Purification and Properties of a Phospholipase A2/Lipase Preferring Phosphatidic Acid, Bis(monoacylglycerol) Phosphate, and Monoacylglycerol from Rat Testis
2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês
10.1074/jbc.m202817200
ISSN1083-351X
AutoresMasafumi Ito, Urbain Tchoua, Mitsuhiro Okamoto, Hiromasa Tojo,
Tópico(s)Neonatal Health and Biochemistry
ResumoPhospholipase A2(PLA2) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca2+ ions for activity and exhibited both phosphatidic acid-preferring PLA2 and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA2 and lipase activities. The sequence of NH2-terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA2 activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA2-resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA2, and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments. Phospholipase A2(PLA2) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca2+ ions for activity and exhibited both phosphatidic acid-preferring PLA2 and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA2 and lipase activities. The sequence of NH2-terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA2 activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA2-resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA2, and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments. Lysophosphatidic acid (LPA) 1The abbreviations used are: LPA, lysophospatidic acid; PLA, phospholipase A; PLD, phospholipase D; ADAM, 9-anthryldiazomethane; HPTLC, performance thin layer chromatography; DTT, dithiothreitol; C12E8, octaethylene glycol dodecyl ether; APMSF, (p-amidinophenyl) methanesulfonyl fluoride; MAFP, methylarachidonyl fluorophosponate; DOC, deoxycholate; 1, 2-DPPA, 1,2-dipalmitoyl-sn-glycero-3-phosphate; 2, 3-DPPA, 2,3-dipalmitoyl-sn-glycero-1-phosphate; 1-O-hexadecyl-OPC, 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine; 1-O-hexadecyl-OPA, 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphate; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; POPS, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine; POPI, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol; POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol; POPA, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate; PAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; SAPA, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphate; PA, phospatidic acid; PG, phospatidylglycerol; LPC, lysophosphatidylcholine; LPS, lysophosphatidylserine; LPE, lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; LPG, lysophospatidylglycerol; LBPA, lysobisphosphatidic acid; MGL, monoacylglycerol lipase; HPLC, high performance liquid chromatography; MES, 4-morpholineethanesulfonic acid; PAPA, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphate; ESI, electrospray ionization; MS, mass spectrometry; MS/MS, tandem MS is a key intermediate for de novo synthesis of phospholipids and triacylglycerol. In addition, recent studies have established that LPA serves as an intercellular signaling molecule that mediates diverse cellular functions, such as cell growth and cytoskeletal remodeling (1Moolenaar W.H. Kranenburg O. Postma F.R. Zondag G.C. Curr. Opin. Cell Biol. 1997; 9: 168-173Crossref PubMed Scopus (474) Google Scholar), via G protein-coupled receptors (2Contos J.J.A. Ishii I. Chun J. Mol. Pharmacol. 2000; 58: 1188-1196Crossref PubMed Scopus (372) Google Scholar). These receptors comprise several isoforms (2Contos J.J.A. Ishii I. Chun J. Mol. Pharmacol. 