Lysophospholipase Activity Research Articles

Properties and regulation of cytosolic phospholipase A2.

Properties and regulation of cytosolic phospholipase A2.

Cloning, Expression, and Catalytic Mechanism of Murine Lysophospholipase I

A lysophospholipase (LysoPLA I) has been purified and characterized from the mouse macrophage-like P388D1 cell line (Zhang, Y. Y, and Dennis, E. A. (1988) J. Biol. Chem. 263, 9965-9972). This enzyme has now been sequenced, cloned, and expressed in Escherichia coli cells. The enzyme contains 230 amino acid residues with a calculated molecular mass of 24.7 kDa. It has a high helical content in its predicated secondary structure, which is also indicated in its CD spectrum. The cloned LysoPLA I was purified to homogeneity from the transformed E. coli cells by a gel filtration column and an ion exchange column. The specific activity of the purified protein is 1. 47 micromol/min.mg toward 1-palmitoyl-sn-glycero-3-phosphorylcholine at pH 8.0 and 40 degrees C, corresponding to the reported value of 1.3-1.7 micromol/min.mg for the protein purified from the P388D1 cells. In addition, the cloned protein cross-reacted with an antibody raised against LysoPLA I also purified from the P388D1 cells. The deduced LysoPLA I sequence contains a well conserved GXSXG motif found in the active site of many serine enzymes, and the activity of the LysoPLA I was irreversibly inhibited by the classical serine protease inhibitor diisopropyl fluorophosphate. Furthermore, site-directed mutagenesis was employed to change Ser-119 in the GXSXG motif to an Ala. The resulting mutant protein lost all of its lysophospholipase activity, even though it had the same overall protein conformation as that of the wild-type LysoPLA I. Therefore, LysoPLA I has been demonstrated to be a serine enzyme with Ser-119 at the active site.

On the mechanism of lysophospholipase activity of secretory phospholipase A 2 (EC 3.1.1.4): deacylation of monoacylphosphoglycerides by intrinsic sn-1 specificity and pH-dependent acyl migration in combination with sn-2 specificity

We show for the first time that secreted low-molecular weight phospholipase A 2 (EC 3.1.1.4) catalyzes the deacylation of monoacylphosphoglycerides directly from the sn-1 position, although at a very low rate: purified phospholipase A 2 enzymes from bee venom, crotalus atrox venom, and porcine pancreas hydrolyze the sn-1 ester bond in 1-palmitoyl-2-O-methyl- sn-glycero-3-phosphorylcholine. Hydrolytic rates with the corresponding isomer, 1-O-methyl-2-palmitoyl- sn-glycero-3-phosphorylcholine, are about 3-4 orders of magnitude higher. The similarities in Ca 2+ requirement and inactivation profiles suggest that deacylation, albeit with different rates, from both sn-1 and sn-2 positions is catalyzed by the same catalytic site of phospholipase A 2. Furthermore, evidence is provided that phospholipase A 2-catalyzed 1-acyl lysophospholipid deacylation is mediated by sn-1-directed action, but above pH 7 acyl migration with subsequent enzyme-catalyzed hydrolytic cleavage from the sn-2 position contributes to the overall deacylation of monoacylphosphoglycerides, acyl migration becoming eventually the rate-limiting factor. © 1997 Elsevier Science B.V. All rights reserved.

Ultrastructural localization of Charcot-Leyden crystal protein in human eosinophils and basophils.

