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PubMed1997

Phospholipase D activity in the intestinal mitochondria: activation by oxygen free radicals.

A prominent feature of cell damage caused by oxidative stress is morphological and functional changes in the mitochondria. The present study looked at the effect of free radical exposure on intestinal mitochondrial lipids. Free radical exposure did not alter neutral lipids, but among the phospholipids, phosphatidylethanolamine (PE) content was decreased on exposure to superoxide anion, generated by xanthine-xanthine oxidase or menadione with a concomitant increase in the level of phosphatidic acid (PA), suggesting activation of phospholipase D (PLD). This enzyme did not show transphosphatidylation activity in the presence of ethanol or butanol, and the product formed was phosphatidic acid (PA). This was confirmed by separation of reaction products by HPLC. This alteration in mitochondrial phospholipid was abolished by the presence of superoxide dismutase. Exposure to H2O2 did not have any significant effect. Activation of PLD by free radicals was further confirmed by quantitation of ethanolamine released from PE. Absence of any change in the content of lysophospholipid or diglyceride following exposure of mitochondria to superoxide ruled out the involvement of phospholipase A2 or C in the altered lipid composition. Moreover, inclusion of phospholipase A2 inhibitors, chlorpromazine, or p-bromophenacyl bromide did not prevent the generation of PA on exposure to free radicals. These findings suggest that superoxide anion stimulates intestinal mitochondrial PLD resulting in PE degradation and PA formation. These alterations in mitochondrial lipids may play a role in causing the functional alteration seen in oxidative stress.

Animals↗
PubMed2000

Rationale and applications of lipids as prodrug carriers.

Lipidic prodrugs, also called drug-lipid conjugates, have the drug covalently bound to a lipid moiety, such as a fatty acid, a diglyceride or a phosphoglyceride. Drug-lipid conjugates have been prepared in order to take advantage of the metabolic pathways of lipid biochemistry, allowing organs to be targeted or delivery problems to be overcome. Endogenous proteins taking up fatty acids from the blood stream can be targeted to deliver the drug to the heart or liver. For glycerides, the major advantage is the modification of the pharmacokinetic behavior of the drug. In this case, one or two fatty acids of a triglyceride are replaced by a carboxylic drug. Lipid conjugates exhibit some physico-chemical and absorption characteristics similar to those of natural lipids. Non-steroidal, anti-inflammatory drugs such as acetylsalicylic acid, indomethacin, naproxen and ibuprofen were linked covalently to glycerides to reduce their ulcerogenicity. Mimicking the absorption process of dietary fats, lipid conjugates have also been used to target the lymphatic route (e.g., L-Dopa, melphalan, chlorambucil and GABA). Based on their lipophilicity and resemblance to lipids in biological membranes, lipid conjugates of phenytoin were prepared to increase intestinal absorption, whereas glycerides or modified glycerides of L-Dopa, glycine, GABA, thiorphan and N-benzyloxycarbonylglycine were designed to promote brain penetration. In phospholipid conjugates, antiviral and antineoplasic nucleosides were attached to the phosphate moiety. After presenting the biochemical pathways of lipids, the review discusses the advantages and drawbacks of lipidic prodrugs, keeping in mind the potential pharmacological activity of the fatty acid itself.

Animals↗
PubMed1996

The fixative lipid of tiger pheromone.

Tigers communicate with one another with the help of Marking Fluid (MF) which is a lipid-rich fluid sprayed upwards and backwards through the urinary channel of both the sexes. The volatile molecules of the MF are made to last longer with the help of lipid 'fixatives' the total amount of which is 1-2 mg/ml. This lipid comprises cholesterol ester, wax ester, triglyceride, free fatty acids, diglyceride, monoglyceride, free sterol and phospholipid as revealed by thin layer chromatography. The gas liquid chromatogram of fatty acid methyl esters of the total lipid of the MF, when compared with the fatty acids of groin and body fat of tiger (analysed by other workers), reveals differences in higher proportions of palmitoleic and myristic acids and of highly unsaturated fatty acids. The palmitoleic acid content of total lipid and triglyceride is high in comparison to the wax esters and cholesterol esters of the MF-fat. The unique feature of the alcohol part of the wax esters is a series of saturated straight chain primary alcohols of C14 to C20 and these are accompanied by the corresponding monoenoic unsaturates.

Animals↗
PubMed2003

Chemical composition of lipophilic extractives released during the hot water treatment of wheat straw.