2000; 58: 1188-1196Crossref PubMed Scopus (372) Google Scholar), which fulfill distinct functions through different signaling pathways depending on isoforms. On the other hand, there is little information on the metabolic pathways and enzymes responsible for LPA synthesis; several pathways have been proposed depending on tissue and cell types (3Gaits F. Fourcade O., Le Balle F. Gueguen G. Gaige B. Gassama-Diagne A. Fauvel J. Salles J.P. Mauco G. Simon M.F. Chap H. FEBS Lett. 1997; 410: 54-58Crossref PubMed Scopus (148) Google Scholar). Phospholipase A2(PLA2) or phospholipase A1 (PLA1) directly produces LPA from phosphatidic acid (PA) generated by combined action of phospholipase C and diacylglycerol kinase or by direct action of phospholipase D (PLD). Diacylglycerol lipase deacylates diacylglycerol produced in response to stimulation and then the product 2-monoacylglycerol can be phosphorylated by monoacylglycerol kinase, generating LPA. Finally, plasma lysophospholipase D can hydrolyze lysophosphatidylcholine (LPC), yielding LPA. Of these enzymes PLA2s are ubiquitous and have been studied most intensively. A variety of isozymes have been known, and some isoforms exhibit specificity for PA, including group IIA PLA2 (4Snitko Y. Yoon E.T. Cho W. Biochem. J. 1997; 321: 737-741Crossref PubMed Scopus (84) Google Scholar), rat brain 58-kDa PLA2 (5Thomson F.J. Clark M.A. Biochem. J. 1995; 306: 305-309Crossref PubMed Scopus (58) Google Scholar), and intracellular Ca2+-independent PLA2 (6Tang J. Kriz R.W. Wolfman N. Shaffer M. Seehra J. Jones S.S. J. Biol. Chem. 1997; 272: 8567-8575Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). They might be candidates for LPA-synthesizing enzymes, but relevance to LPA synthesis in vivo has not yet been established, although it was suggested that group IIA PLA2 attacks PA-containing microvesicles, shed from damaged cells in inflammation, to produce LPA (7Fourcade O. Simon M.F. Viode C. Rugani N. Leballe F. Ragab A. Fournie B. Sarda L. Chap H. Cell. 1995; 80: 919-927Abstract Full Text PDF PubMed Scopus (500) Google Scholar). In the course of a study on tissue distribution of PLA2 activity toward mixed micelles of acidic phospholipid and cholate, we found that rat testis contained appreciable amounts of Ca2+-independent PLA1 and PLA2 (8Tojo H. Ono T. Okamoto M. J. Lipid Res. 1993; 34: 837-844Abstract Full Text PDF PubMed Google Scholar). Recently, PA-preferring PLA1 was purified and cloned from bovine testis (9Higgs H.N. Glomset J.A. J. Biol. Chem. 1996; 271: 10874-10883Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 10Higgs H.N. Han M.H. Johnson G.E. Glomset J.A. J. Biol. Chem. 1998; 273: 5468-5477Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In this study, we purified to homogeneity a PLA2 with a substantial specificity to acidic phospholipids, PA and phosphatidylglycerol (PG) from rat testis supernatant and characterized it enzymatically. The purified enzyme (PA-PLA2/MGL) exhibited both PLA2 and monoacylglycerol lipase activities as a single 63-kDa molecule but did not exhibit PLA1 and lysophospholipase activities. The PLA2 activity of the enzyme toward PA showed acidic pH optimum of 5.5, suggesting that the enzyme might work in cellular acidic compartments including endosome/lysosome system. The late endosomes specifically contain another acidic phospholipid, bis(monoacylglycerol) phosphate (lysobisphosphatidic acid, LBPA), a structural isomer of PG, that takes part in protein and cholesterol sorting in this system (11Kobayashi T. Stang E. Fang K.S. de Moerloose P. Parton R.G. Gruenberg J. Nature. 1998; 392: 193-197Crossref PubMed Scopus (669) Google Scholar, 12Simons K. Gruenberg J. Trends Cell Biol. 2000; 10: 459-462Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The acidic pH optimum of the enzyme led us to examine its ability to hydrolyze LBPA, which has been known as a unique PLA2-resistant phospholipid because of itssn-1:sn-1′ stereo-configuration (13Amidon B. Schmitt J.D. Thuren T. King L. Waite M. Biochemistry. 