The Charcot-Leyden crystal (CLC) protein with lysophospholipase activity and carbohydrate-binding properties is a characteristic constituent of eosinophils and basophils. We investigated its subcellular distribution using immunoelectron microscopy. Eosinophil progenitors, mature eosinophils and basophils all contained CLC protein in their cytosol and in the euchromatin of the nucleus. A minor population of granules in eosinophils, increasing in number with maturation, and a more abundant granule-population in basophils, were found to contain CLC protein. Double-labeling experiments showed, in eosinophils, that CLC protein-containing granules contain also eosinophil peroxidase, a characteristic specific granule protein. This suggests a relationship between the CLC protein-containing organelle and the specific granule. In basophils both the CLC protein positive and the negative granules showed the same characteristic particulate-like structure of the granular matrix and both share the same membrane marker CD63. In nasal polyps, macrophages were observed phagocytosing necrotic eosinophils. In these macrophages CLC protein-containing vesicles were observed, probably representing late endosomes. The dual (cytosolic/nuclear and granular) localization of CLC protein suggests that this protein enters both a secretory and a nonsecretory pathway during its biosynthesis, indicating functional roles for this protein both within the cell and extracellularly.

Expression, Purification, and Kinetic Characterization of a Recombinant 80-kDa Intracellular Calcium-independent Phospholipase A2

A CHO cell-derived 80-kDa recombinant polypeptide (GenBank number I15470I15470) putatively encoding a calcium-independent phospholipase A2 was expressed in S. frugiperda cells resulting in over a 15-fold increase in a calcium-independent phospholipase A1/A2 activity which was entirely inhibitable by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. The recombinant polypeptide was purified from cytosol by sequential tandem affinity chromatographies employing ATP-agarose and calmodulin-Sepharose stationary phases. This strategy resulted in the rapid purification (36 h) of recombinant phospholipase A2 activity in 56% overall yield to a single intense 80-kDa protein band on SDS-polyacrylamide gel electrophoresis after silver staining. The purified protein possessed phospholipase A1, phospholipase A2, and lysophospholipase activities. Microbore anion exchange chromatography demonstrated that the 80-kDa protein band was comprised of multiple distinct isoforms including an anionic isoform which possessed over a 5-fold higher specific activity (5 micromol/mg.min) than earlier eluting isoforms. Collectively, these results unambiguously demonstrate that: 1) the 80-kDa polypeptide catalyzes phospholipase A1/A2 and lysophospholipase activities with distinct kinetic parameters; 2) calmodulin and ATP both interact with the catalytic polypeptide independent of regulatory proteins; and 3) distinct isoforms of this polypeptide exist which possess markedly different specific activities.

Guinea pig intestinal phospholipase B: protein expression during enterocyte maturation and effects of N-oligosaccharide removal on enzymatic activities and protein stability.

Guinea pig phospholipase B (PLB) is an intestinal brush-border hydrolase displaying a broad substrate specificity towards various dietary lipids. PLB was detected by immunoblotting as a single 140-kDa polypeptide in all cell populations isolated from guinea pig intestinal mucosa, but increased in parallel to its activity from undifferentiated to mature cells, the specific activity of the enzyme remaining constant. Moreover, N-glycosylation, which contributed to 23% of the apparent molecular mass, was identical along the cell differentiation axis. In all cell fractions, N-linked sugar chains were of the complex type, since they were removed by N-glycosidase F, whereas PLB remained insensitive to endoglycosidase H. Moreover, lack of O-glycosylation was demonstrated by the insensitivity of PLB to O-glycosidase and by its failure to interact with Helix pomatia lectin after prior treatment with neuraminidase or alpha-fucosidase. Enzymatic removal of sugar chains reduced phospholipase A2, lysophospholipase and diacylglycerol lipase activities by 27-35%, kinetic analysis indicating a decrease in apparent Vmax values for the three enzymatic activities, whereas the Km remained unchanged. Finally, the carbohydrate-depleted form of PLB did not display gross changes in thermal stability, in contrast to PLB from microorganisms previously investigated. Our data indicate that the high level of PLB N-glycosylation is poorly related to its biological function. Whether carbohydrate chains are involved in proper targeting of the enzyme to the brush-border membrane remains to be established.