Treatment of wheat straw with hot water at 80-95 degrees C for 0.5 h at pH 6.0-8.0 released 41.0-53.0% of the original lipophilic extractives. The chemical compositions of six lipophilic extractives were determined by GC on a medium-length high-temperature capillary column without derivatization, thus giving a method for direct determination of individual components of free fatty and resin acids, sterols, waxes, sterol esters, and triglycerides. The extracts contained 68.7-75.8% lipophilic substances, comprising mainly free fatty acids (25.8-48.4%), waxes (9.4-27.0%), sterols (4.1-8.0%), triglycerides (3.3-11.0%), and sterol esters (2.6-5.1%). Minor amounts of diglycerides (0.3-0.5%), resin acid (0.5-3.1%), and phenolic compounds (0.9-3.6%) were also quantitatively determined in the extractives.

Chromatography, Gas↗
PubMed1997

Cerium-based ultracytochemical localization of aspartate transcarbamylase activity in the cell membrane complex of Saccharomyces cerevisiae.

Aspartate transcarbamylase (ATCase) activity was localized ultracytochemically in the yeast Saccharomyces cerevisiae by precipitation of its reaction product orthophosphate as cerium phosphate. We prefixed yeast cells with ice-cold 1% glutaraldehyde for 30 min which preserved 80% of ATCase activity. Cells were washed and incubated with ATCase substrates (aspartate, carbamyl phosphate) plus cerium chloride, and postfixed by osmium tetroxide. In cells from exponential batch cultures, deposits of cerium phosphate delineated simultaneously or alternatively membranes of the secretory pathway: nuclear envelope, endoplasmic reticulum, Golgi complex and the plasmalemma; mitochondrial membranes and intramitochondrial fibrous component were labelled as well. Deposits of cerium phosphate were never observed in the nucleoplasm. Cells incubated in the absence of cerium or ATCase substrates and mutant S. cerevisiae cells lacking ATCase activity served as controls. Small round electron-dense condensates were found to be randomly distributed within some cells, both in control and experimental runs, in the nucleoplasm, cytoplasm and mitochondrial matrix and represented undefined osmicated endogenous compounds. Our results suggest that the synthesis of pyrimidine precursors occurs in membranes, where compounds such as UDP-glucose and CDP-diglycerides are needed for membrane and/or yeast cell wall synthesis. The possible contribution of ATCase activity found in the nuclear envelope to nucleic acid synthesis remains to be clarified.

Aspartate Carbamoyltransferase↗
PubMed1999

Phospholipase D, tumor promoters, proliferation and prostaglandins.

Phosphatidylcholine hydrolysis by phospholipase D is a widespread response to cellular stimulation. However, the downstream signaling events subsequent to phosphatidylcholine hydrolysis are just beginning to be determined. Initially it was proposed that diglyceride formation by phospholipase D and phosphatidate phosphohydrolase resulted in long-term stimulation of protein kinase C. However, recent studies indicate that phosphatidic acid is the relevant signaling molecule in some signaling pathways. The present review will summarize studies of phospholipase D in the response of cells to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, which causes cells to mimic the phenotype of oncogenic transformation. The role of phospholipase D in stimulation of Raf-1 and prostaglandin H synthase type-2 is emphasized.

Animals↗
PubMed1999

Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent.

A new enzymatic method of synthesizing methyl esters from plant oil and methanol in a solvent-free reaction system was developed. It is anticipated that such plant oil methyl esters can be used as a biodiesel fuel in the future. Lipase from Rhizopus oryzae efficiently catalyzed the methanolysis of soybean oil in the presence of 4-30 wt% water in the starting materials; however the lipase was nearly inactive in the absence of water. The methyl ester (ME) content in the reaction mixture reached 80-90 wt% by stepwise additions of methanol to the reaction mixture. The kinetics of the reaction appears to be in accordance with the successive reaction mechanism. That is, the oil is first hydrolyzed to free fatty acids and partial glycerides, and the fatty acids produced are then esterified with methanol. Although R. oryzae lipase is considered to exhibit 1(3)-regiospecificity, a certain amount of 1,3-diglyceride was obtained during the methanolysis and hydrolysis of soybean oil by R. oryzae lipase solution. Therefore, the high ME content in the reaction mixture is probably attributable to the acyl migration from the sn-2 position to the sn-1 or sn-3 position in partial glycerides.

Journal Article↗
PubMed2002

DNA-bound lipids of normal and tumor cells: retrospective and outlooks for functional genomics.