1995; 34: 5554-5560Crossref PubMed Scopus (33) Google Scholar). In the present study, we assayed LBPA-hydrolyzing activity of the purified enzyme using LBPA-rich lipid extracts prepared from liver lysosomal fractions of the rats treated with chloroquine (14Yamamoto A. Adachi S. Matsuzawa Y. Kitani T. Hiraoka A. Seki K. Lipids. 1976; 11: 616-622Crossref PubMed Scopus (50) Google Scholar) as substrate. LBPA and the reaction products were separated and identified by microbore normal phase HPLC/electrospray ionization ion-trap mass spectrometry. The following glycerolipids were obtained from Avanti Polar Lipids, Inc.: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol (POPI), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine (1-O-hexadecyl-OPC), LPC, lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), and lysophosphatidylinositol (LPI). Bisphosphatidic acid was purchased from Doosan Serdary Research Laboratories. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, 2,3-dipalmitoyl-sn-glycero-1-phosphocholine, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphate (SAPA), dithiothreitol (DTT), PLD, and LPA were obtained from Sigma. Monostearoylglycerol, monooleoylglycerol, monolinoleoylglycerol, monolinolenoylglycerol, monoarachidonoylglycerol, 1,3-dioleoylglycerol, 1,3-diarachidonoylglycerol, and triarachidonoylglycerol were obtained from Nu-Check Prep, Inc. Trioleoylglycerol and octaethylene glycol dodecyl ether (C12E8) were purchased from Nacalei Tesque, Ltd. (Kyoto, Japan). Silica gel 60 HPTLC plates were obtained from Merck. HPLC-grade acetonitrile was purchased from Katayama Chemical Industries, Ltd. (Osaka, Japan). (p-Amidinophenyl) methanesulfonyl fluoride (APMSF) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Methylarachidonyl fluorophosphonate (MAFP) was purchased from Cayman Chemical Co. (Ann Arbor, MI). Platelets were prepared as described previously (8Tojo H. Ono T. Okamoto M. J. Lipid Res. 1993; 34: 837-844Abstract Full Text PDF PubMed Google Scholar). PLA2, lysophospholipase, and lipase activities were determined by a non-radiometric HPLC method based on precolumn derivitization with 9-anthryldiazomethane (ADAM) as described previously (8Tojo H. Ono T. Okamoto M. J. Lipid Res. 1993; 34: 837-844Abstract Full Text PDF PubMed Google Scholar). Individual fatty acids released from mixed-acyl glycerophospholipids and tri-, di-, and monoacylglycerols were determined simultaneously by this method. Substrate stock solutions used were as follows: mixed micelles of 5 mm diradyl-phospholipid with various concentrations of taurocholate, cholate, deoxycholate (DOC), or Triton X-100; emulsions of 5 mm triacylglycerol or diacylglycerol; and 5% gum arabic for lipase. In a typical experiment, the assay mixtures contained 10 mm EDTA, substrate micelles, or emulsion (10-μl stock solution), 0.1 m NaCl, 0.1 mTris-HCl (pH 8.5 for lipase activity), or MES (pH 5.5 for PLA2 and LBPA activity), 6 mm DTT, and the enzyme sample in a final volume of 50 μl. The addition of DTT was essential for activity. In inhibition studies, enzyme solutions were preincubated for 1 h with APMSF and MAFP (the final concentrations of 500 and 50 μm, respectively, in the assay mixture) and then enzyme activity was assayed. Cholesterol esterase activity was also determined by the ADAM method. Cholesterol oleate (5 mm) was emulsified with 3.6 mg/ml mineral oil (15Tojo H. Ichida T. Okamoto M. J. Biol. Chem. 1998; 273: 2214-2221Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), 7.5 mm POPC, or 5% arabic gum. POPC was used, because PA-PLA2/MGL has virtually no activity for POPC. The