Lysophospholipase L 2 of Vibrio cholerae O1 affects cholera toxin production

The implication in cholera toxin (CT) production of the newly identified gene, lypA, that encodes the lysophospholipase L 2 of Vibrio cholerae, was investigated. Introduction of lypA into the V. cholerae O1 mutant (NF404), which has a Tn5-insertion in lypA and has lost CT as well as haemolysin production, restored the lysophospholipase activity and CT production but not the haemolytic activity. Inactivation of the lypA gene of the wild-type strain by chromosomal integration of a plasmid containing a portion of the lypA gene decreased the lysophospholipase L 2 activity and the production of CT but not the haemolytic activity. Furthermore, constructed mutants of El Tor-biotype and Classical-biotype strains which have a defective lypA failed to produce CT and exhibited decreased enterotoxicity in the ligated rabbit ileal loop test. These results suggest that lypA is possibly required for the expression of CT and may play a role in pathogenicity of V. cholerae.

Lysophospholipase L2 of Vibrio cholerae O1 affects cholera toxin production.

The implication in cholera toxin (CT) production of the newly identified gene, lypA, that encodes the lysophospholipase L2 of Vibrio cholerae, was investigated. Introduction of lypA into the V. cholerae O1 mutant (NF404), which has a Tn5-insertion in lypA and has lost CT as well as haemolysin production, restored the lysophospholipase activity and CT production but not the haemolytic activity. Inactivation of the lypA gene of the wild-type strain by chromosomal integration of a plasmid containing a portion of the lypA gene decreased the lysophospholipase L2 activity and the production of CT but not the haemolytic activity. Furthermore, constructed mutants of E1 Tor-biotype and Classical-biotype strains which have a defective lypA failed to produce CT and exhibited decreased enterotoxicity in the ligated rabbit ileal loop test. These results suggest that lypA is possibly required for the expression of CT and may play a role in pathogenicity of V. cholerae.

Regulation of Lysophospholipase Activity of the 85-kDa Phospholipase A2 and Activation in Mouse Peritoneal Macrophages

The regulation of the lysophospholipase activity of the 85-kDa cytosolic phospholipase A2 (PLA2) was studied in vitro and in stimulated macrophages. Bovine serum albumin was found to inhibit lysophospholipase activity of the recombinant 85-kDa PLA2 when assayed at a relatively low substrate concentration. Inhibition could be reversed if the substrate concentration was increased or if Ca2+ was present in the assay. Incubation of recombinant enzyme with macrophage membranes and lipid extracts from macrophage membranes resulted in the release of arachidonic acid, as well as, stearic acid, which is enriched at the sn-1 position of macrophage phospholipids. This suggests that with a bilayer substrate the PLA2 can sequentially deacylate the sn-2 then sn-1 acyl groups. This was verified by demonstrating that the phospholipids, phosphatidylcholine and phosphatidylinositol, were hydrolyzed to glycerophosphocholine and glycerophosphoinositol by incubation with recombinant 85-kDa PLA2. The 85-kDa enzyme was identified as the main lysophospholipase activity in mouse peritoneal macrophage cytosols. Addition of Ca2+ to the assay enhanced activity, but this effect decreased as the substrate concentration was increased. Incubation of macrophages with zymosan increased the lysophospholipase activity of the 85-kDa PLA2 in cytosols. Phosphorylation of recombinant PLA2 with mitogen-activated protein kinase resulted in an increase in lysophospholipase, as well as, PLA2 activity. In macrophages stimulated with zymosan release of stearic acid (18:0) and palmitic acid (16:0) was observed in addition to arachidonic acid (20:4). These results are consistent with a role of the 85-kDa PLA2 in regulating lysophospholipid levels in macrophages during zymosan stimulation.

Presence in human eosinophils of a lysophospholipase similar to that found in the pancreas.