By very soft phenol method, the high-molecular-mass natural DNA complexes (10(8)-10(9) Da), which contain 1-3% specific lipids, were isolated from different eukaryotic and prokaryotic cells. Two pools of DNA-bound lipids were isolated: loosely bound (extracted with 35% ethanol) and tightly bound lipids (extracted after additional treatment DNAse I). The composition of these two lipid pools of different sources (rat thymus, liver, regenerating liver, loach sperm, pigeon erythrocytes, Zajdel ascites hepatoma, Ehrlich ascites carcinoma, sarcoma 37, Escherichia coli B, T2 phage) was studied. The DNA-bound lipid pools consist of neutral lipids (NL) and phospholipids (PL), moreover NL is always in a few fold more than PL. The composition of these lipid pools of eukaryotes distinguishes between themselves, mainly, by free cholesterol (minor fraction), cardiolipin (major fraction), and by phosphatidylcholine. Only the tightly bound lipid pool was present in T2 phage DNA. The dramatic redistribution effect between all fractions of NL pools (free and ester cholesterol, free fatty acids, diglycerides) was observed in DNA synthesis phase of cell cycle on the background of the unchanged composition of PL pools. Comparative analysis of DNA-bound lipid pools of normal and cancer cells was carried out. The DNA-bound lipid pools of transformed cells significantly differ from the same normal cells both by PL composition (cardiolipin) and by the presence of additional fractions (mono- and triglycerides) as well. The possible functions of DNA-bound lipid pools, especially of cardiolipin and cholesterol at the attachment of DNA loops to the nuclear matrix, DNA replicon organization, replication, and transcription are discussed.

Animals↗
PubMed2004

Changes in lipid fractions and sensory properties of Idiazabal cheese induced by lipase addition.

This work studied the addition of an adequate lipase to enhance lipolysis reactions and the development of piquant flavour and sharp odour in Idiazabal cheese, as an alternative to the use of lamb rennet paste. Cheeses were manufactured from bulk raw ewes' milk in 50 l vats with commercial bovine rennet and 80 lipase units of pregastric or 180 lipase units of fungal lipase and ripened for 180 days. A higher lipolytic activity was induced by lipase addition promoting strong changes in odour and flavour attributes. Both fungal and pregastric lipases increased the content of total free fatty acids (FFA), but the fungal lipase released mainly medium- and long-chain FFA. In contrast, the pregastric lipase preferably released short-chain FFA. Diglyceride (DG) content was considerably higher in cheeses made with added pregastric lipase compared with those made with fungal lipase or with no lipase. Monoglycerides (MG) were detected only in cheeses made with either lipase added, reaching comparable concentrations after ripening for 180 days. The cheeses made with pregastric lipase had the highest scores for odour and flavour intensity, and sharp and rennet odours, desirable attributes for the Idiazabal cheese made with lamb rennet paste. None of the texture attributes were significantly influenced by the concentrations of MG and DG in the cheeses made with either lipase. Thus, the pregastric lipase was more appropriate than the fungal lipase to develop a more traditionally-flavoured Idiazabal cheese.

Animals↗
PubMed1989

Lipids in hydatid fluid collected from lungs and livers of sheep and man.

Hydatid fluid collected from the lungs and livers of sheep and humans was analysed for protein and lipid composition. There were no marked differences in the composition of these parameters and the major lipids were triglycerides and diglycerides. The phospholipids, which formed the minor fraction, were mainly phosphatidyl choline and phosphatidyl inositol. Cholesterol present was in the free form.

Animals↗
PubMed1990

Characterization of underivatized lipid biomarkers from microorganisms with pyrolysis short-column gas chromatography/ion trap mass spectrometry.

A microvolume Curie-point pyrolysis short-column (5 m) gas chromatography/mass spectrometry (Py-GC/MS) procedure was developed for the characterization of various lipid moieties in microorganisms. High linear flow rates (approximately 175 cm/s) characterized the GC conditions in order to effect an efficient chromatographic transfer and elution of the underivatized diglycerides and monoglycerides, and small modifications were necessary to the ion trap MS system in order for it to accommodate the relatively high gas load. During a typical analysis run anhydrodiacylglycerides eluted within a 5-6-min time frame. Gram-positive bacilli and Gram-negative species were differentiated from each other by the pyrolysis patterns of their lipid components. In spite of the complexity of the analyte, a straightforward visual analysis was achieved with the aid of simple computerized data display procedures. These procedures included examination of (1) total ion current (TIC) profiles of the lipid region of the reconstructed chromatogram, (2) the integrated mass spectrum of this region, (3) selected reconstructed ion chromatograms (RICs), (4) RIC intensity distributions, and (5) corresponding mass spectra. An appealing aspect of the lipid data reduction procedure is that most of it can be accomplished visually without requiring computerized pattern recognition techniques.