assay mixtures contained cholesterol ester emulsion (10 μl), either 10 mm DOC or 30 mm Triton X-100, 6 mm DTT, and 0.1 m NaCl, either 0.1m Tris-HCl (pH 8.5) or MES (pH 5.5), and enzyme sample. The transacylase activity was measured with mixed micelles of 6 mmtaurocholate and the following acyl donor and acceptor combinations: mixtures of 1 mm LPC and 1 mm LPE as acceptor and either 1 mm SAPA or 1 mm POPA as donor; 2 mm LPA as acceptor and 1 mm POPG as donor. The assay mixtures contained above substrates and 0.1 m NaCl, 0.1 m MES (pH 5.5), 6 mm DTT, 10 mmEDTA, and enzyme sample in a final volume of 100 μl. These mixtures were incubated for 3 h and extracted by a modified Bligh and Dyer method (16Bligh E.J. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (44150) Google Scholar). The reaction products were analyzed by HPLC/electrospray ionization ion-trap mass spectrometry as described below. Esterase activities were spectrophotometrically determined with p-nitrophenyl valerate and p-nitrophenyl myristate as substrates. The assay mixtures contained 0.5 mm substrate, 10 mm deoxycholate, 0.1 m Tris-HCl (pH 8.0), 6 mm DTT, and enzyme sample in a final volume of 0.5 ml. After the addition of enzyme solution an increase in absorbance at 400 nm was monitored continuously with a Jasco M550 spectrophotometer at 25 °C. The frozen rat testes (50 g) were homogenized in 500 ml of 20 mm Tris-HCl (pH 7.5) containing 2 mm EDTA, 0.5 mm APMSF, and 1 mm DTT and then sonicated for 5 min on ice. After filtrating the resultant homogenate with a stainless mesh, the extract was centrifuged at 23,000 × g for 90 min. The pH of the supernatant was adjusted to 8.5 and then the solution was applied to a TEAE-cellulose column (6 × 15 cm) pre-equilibrated with 20 mm Tris-HCl (pH 8.5) containing 0.5% Triton X-100 and 1 mm DTT. The PLA2activity was eluted with 20 mm Tris-HCl (pH 7.5) containing 0.1% Triton X-100 and 1 mm DTT by increasing the NaCl concentration from 0 to 1 m. To the pooled PLA2fractions was added 1 m lithium sulfate and then the solutions were applied to a phenyl-Sepharose column (3 × 10 cm) pre-equilibrated with 20 mm Tris-HCl (pH 7.5) containing 1m lithium sulfate and 1 mm DTT. The PLA2 activity bound to this column under these conditions and was eluted with 10 mm Tris-HCl containing 10% ethylene glycol, 0.1%Triton X-100, and 1 mm DTT. The pooled PLA2 fractions were applied on a SuperQ-Toyopearl column (1 × 15 cm) after pre-equilibration in 20 mm Tris-HCl (pH 8.5) containing 0.1% Triton X-100. The column was washed with 20 mm Tris-HCl (pH 8.5) containing 0.1% C12E8 to remove strongly UV-absorbing Triton X-100, then connected to a HPLC system, and developed with a linear gradient of NaCl concentration in 20 mm Tris-HCl (pH 8.5) containing 0.1% C12E8 and 1 mm DTT (Buffer A), from 0 to 0.15 m for 120 min, and then from 0.15 to 1 m for 125 min. The flow rate was 0.7 ml/min, and 1.4-ml fractions were collected. The resultant PLA2fractions were loaded onto a Biogel A-0.5m column (2 × 50 cm) pre-equilibrated with 20 mm Tris-HCl (pH 7.5) containing 0.3 m NaCl, 1 mm DTT, and 0.1% C12E8. The active PLA2 fractions were purified further by HPLC on a Cosmogel QA column (7.5 × 75 mm; Nacalai Tesque) pre-equilibrated with Buffer A. The PLA2 activity was eluted with a concentration gradient of NaCl in Buffer A from 0 to 0.2 m for 60 min and then from 0.2 to 1 m for 80 min. The pooled PLA2 fraction was rechromatographed on the same column; the pH of the eluent was decreased to 8.0, and a shallower gradient of NaCl from 0 to 0.1m for 90 min was used. The flow rate was 0.5 ml/min, and 1-ml fractions were collected. The active PLA2 pools were purified further by HPLC on a Super SW 3000 column (4.6 × 300 mm; Tosoh Corporation) pre-equilibrated with 20 mmTris-HCl (pH 7.0) containing 300 mm NaCl, 0.1% C12E8, and 1 mm DTT at 0.1 ml/min. The enzyme activity was eluted at the retention time of 29 min, coinciding well with a 280-nm peak. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphate (PAPA), 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphate (1-O-hexadecyl-OPA), and 1,2-dipalmitoyl-sn-glycero-3-phosphate (1,2 DPPA) and its enantiomer (2,3-DPPA) were produced from the corresponding phosphatidylcholines by the action of PLD as described previously (17Yang S.F. Freer S. Benson A.A. J. Biol. Chem. 1967; 242: 477-484Abstract Full Text PDF PubMed Google Scholar). Briefly, substrates (25 mg each) were resuspended in 2 ml of diethyl ether and then 100 units of Streptomyces sp.PLD (Sigma) were added in 1 ml of 0.2 m sodium acetate (pH 5.6) and 40 mm CaCl2 at room temperature. After a 12-h incubation with vigorous shaking, the diethyl ether was evaporated and then lipids were extracted by the acidic Bligh and Dyer method (16Bligh E.J. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (44150) Google Scholar). The extracted lipids were resuspended in 0.2 ml of hexane/2-propanol (3:2) (v/v). Aliquots were purified by normal-phase HPLC on a Dynamax-60A Si column (0.4 × 25 cm; Rainin Instrument Company, Inc.). The column was pre-equilibrated with Solvent A, hexane/2-propanol (4:6) (v/v) and developed with a linear gradient of Solvent B and Solvent A/20 mm potassium phosphate in water (pH 7.5)/water (100:6:6) (v/v/v) from 30 to 100% for 40 min at the flow rate of 1 ml/min. 1-O-Hexadecyl-OPA and PAPA were detected at 210 nm and 1,2-DPPA or 2,3-DPPA at 200 nm. The collected solutions were evaporated and resuspended in 1 ml of hexane/2-propanol (3:2) (v/v) and washed with 0.5 ml of 6.7% Na2SO4. Male albino rats of the Sprague-Dawley strain were given an aqueous solution of chloroquine at 100 mg per kg of body weight per day through a stomach tube for 1 week (14Yamamoto A. Adachi S. Matsuzawa Y. Kitani T. Hiraoka A. Seki K. Lipids. 1976; 11: 616-622Crossref PubMed Scopus (50) Google Scholar). The rats were anesthetized with pentobarbital and killed by drawing blood from the abdominal aorta. The liver was removed and homogenized with Buffer B (0.25 m sucrose containing 5 mm Tris-HCl, 1 mm MgCl2, and 2 mm EDTA (pH 7.4)). The homogenate was centrifuged at 1,000 × g for 10 min and then the supernatant was centrifuged at 10,500 × g for 20 min. Lipids in the resultant pellets (crude lysosomal fractions) were extracted by the same method as used for extracting synthesized PAs as described above, and their phosphorus contents were determined and then used for assays as soon as possible. In some experiments the extracted lipids were analyzed by HPTLC with a one-dimensional two-solvent system using chroloform/methanol/30% ammonium hydroxide (65:35:8) (v/v/v) followed by hexane/diethyl ether/acetic acid (16:4:2) (v/v/v). LBPA and its molecular species were identified by a normal phase HPLC/ESI ion-trap mass spectrometry. An aliquot of extracted lipids was injected to a Lichroshere Si-100 column (1 × 150 mm) pre-equilibrated with Solvent A at the flow rate of 50 μl/min, and the column was developed with a linear gradient of Solvent C, Solvent A/1 m ammonium formate/water (100:2.24:9.76) (v/v/v). The effluent was monitored with a ThermoFinnigan LCQ mass spectrometer equipped with an ESI ion source in alternate positive and negative ion full scan, and data-dependent negative ion MS/MS modes on a single run. The mixed micelles of freshly reprepared LBPA-rich lipids (1 mm of total phospholipids) and 6 mm taurocholate was used for substrate. Because LBPA is not a substrate for pancreatic PLA2, almost all phospholipids other than LBPA included in the micelles were first hydrolyzed thoroughly by pancreatic PLA2 (4.8 μg/ml) purified from guinea pig stomach (18Tojo H. Ying Z. Okamoto M. Eur. J. Biochem. 