The supernatant fraction from lysed human eosinophils, when separated by gel-filtration chromatography, contains a protein with lysophospholipase activity of approximate molecular mass 74 kDa. This mass differs substantially from the 17 kDa of a previously cloned eosinophil lysophospholipase (Charcot-Leyden crystal protein), but is similar to that reported for a pancreatic enzyme. We have therefore further characterized this pancreatic-like lysophospholipase in human eosinophils. A rabbit polyclonal antibody was produced against a synthetic peptide consisting of amino acids 325-349 from the 74 kDa rat pancreatic lysophospholipase. Western-blot analysis of eosinophil extracts indicate that this antibody recognizes a single 74 kDa band in these preparations. Incubation of the supernatant fraction from sonified eosinophils with this antibody, followed by precipitation of antibody-antigen complexes with Protein A, removes the majority of the lysophospholipase activity. Indirect immunofluorescence examination with this antibody indicates this protein to be localized to granules of eosinophils and not in other leucocytes. Moreover, reverse transcriptase PCR of polyadenylated RNA from eosinophils and from rat pancreatic tissue with primers to rat pancreatic lysophospholipase resulted in readily detectable 1 kb DNA products in both samples. Sequencing revealed this DNA fragment to be identical with the human pancreatic lysophospholipase cDNA sequence. Taken together, these data indicate that eosinophils contain a lysophospholipase that is similar to the human pancreatic enzyme.

Phospholipid Degradation in Rat Calcium Ionophore-Activated Platelets Is Catalyzed Mainly by Two Discrete Secretory Phospholipase As

An "A1 type" phospholipase activity with serine-phospholipid preference was released by rat activated platelets. It was distinct from the secretory type II phospholipase A2 [Horigome, K., Hayakawa, M., Inoue, K., and Nojima, S. (1987) J. Biochem. 101, 625-631] and co-purified with the secretory lysophosphatidylserine-selective lysophospholipase activity [Higashi, S., Kobayashi, T., Kudo, I., and Inoue, K. (1988) J. Biochem. 103, 442-447]. Several lines of evidence indicated that a single protein was responsible for the phospholipase A1 and lysophospholipase activities. Marked accumulation of lysophospholipids was observed in rat calcium ionophore-activated washed platelets and both phospholipase A1/lysophospholipase and type II phospholipase A2 were shown to contribute to this phospholipid degradation. A selective inhibitor of type II phospholipase A2 reduced the phospholipid degradation and enhanced the clotting time and prothrombinase activity. These results indicate that secretory platelet phospholipases may play a role in regulation of blood clotting.

Purification, properties, and specificity of rat brain cytosolic fatty acyl coenzyme A hydrolase.

Rat brain cytosolic acyl-CoA hydrolase has been purified 3,500-fold to apparent homogeneity using heat treatment, ammonium sulfate fractionation followed by anion exchange, hydrophobic interaction, and hydroxyapatite c chromatography. The purified enzyme remains stable only in the presence of a high concentration (30%, vol/vol) of ethylene glycol. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified enzyme shows a single band of 40.9 kDa. However, on high-performance size-exclusion chromatography the migration rate of the enzyme corresponds with an apparent molecular mass of 148 kDa, indicating that the native enzyme may be a tetramer. The enzyme catalyzes the hydrolysis of fatty acyl-CoAs from six to 18 carbon chains long, having the highest activity for lauroyl (12:0)-CoA. For the purified enzyme the Km for palmitoyl-CoA is 5.8 microM and the Vmax is 1,300 mumol/min/mg of protein. The enzyme is inhibited by bovine serum albumin, various detergents, lysophosphatidylcholine, and palmitoyl carnitine. Among the sulfhydryl agents, only p-hydroxymercuribenzoate inhibited the enzyme. The enzyme is also inactivated by treatment with a high concentration of diethyl pyrocarbonate, an active center histidine-reacting agent, but not by phenylmethylsulfonyl fluoride (10 mM), a serine esterase inhibitor. The purified enzyme does not appear to possess any O-ester hydrolase, lysophospholipase, transacylase, or acyltransferase activity.