Bacteria↗
PubMed2006

Comparison of atmospheric pressure photoionization, atmospheric pressure chemical ionization, and electrospray ionization mass spectrometry for analysis of lipids.

In this work, we compare the quantitative accuracy and sensitivity of analyzing lipids by atmospheric pressure photoionization (APPI), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI) LC/MS. The target analytes include free fatty acids and their esters, monoglyceride, diglyceride, and triglyceride. The results demonstrate the benefits of using LC/APPI-MS for lipid analysis. Analyses were performed on a Waters ZQ LC/MS. Normal-phase solvent systems were used due to low solubility of these compounds in aqueous reversed-phase solvent systems. By comparison, APPI offers lower detection limits, generally highest signal intensities, and the highest S/N ratio. APPI is 2-4 times more sensitive than APCI and much more sensitive than ESI without mobile-phase modifiers. APPI and APCI offer comparable linear range (i.e., 4-5 decades). ESI sensitivity is dramatically enhanced by use of mobile phase modifiers (i.e., ammonium formate or sodium acetate); however, these ESI adduct signals are less stable and either are nonlinear or have dramatically reduced linear ranges. Analysis of fish oils by APPI shows significantly enhanced target analyte intensities in comparison with APCI and ESI.

Atmospheric Pressure↗
PubMed2000

(R)-3-hydroxybutyrate dehydrogenase: selective phosphatidylcholine binding by the C-terminal domain.

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme that has a specific requirement of phosphatidylcholine (PC) for function. The C-terminal domain (CTBDH) of human heart BDH (residues 195-297) has now been expressed in Escherichia coli as a chimera with a soluble protein, glutathione S-transferase (GST), yielding GST-CTBDH, a novel fusion protein that has been purified and shown to selectively bind to PC vesicles. Both recombinant human heart BDH (HH-Histag-BDH) and GST-CTBDH (but not GST) form well-defined protein-lipid complexes with either PC or phosphatidylethanolamine (PE)/diphosphatidylglycerol (DPG) vesicles (but not with digalactosyl diglyceride vesicles) as demonstrated by flotation in sucrose gradients. The protein-PC complexes are stable to 0.5 M NaCl, but complexes of either HH-Histag-BDH or GST-CTBDH with PE/DPG vesicles are dissociated by salt treatment. Thrombin cleavage of GST-CTBDH, either before or after reconstitution with PC vesicles, yields CTBDH (12 111 Da by MALDI mass spectrometry) which retains lipid binding without attached GST. The BDH activator, 1-palmitoyl-2-(1-pyrenyl)decanoyl-PC (pyrenyl-PC), at <2.5% of total phospholipid in vesicles, efficiently quenches a fraction (0.36 and 0.47, respectively) of the tryptophan fluorescence of both HH-Histag-BDH and GST-CTBDH with effective Stern-Volmer quenching constants, (K(Q))(eff), of 11 and 9.3 (%)(-)(1), respectively (half-maximal quenching at approximately 0.1% pyrenyl-PC). Maximal quenching by pyrenyl-PC obtains at approximately stoichiometric pyrenyl-PC to protein ratios, reflecting high-affinity interaction of pyrenyl-PC with both HH-Histag-BDH and GST-CTBDH. The analogous pyrenyl-PE effects a similar maximal quenching of tryptophan fluorescence for both proteins but with approximately 15-fold lower (K(Q))(eff) (half-maximal quenching at approximately 1.5% pyrenyl-PE) referable to nonspecific interaction of pyrenyl-PE with HH-Histag-BDH or GST-CTBDH. Thus, the 103-residue CTBDH constitutes a PC-selective lipid binding domain of the PC-requiring BDH.

Fluorescent Dyes↗
PubMed1993

Processive interfacial catalysis by mammalian 85-kilodalton phospholipase A2 enzymes on product-containing vesicles: application to the determination of substrate preferences.

