as a mechanism for the compartmentalization of PI(4,5)P(2) in the plasma membrane. Water/bilayer partition of PI(4,5)P(2) is also shown to be dependent on the protonation state of the lipid.

Light-induced Oxidation of Photoreceptor Outer Segment Phospholipids Generates Ligands for CD36-mediated Phagocytosis by Retinal Pigment Epithelium: A POTENTIAL MECHANISM FOR MODULATING OUTER SEGMENT PHAGOCYTOSIS UNDER OXIDANT STRESS CONDITIONS

Clearance by the retinal pigment epithelium (RPE) of shed photoreceptor outer segments (OSs), a tissue with one of the highest turnover rates in the body, is critical to the maintenance and normal function of the retina. We hypothesized that there is a potential role for photo-oxidation in OS uptake by RPE via scavenger receptor-mediated recognition of structurally defined lipid peroxidation products. We now demonstrate that specific structurally defined oxidized species derived from arachidonyl, linoleoyl, and docosahexanoyl phosphatidylcholine may serve as endogenous ligands on OSs for uptake by RPE via the scavenger receptor CD36. Mass spectrometry studies of retinal lipids recovered from dark-adapted rats following physiological light exposure demonstrate in vivo formation of specific oxidized phosphatidylcholine molecular species possessing a CD36 recognition motif, an oxidatively truncated sn-2 acyl group with a terminal gamma-hydroxy(or oxo)-alpha,beta-unsaturated carbonyl. Cellular studies using RPE isolated from wild-type versus CD36 null mice suggest that CD36 plays a role in engulfment, but not initial binding, of OSs via these oxidized phospholipids. Parallel increases in OS protein-bound nitrotyrosine, a post-translational modification by nitric oxide (NO)-derived oxidants, were also observed, suggesting a possible role for light-induced generation of NO-derived oxidants in the initiation of OS lipid peroxidation. Collectively, these studies suggest that intense light exposure promotes "oxidative tagging" of photoreceptor outer segments with structurally defined choline glycerophospholipids that may serve as a physiological signal for CD36-mediated phagocytosis under oxidant stress conditions.

Fluorescent, Acyl Chain-labeled Phosphatidylcholine Analogs Reveal Novel Transport Pathways across the Plasma Membrane of Yeast

Acyl chain-labeled NBD-phosphatidylcholine (NBD-PC) has been used to identify three gene products (Lem3p, Dnf1p, and Dnf2p) that are required for normal levels of inward-directed phospholipid transport (flip) across the plasma membrane of yeast. Although the head group structure of acyl chain-labeled NBD phospholipids has been shown to influence the mechanism of flip across the plasma membrane, the extent to which the acyl chain region and the associated fluorophore affect flip has not been assessed. Given the identification of these proteins required for NBD-PC flip, it is now possible to determine whether the fluorophore attached to a phospholipid acyl chain influences the mechanism of flip. Thus, flip of phosphatidylcholine molecules with three different Bodipy fluorophores (Bodipy FL, Bodipy 530, and Bodipy 581) was tested and compared with that of NBD-PC in strains carrying deletions in LEM3, DNF1, and DNF2. Deletion of these genes significantly reduced the flip of NBD-PC and Bodipy FL-PC but had no effect on that of Bodipy 581-PC and Bodipy 530-PC. These data, in combination with comparisons of the effect of ATP depletion, collapse of the proton electrochemical gradient across the plasma membrane, and culture density led to the conclusion that at least three different flip pathways exist in yeast that are selective for the structure of the fluorophore attached to the acyl chain of phosphatidylcholine molecules.

Ultraviolet B Radiation Generates Platelet-activating Factor-like Phospholipids underlying Cutaneous Damage

Ultraviolet B light (UVB) causes cutaneous inflammation and cell death, but the agents responsible are not defined. These studies examined the role of the platelet-activating factor (PAF) signaling system in UVB-mediated effects. Expression of the PAF receptor in the PAF receptor-negative epidermoid cell line KB augmented apoptosis in response to UVB irradiation. Overexpression of the PAF receptor in primary human keratinocytes also enhanced UVB-mediated apoptosis in vitro, and it enhanced apoptosis in an in vivo model of human keratinocytes grafted onto severe combined immune-deficient (SCID) mice. To define the mechanism by which UVB activates the PAF receptor, we used mass spectrometry to demonstrate significant amounts of the C4 PAF analogs 1-alkyl-2-(butanoyl and butenoyl)-sn-glycero-3-phosphocholine, as well as native PAF in an epidermal cell line after UVB irradiation. Supplementing the cells with the precursor phospholipid 1-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (HAPC) increased the amount of C4 PAF analogs recovered after UVB exposure. We irradiated HAPC directly and found, even in the absence of a photosensitizer, fragmentation to C4-PAF receptor ligands. We conclude UVB photo-oxidizes cellular phospholipids, creating PAF analogs that stimulate the PAF receptor to induce further PAF synthesis and apoptosis. PAF signaling may participate in the cutaneous inflammation that occurs during photo-aggravated dermatoses.

Characterisation of acyltransferases from Synechocystis sp. PCC6803

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C 16 acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.