Active Recombinant Human Cytosolic Phospholipase A 2 Is Expressed in Escherichia coli

The cDNA encoding human cytosolic phospholipase A 2 (cPLA 2) has been subcloned into a prokaryotic pET16b expression vector which also encodes an amino-terminal deca-histidine affinity tag to facilitate purification of the recombinant enzyme. Soluble, active fusion protein, designated His-cPLA 2, has been obtained reproducibly from this expression system using the E. coli strain BL21 (DE3). The protein has been purified to homogeneity in four steps and the mass confirmed by electrospray mass spectrometry. His-cPLA 2 was characterized by kinetic analysis which demonstrated that the enzyme is similar to native cPLA 2 in all respects investigated. Specifically, the enzyme binds to anionic vesicles containing substrate, and acts processively on these vesicles. Enzymatic activity is supported by the presence of Ca 2+ and several other divalent metal ions, and is inhibited by several transition metal ions. Finally, the enzyme demonstrates lysophospholipase activity and exhibits a high selectivity for sn-2 arachidonyl esters. This prokaryotic expression system yields moderate amounts of unmodified recombinant His-cPLA 2, and is advantageous for rapid production of protein and mutational analyses.

Identification and Characterization of Carboxyl Ester Hydrolase as a Phospholipid Hydrolyzing Enzyme of Zymogen Granule Membranes from Rat Exocrine Pancreas

Salt-washed (0.6 m NaCl) zymogen granule membranes (ZGM) of rat pancreatic acinar cells were utilized to identify and characterize membrane protein(s) responsible for phospholipase and lysophospholipase activities. Five major bands were identified in salt-washed ZGM by Coomassie Brilliant Blue. A 70-kDa protein with enzymatic activity was retained in significant quantities after several washes with 0.6 M NaCl but could be displaced from ZGM by 2 m NaCl or by 100 mg/ml heparin. By contrast, GP2, an integral membrane protein, was not displaced under these conditions. These findings suggest that the enzyme is a peripheral membrane protein of ZGM. Renaturation of ZGM proteins following electrophoresis revealed that the 70-kDa protein possessed phospholipase activity. Identification of the 70-kDa protein as a membrane-associated carboxyl ester hydrolase was based upon: (a) the use of a specific polyclonal antiserum, (b) N-terminal sequence, (c) two-dimensional gel analysis, (d) enzymatic characterization, and (e) co-localization to an area of a non-reducing gel containing significant phospholipase activity. Other ZGM proteins, namely GP2 and GP3, could not be demonstrated to possess phospholipase activity under the experimental conditions employed. Our finding that carboxyl ester hydrolase from ZGM exhibits PLA1 and lysophospholipase activities represents the first identification and characterization of a protein responsible for phospholipase activity in secretory granule membranes.

Osmotic regulation of synthesis of glycerophosphocholine from phosphatidylcholine in MDCK cells

Glycerophosphocholine (GPC) is osmotically regulated in renal medullary cells and in cultured Madin-Darby canine kidney (MDCK) cells. Previously, it was shown that a high extracellular concentration of urea or NaCl causes these cells to accumulate large amounts of GPC. GPC is known to be a product of phosphatidylcholine (PC) catabolism. The purpose of the present experiments was to examine the role of changes in the rate of GPC synthesis from PC in hyperosmotically induced GPC accumulation. When 1-palmitoyl-2-lysophosphatidyl-[methyl-3H]choline ([3H]LPC) is added to the medium, it is taken up by the cells and most of it is rapidly converted to PC. During a chase, 3H lost from PC appears almost exclusively in GPC and sphingomyelin. The rate of catabolism of PC is twofold greater in cells exposed to high NaCl (200 mosmol/kgH2O, added for 2 days) than in control or high-urea medium. Increased PC catabolism in NaCl-treated cells is associated with a 2.6-fold increase in GPC synthesis from PC; sphingomyelin synthesis decreases, and total cell PC does not change. Also, neither total mass nor specific radioactivity of lysophosphatidylcholine changes. PC catabolism is unaffected by short (2 h) exposure to high NaCl or urea. To investigate the enzymatic basis for the increased PC catabolism in response to high NaCl, phospholipase activity was measured in cell homogenates with 1-palmitoyl-2-[1-14C]palmitoyl-PC as a substrate. Exposure of cells to high NaCl for 2 days (but not 2 h) increases activity 2.8-fold compared with control or high-urea medium. Lysophospholipase activity (measured with [3H]LPC as the substrate) is unchanged. The increased phospholipase activity occurs with dipalmitoyl PC, but not sn-2-arachidonyl PC, as a substrate. Collectively, these data suggest a role for a phospholipase, unrelated to the arachidonyl-selective enzyme, in the regulation of PC catabolism during accumulation of GPC induced by prolonged exposure to high extracellular NaCl.