Substrate specificities of the human and rat kidney 85-kDa phospholipase A2 enzymes (hmw-PLA2) have been determined under conditions in which hydrolysis of substrate vesicles occurs without the desorption of enzyme from the interface (scooting mode catalysis). The rat kidney enzyme binds to vesicles of 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC), which contain the substrate 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine (SAPC) and 10 mol% arachidonic acid (20:4) and 1-stearoyl-sn-glycero-3-phosphocholine (S-lyso-PC) as the hydrolysis reaction products, with a second-order rate constant k(on) approximately equal to 2 x 10(7) M-1 s-1. Upper limits of k(off) < or = 3 x 10(-4) s-1 and KD < = or 15 pM for the dissociation rate and equilibrium constants, respectively, are estimated from the vesicle binding measurements. The initial rates of hydrolysis of either radiolabeled 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphoserine (3H-SAPS), -phosphoethanolamine (3H-SAPE), -phosphoinositol (14C-SAPI), or -phosphate (3H-SAPA) and either 3H-SAPC or 14C-SAPC, which were incorporated into product-containing OPPC vesicles, were simultaneously measured with dual isotope radiometric assays. The plasmenylcholine 1-O-(Z-hexadec-1'-enyl)-2-arachidonyl-sn-glycero-3- phosphocholine (3H-PlasAPC) was also tested. Relative substrate specificity constants (Kcat/KM* values) were determined from the concentrations and initial rates of hydrolysis of the labeled substrates; the rank order of the values is SAPC approximately equal to SAPI approximately equal to PlasAPC > SAPE > SAPA approximately equal to SAPS. The maximal difference in specificity constants is 3.5-fold, indicating that the hmw-PLA2 does not significantly discriminate between phospholipids with different polar head groups. The diglyceride 1-stearoyl-2-arachidonyl-sn-glycerol is not a substrate for the human hmw-PLA2. Two mixtures of 1-stearoyl-2-acyl-sn-glycero-3-phosphocholine, which have different sn-2 acyl chains, were prepared and compared to SAPC as substrates. One mixture contained naturally-occurring unsaturated fatty acyl chains and the other contained a mixture of 20:4, all of its partially hydrogenated analogues (20:3, 20:2, and 20:1), and arachidic acid (20:0). The order of preference for the human hmw-PLA2 is sn-2-20:4 > sn-2-alpha-linolenoyl > sn-2-linoleoyl > sn-2-oleoyl > or = sn-2-palmitoyleoyl.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Carbon-13 nuclear magnetic resonance spectroscopy of lipids: differential line broadening due to cross-correlation effects as a probe of membrane structure.

We have obtained proton-coupled carbon-13 nuclear magnetic resonance (NMR) spectra of a variety of lipid-water and lipid-drug-water systems, at 11.7 T, as a function of temperature, using the "magic-angle" sample-spinning (MAS) NMR technique. The resulting spectra show a wide range of line shapes, due to interferences between dipole-dipole and dipole-chemical shielding anisotropy interactions. The differential line-broadening effects observed are particularly large for aromatic and olefinic (sp2) carbon atom sites. Coupled spectra of the tricyclic antidepressants desipramine and imipramine, in 1,2-dimyristoyl-sn-glycero-3-phosphocholine-water mesophases, show well-resolved doublets having different line shapes for each of the four aromatic methine groups, due to selective averaging of the four C-H dipolar interactions due to rapid motion about the director (or drug C2) axis. 2H NMR spectra of [2,4,6,8-2H4]desipramine (and imipramine) in the same 1,2-dimyristoyl-sn-glycero-3-phosphocholine-water mesophase exhibit quadrupole splittings of approximately 0-2 and approximately 20 kHz, indicating an approximate magic-angle orientation of the C2-2H(1H) and C8-2H(1H) vectors with respect to an axis of motional averaging, in accord with the 13C NMR results. Selective deuteration of imipramine confirms these ideas. Spectra of digalactosyl diglyceride [primarily 1,2-di[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl ]-3- (alpha-D-galactopyranosyl-1-6-beta-D-galactopyranosyl)-sn-glycerol]-H2O (in the L alpha phase) show a large differential line broadening for C9 but a reduced effect for C10, consistent with the results of 2H NMR of specifically 2H-labeled phospholipids [Seelig, J., & Waespe-Saracevic, N. (1978) Biochemistry 17, 3310-3315].(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Isotopes↗
PubMed1982

Phospholipid molecular species alterations in Tetrahymena ciliary membranes following low-temperature acclimation.