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 for altered positional specificity of LCAT in vivo: studies with docosahexaenoic acid feeding in humans

The percentage of saturated cholesteryl esters (CEs) synthesized by human LCAT is several times higher than expected from the sn-2 acyl composition of plasma phosphatidylcholine (PC), whereas the synthesis of 20:4 CE and 22:6 CE is much lower than expected. To explain these discrepancies, we proposed that LCAT transfers some saturated fatty acids from the sn-1 position of PC species that contain 20:4 or 22:6 at sn-2. The present studies provide in vivo evidence for this hypothesis. We determined the composition and synthesis of CE species in plasma of volunteers before and after a 6 week dietary supplementation with docosahexaenoic acid (22:6; DHA). In addition to an increase in the DHA content of all plasma lipids, there was a significant (+12%; P <0.005) increase of 16:0 CE, although there was no increase in 16:0 at sn-2 of PC. The increase of DHA in CE was much lower than its increase at sn-2 of PC. Ex vivo synthesis of CE species in plasma showed a significant (+24%; P <0.005) increase in the synthesis of 16:0 CE after DHA supplementation, which correlated positively with the increase of 22:6, but not of 16:0, at sn-2 of PC. These results show that the positional specificity of human LCAT is altered when the concentration of 16:0-22:6 PC is increased by DHA supplementation.

Fragmentation study of short‐chain products derived from oxidation of diacylphosphatidylcholines by electrospray tandem mass spectrometry: identification of novel short‐chain products

Lineloyl-palmitoyl (PLPC) and arachidonoyl-palmitoyl (PAPC) phosphatidylcholine were oxidized under Fenton reaction conditions (H2O2 and Fe2+), and the short-chain products formed were identified by electrospray ionization mass spectrometry (ESI-MS). The short-chain products resulted from beta-cleavage of oxygen-centered radicals and comprised aldehydes, hydroxyaldehydes and dicarboxylic acids that yielded both [MH]+ and [MNa]+ ions. The fragmentation of the [MH]+ and [MNa]+ ions of the peroxidation products was studied by tandem mass spectrometry (MS/MS). The MS/MS spectra of both ions showed ions resulting from characteristic losses of glycerophosphatidylcholine. Other product ions, resulting from C-C cleavages occurring in the vicinity of the functional group, and fragmentations involving the hydroxy groups, were the most informative since they allowed us to obtain structural information relating to the sn-2 acyl residue. Both fragmentation pathways are due to charge-remote fragmentation occurring by a 1,4-hydrogen elimination mechanism and/or by homolytic cleavage. Furthermore, the fragmentation pathway of some ions observed in the ESI-MS spectrum was not consistent with the fragmentation behavior expected for some of the short-chain species identified in the literature and allowed the reassignment of the ions as different structures. Isobaric ions were observed in the ESI-MS spectra of both oxidized phospholipids, and were differentiated based on distinct fragmentation. The detailed knowledge of lipid peroxidation degradation products is of major importance and should be very valuable in providing new markers for oxidative stress signaling and for disease states monitoring.

Atomic resolution (0.97 Å) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2

The enzyme phospholipase A2 catalyzes the hydrolysis of the sn-2 acyl chain of phospholipids, forming fatty acids and lysophospholipids. The crystal structure of a triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 in which the lysine residues at positions 53, 56 and 121 are replaced recombinantly by methionines has been determined at atomic resolution (0.97 A). The crystal is monoclinic (space group P2), with unit-cell parameters a = 36.934, b = 23.863, c = 65.931 A, beta = 101.47 degrees. The structure was solved by molecular replacement and has been refined to a final R factor of 10.6% (Rfree = 13.4%) using 63,926 unique reflections. The final protein model consists of 123 amino-acid residues, two calcium ions, one chloride ion, 243 water molecules and six 2-methyl-2,4-pentanediol molecules. The surface-loop residues 60-70 are ordered and have clear electron density.

Crystal structure of an acidic platelet aggregation inhibitor and hypotensive phospholipase A 2 in the monomeric and dimeric states: insights into its oligomeric state

Phospholipases A 2 belong to the superfamily of proteins which hydrolyzes the sn-2 acyl groups of membrane phospholipids to release arachidonic acid and lysophospholipids. An acidic phospholipase A 2 isolated from Bothrops jararacussu snake venom presents a high catalytic, platelet aggregation inhibition and hypotensive activities. This protein was crystallized in two oligomeric states: monomeric and dimeric. The crystal structures were solved at 1.79 and 1.90 Å resolution, respectively, for the two states. It was identified a Na + ion at the center of Ca 2+-binding site of the monomeric form. A novel dimeric conformation with the active sites exposed to the solvent was observed. Conformational states of the molecule may be due to the physicochemical conditions used in the crystallization experiments. We suggest dimeric state is one found in vivo.

Studies of lipid turnover in cells with stable isotope and gas chromatograph–mass spectrometry

Phospholipids are major building blocks for biological membranes. In addition, metabolites derived from their degradation are important signals in major cellular events, such as proliferation and apoptosis. The concept of lipid signaling in cells is derived mainly from the measurement of change in the concentration of lipid molecules. However, these changes in concentration are only a small part of the underlying metabolic change induced by a perturbation in the cell. In contrast, metabolic kinetic studies documenting product–precursor relationships and turnover rates are useful in elucidating the responsible mechanisms. Historically, metabolic studies of phospholipids in cells have been carried out with pulse or pulse–chase methods using radioactive isotopes. While these studies provide valuable information, their scope is restricted by inherent limitations. In this paper we describe a method using [1,2,3,4- 13C 4]palmitate as the tracer for studying the metabolic kinetics of the molecular species of diacylglycerol, ceramide, phosphatidylcholine, and sphingomyelin. After growing cells in the presence of labeled palmitate complexed to serum albumin, the lipids are extracted and separated into lipid classes. After enzymatic hydrolysis, diacylglyerols and ceramides as bis-trimethylsilyl derivatives are determined quantitatively with capillary column gas chromatography. Internal standards for each lipid class are used in the procedure. In addition, the isotopic enrichments of the lipid molecular species are determined with gas chromatograph–mass spectrometry. We applied this method to the study of HL60 cells. Different turnover rates were found for various molecular species. In addition, the sn-1 and sn-2 acyl groups appear to be synthesized at different rates for different molecular species. Other information, such as chain elongation and desaturation, might also be derived through the use of this method.