Conodipine-M, a Novel Phospholipase A2 Isolated from the Venom of the Marine Snail Conus magus

We describe the purification and first biochemical characterization of an enzymatic activity in venom from the marine snail Conus magus. This enzyme, named conodipine-M, is a novel phospholipase A2 with a molecular mass of 13.6 kDa and is comprised of two polypeptide chains linked by one or more disulfide bonds. The amino acid sequence of conodipine-M shows little if any homology to other previously sequenced phospholipase A2 enzymes (PLA2s). Conodipine-M thus represents a new group of PLA2s. This is remarkable, since conodipine-M displays a number of properties that are similar to those of previously characterized 14-kDa PLA2s. The enzyme shows little, if any, phospholipase A1, diacyglycerol lipase, triacylglycerol lipase, or lysophospholipase activities. Conodipine-M hydrolyzes the sn-2 ester of various preparations of phospholipid only in the presence of calcium and with specific activities that are comparable to those of well known 14-kDa snake venom and pancreatic PLA2s. The Conus enzyme binds tightly to vesicles of the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol and catalyzes the hydrolysis of this substrate in a processive fashion. Conodipine-M does not significantly discriminate against phospholipids with unsaturated versus saturated fatty acids at the sn-2 position or with different polar head groups. Linoleoyl amide and a phospholipid analog containing an alkylphosphono group at the sn-2 position are potent inhibitors of conodipine-M. We suggest that the functional resemblance of conodipine-M to other PLA2s might be explained by the utilization of similar catalytic residues.

Identification of Entamoeba histolytica intracellular phospholipase A and lysophospholipase L1 activities.

Entamoeba histolytica phospholipase A and lysophospholipase activities from a vesicular subcellular fraction (P30) were analyzed. The products, obtained using specific substrates labeled with 14C or 3H, indicated the presence of phospholipase A1 and A2 as well as lysophospholipase L1 activities. The enzymes detected could participate in phospholipid metabolism and the alkaline phospholipase A2 may contribute to E. histolytica cytopathogenicity.

Comparison of lipases and phospholipases in the hydrolysis of phospholipids

Two commercial fungal lipase preparations from Aspergillus niger and Penicillium cyclopium, two fungal phospholipases from A. niger (A 1 and A 2) and one phospholipase from porcine pancreas (A 2, Lecitase) were compared in the hydrolysis of soybean phospholipids. The isoelectric points, pH and temperature optima and temperature stabilities of the phospholipases in these enzyme preparations were also determined. Lipase preparations which also contained phospholipase and lysophospholipase activities were more efficient than phospholipase preparations in the hydrolysis of soybean phospholipids. Phospholipase A 1 and lipase preparations released fatty acids from both positions of the substrate molecule and as a result glyceryl-phosphoryl-choline was formed. Both the A 2 phospholipase preparations tested produced lysophospholipids through partial selective hydrolysis of phospholipids.

Characterization of lysophospholipid metabolizing enzymes in human brain.