1993; 215: 81-90Crossref PubMed Scopus (18) Google Scholar) for 30 min in the presence of 5 mm CaCl2 and then the resulting solution was immediately used for an LBPA assay. The assay mixtures contained the pancreatic PLA2-treated mixed micelles (20 μl) and 0.1 m NaCl, 0.1 m MES (pH 5.5), 6 mm DTT, 10 mm EDTA, and purified PA-PLA2/MGL in a final volume of 100 μl. The mixtures were incubated at 37 °C and then extracted as reported (16Bligh E.J. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (44150) Google Scholar). Fatty acid release from LBPA was determined by the ADAM method described above. Another reaction product, lysophosphatidylglycerol, and its molecular species were identified by HPLC/negative ion MS/MS spectrometry as described above. The HPLC system consisted of two Gilson model 302 liquid delivery modules, a Gilson model 811 dynamic mixer, and a model 611 programmable UV detector. For mass spectrometry, the UV detector was not used, but the column was connected directly to a mass spectrometer. For a column of 1-mm diameter, the use of Accurate (LC Packings) as a mixer greatly improved mixing efficiency. Lichrosphere Si-100 (Merck) was slurry-packed into a column (1 × 150 mm) in our laboratory. The purified enzyme was reduced,S-carboxymethylated, and purified as described in the legend for Fig. 4. The amino acid sequences were analyzed with an Applied Biosystems 477A sequencer and a 120A PTH analyzer. To identify a phenylthiohydantoin S-carboxymethylated Cys precisely, bothS-carboxymethylated and unmodified proteins were analyzed, and the resultant data were compared. We purified to homogeneity PA-PLA2/MGL as described under "Experimental Procedures," and the results of purification are summarized in Table I. We focused on and purified the PLA2 activities with preference to PA. Rat testis contains a PLA1 activity toward PA (9Higgs H.N. Glomset J.A. J. Biol. Chem. 1996; 271: 10874-10883Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), which was co-purified with PLA2 at earlier steps of purification but was not detectable after a SuperQ Toyopearl chromatography presumably because of its nonspecific adsorption onto the column. Among several chromatographic steps involved in this purification strategy, Cosmogel QA HPLC at pH 8.0 was the most effective. At the final step of purification gel chromatography on a Super SW 3000 column allowed us to remove minor contaminants reproducibly. To ensure its high resolution, the HPLC was operated at a low flow rate of 0.1 ml/min, leading to the overall purification of 538-fold (see Table I and Fig.1 B). Inclusion of DTT and a non-ionic detergent in the eluent of chromatographies was essential for improving the recovery.Table IPurification of PA-PLA2 /MGL from rat testisStepsSpecific activityMonoacylglycerol lipase/PLA2aThe ratios of monoacylglycerol lipase to PLA2 activities.PurificationbBased on the specific activity of PLA2.PLA2Monoacylglycerol lipasenmol/min/mg-FoldSupernatant1.618.711.7TEAE-cellulose0.88.610.81Phenyl-Sepharose0.97.58.31.1SuperQ-Toyopearl0.924.627.31.1Biogel A-0.5 m12.021017.515.0Cosmogel QA (pH 8.5)20.045923.025.0Cosmogel QA (pH 8.0)2204,00018.2275Super SW 30004308,21019.1538The PLA2 and monoacylglycerol lipase activities were determined as described under "Experimental Procedures" using 1 mmPOPA/6 mm taurocholate and 1 mmmonooleoylglycerol/6 mm taurocholate mixed micelles as substrates.a The ratios of monoacylglycerol lipase to PLA2 activities.b Based on the specific activity of PLA2. Open