The molecular species compositions of phosphatidylcholine, phosphatidylethanolamine, and 2-(aminoethyl)-phosphonolipid, the three principal phosphoglycerides of Tetrahymena pyriformis ciliary membranes, have been determined by coupled gas chromatography-mass spectrometry of the tert-butyldimethylsilyl derivatives of their phospholipase C derived diglycerides. There were striking changes in the molecular species composition of phosphatidylcholine and phosphatidylethanolamine after the cells were chilled from 39 to 15 degrees C. The low temperature induced changes occurring in each phospholipid class were markedly different from those taking place in the other two. The molecular species patterns of the ciliary phosphoglycerides resembled in a qualitative way the patterns found in their microsomal counterparts. However, the relative proportions of many molecular species within each ciliary phospholipid class were very different from those of the corresponding microsomal component, and changes induced in a particular microsomal phospholipid class by chilling were often absent or even reversed in the equivalent ciliary phospholipid. Compositional changes in ciliary phospholipids during low-temperature acclimation were greater in nearly all respects than were concurrent changes in microsomal phospholipids. The molecular species data confirm that the cell possesses a variety of highly selective mechanisms allowing those organelles which accept phospholipids of microsomal origin to retain a surprising degree of independence in responding to environmental stress.

Acclimatization↗
PubMed1983

Synthesis and properties of radioiodinated phospholipid analogues that spontaneously undergo vesicle-vesicle and vesicle-cell transfer.

An efficient method for the synthesis and purification of a variety of iodinated phospholipid analogues is described. 1-Acyl-2-[[[3-(3-[125I]iodo-4-hydroxyphenyl)- propionyl]amino]caproyl]phosphatidylcholine (125I-PC) was prepared by alkylation of 1-acyl-2-(aminocaproyl)phosphatidylcholine with monoiodinated Bolton-Hunter reagent. 125I-Labeled phosphatidic acid, phosphatidylethanolamine, and phosphatidylserine were produced from 125I-PC by phospholipase D catalyzed base exchange in the presence of ethanol-amine or L-serine. All of these lipid analogues transferred readily from donor vesicles into recipient membranes. When an excess of acceptor vesicles was mixed with a population of donor vesicles containing the iodinated analogues, approximately 50% of the 125I-labeled lipids transferred to the acceptor vesicle population. In addition, under appropriate incubation conditions, these lipids were observed to transfer from vesicles to mammalian cells. Autoradiographic analysis of 125I-labeled lipids extracted from the cells after incubation with vesicles at 2 degrees C for 60 min revealed that a large proportion of the 125I-labeled phosphatidic acid was metabolized to 125I-labeled diglyceride and 125I-labeled phosphatidylcholine, whereas no metabolism of exogenously supplied 125I-labeled phosphatidylethanolamine or 125I-labeled phosphatidylcholine could be detected.

Animals↗
PubMed1989

Kinetic characterization of Escherichia coli outer membrane phospholipase A using mixed detergent-lipid micelles.

The substrate specificity of Escherichia coli outer membrane phospholipase A was analyzed in mixed micelles of lipid with deoxycholate or Triton X-100. Diglycerides, monoglycerides, and Tweens 40 and 85 in Triton X-100 are hydrolyzed at rates comparable to those of phospholipids and lysophospholipids. p-Nitrophenyl esters of fatty acids with different chain lengths and triglycerides are not hydrolyzed. The minimal substrate characteristics consist of a long acyl chain esterified to a more or less hydrophilic headgroup as is the case for the substrate monopalmitoylglycol. Binding occurs via the hydrocarbon chain of the substrate; diacyl compounds are bound three to five times better than monoacyl compounds. When acting on lecithins, phospholipase A1 activity is six times higher than phospholipase A2 activity or 1-acyl lysophospholipase activity. Activity on the 2-acyl lyso compound is about two times less than that on the 1-acyl lysophospholipid. The enzyme therefore has a clear preference for the primary ester bond of phospholipids. In contrast to phospholipase A1 activity, phospholipase A2 activity is stereospecific. Only the L isomer of a lecithin analogue in which the primary acyl chain was replaced by an alkyl ether group is hydrolyzed. The D isomer of this analogue is a competitive inhibitor, bound with the same affinity as the L isomer. On these ether analogues the enzyme shows the same preference for the primary acyl chain as with the natural diester phospholipids. Despite its broad specificity, the enzyme will initially act as a phospholipase A1 in the E. coli envelope where it is embedded in phospholipids.

Cell Membrane↗