Lysophospholipids are generated during the turnover and breakdown of membrane phospholipids. We have identified and partially characterized three enzymes involved in the metabolism of lysophospholipids in human brain, namely, lysophospholipase, lysophospholipid:acyl-CoA acyltransferase (acyltransferase), and lysophospholipid:lysophospholipid transacylase (transacylase). Each enzyme displayed comparable levels of activity in biopsied and autopsied human brain, although in all cases the activity was somewhat lower in human than that in rat brain. All three enzymes were localized predominantly in the particulate fraction, with lysophospholipase possessing the greatest activity followed by acyltransferase and transacylase. Lysophosphatidylcholine possessed a Km in the micromolar range for lysophospholipase and transacylase, and in the millimolar range for acyltransferase, whereas arachidonyl-CoA displayed a Km in the micromolar range for acyltransferase. The three enzymes differed in their pH optima, with lysophospholipase being most active at pH 8.0, transacylase at pH 7.5, and acyltransferase at pH 6.0. Both bromophenacyl bromide and N-ethylmaleimide inhibited lysophospholipase activity and, to a lesser extent, that of acyltransferase and transacylase. None of the enzyme activities were affected by the presence of dithiothreitol or EDTA, although particulate lysophospholipase was activated approximately two-fold by the addition of 5 mM MgCl2 or CaCl2 but not KCl. Transacylating activity was stimulated by CoA, the EC50 of activation being 6.8 microM. Acyltransferase displayed an approximately threefold preference for arachidonyl-CoA over palmitoyl-CoA, whereas the acylation rate of different lysophospholipids was in the order lysophosphatidylinositol > 1-palmitoyl lysophosphatidylcholine > 1-oleoyl lysophosphatidylcholine >> lysophosphatidylserine > lysophosphatidylethanolamine. This, and the preference of human brain phospholipase A2 for phosphatidylinositol, suggests that this phospholipid may possess a higher turnover rate than the other phospholipid classes examined. Human brain homogenates also possessed the ability to transfer fatty acid from lysophosphatidylcholine to lysophosphatidylethanolamine. In addition, we also present evidence that diacylglycerophospholipids can act as acyl donors for the transacylation of lysophospholipids. We have therefore demonstrated the presence of, and partially characterized, three enzymes that are involved in the metabolism of lysophospholipids in human brain. Our results suggest that lysophospholipase may be the major route by which lysophospholipids are removed from the cell membrane in human brain. However, all three enzymes likely play an important role in the remodeling of membrane composition and thereby contribute to the overall functioning of membrane-associated processes.

A Continuous Fluorescence-Based Assay for the Human High-Molecular-Weight Cytosolic Phospholipase A2

A sensitive method for continuously monitoring the activity of the human cytosolic phospholipase A2 (cPLA2) is described. Recombinant cPLA2 efficiently hydrolyzes fatty acid esters of 7-hydroxycoumarin, producing the free fatty acid and the highly fluorescent 7-hydroxycoumarin. All of the observed 7-hydroxycoumarinyl ester hydrolase activity (7-HCEase) in a preparation of the purified recombinant cPLA2 was due to this enzyme since: (1) all of the ester hydrolase activity comigrated on nondenaturing polyacrylamide gel with a protein characterized as the cPLA2 by Western analysis; (2) the immunoreactive protein also possessed both phospholipase A2 and lysophospholipase activities; and (3) arachidonyl trifluoromethyl ketone, a potent inhibitor of the phospholipase A2 activity of cPLA2, also inhibited the 7-HCEase activity. A study of the 7-HCEase activity demonstrated that when 7-hydroxycoumarinyl γ-linolenate was dispersed in a phospholipid matrix it was hydrolyzed by cPLA2 at a rate comparable to that of an arachidonyl-containing phospholipid substrate and with an identical reaction progress curve. In the presence of phospholipid vesicles, the cPLA2-catalyzed hydrolysis of hydrophobic 7-hydroxycoumarinyl esters was stimulated by submicromolar concentration of free calcium and showed a preference for polyunsaturated substrates. The cPLA2-catalyzed hydrolysis of the water-soluble substrate 7-hydroxycoumarinyl 6-heptenoate was catalyzed by cPLA2 in the absence of calcium and other lipids.