table in a new tab The PLA2 and monoacylglycerol lipase activities were determined as described under "Experimental Procedures" using 1 mmPOPA/6 mm taurocholate and 1 mmmonooleoylglycerol/6 mm taurocholate mixed micelles as substrates. During purification we found that monoacylglycerol lipase activity was co-purified with PLA2 activity. Almost all of PLA2 activity toward PA bound to a TEAE-cellulose column, whereas a significant fraction of monoacylglycerol lipase activity flew through. The ratios of lipase to PLA2 activities after Biogel A-0.5m gel chromatography were rather constant (Table I). Silver-stained SDS-PAGE analysis showed a single band of 63-kDa protein (Fig. 1 C). This molecular mass was similar to that estimated by gel chromatography on a Super SW 3000 column (72 kDa). To check whether this 63-kDa protein represented PA-PLA2/MGL, aliquots of purified enzyme were separated by polyacrylamide gel electrophoresis on two adjacent lanes of a native gel. A gel strip of one lane was stained with Coomassie Brilliant Blue, and the other lane was sliced into pieces of 3-mm length. Monoacylglycerol lipase and PLA2 activities were assayed with materials extracted from these gel slices, and both activities were recovered in the same strip containing a protein band. The omission of DTT from the eluent on gel chromatography at the final step of purification caused almost complete losses of both PLA2 and monoacylglycerol lipase activities. Similarly, the respective specific activities of purified enzyme for POPA and monooleoylglycerol decreased significantly in a similar extent, from 0.22 and 4.0 μmol/min/mg (the specific activity ratio of lipase/PLA2 of 18.2) in the presence of DTT to 0.13 and 2.2 μmol/min/mg (the ratio of 16.9) in its absence. These results indicate that a single enzyme catalyzes PLA2 and monoacylglycerol lipase activity. The addition of either Ca2+ ions up to 50 or 10 mm EDTA in the assay mixtures did not affect the enzyme activities toward POPA and monoolein at pH 5.5 and 8.5, respectively. The addition of DTT in the assay mixtures enhanced both PLA2 and lipase activities to a similar extent (1.7-fold for PLA2 and 1.8-fold for lipase). Anionic detergents enhanced both PLA2 and monoacylglycerol lipase activities greater (taurocholate > cholate > DOC) than non-ionic Triton X-100; activity was measured at pH 8.5 to ensure solubility of cholate and DOC. The pH dependence of the enzyme action toward POPA and monoolein was intriguingly different; the optimal pH values were 5.5 for POPA and 8.5 for monoolein (Fig.2 B), but both activities extended broadly over a physiologically relevant pH range from pH 5.5 to 7.5. Fig. 3 shows the substrate specificity of the purified enzyme. The enzyme preferred anionic phospholipids POPA and POPG in this order but hardly hydrolyzed zweiterionic POPE and POPC. It released oleic acid from the sn-2 position of POPG, but not palmitic acid esterified at its sn-1 position, and can hydrolyze 1-O-hexadecyl-OPA as efficiently as POPA, confirming the A2 regiospecificity for these phospholipids (see Fig.2 A and Fig. 3 A). As to the specificity for thesn-2 acyl groups, PA-PLA2/MGL much preferred unsaturated than saturated acyl chains (Fig. 3 A). The enzyme also hydrolyzed bisphosphatidic acid, an anionic and more bulky substrate, to the extent similar to POPA, but we did not address the regiospecificity for this substrate. PA-PLA2/MGL hardly exhibited lysophospholipase activities toward 1-acyl lysophospholipids including LPA, LPC, LPI, and LPS at pH 5.5.Figure 3Substrate specificity of PA-PLA2/MGL for phospholipids (A) and for monoacylglycerols (B). Enzyme activities were assayed as described under "Experimental Procedures." Substrates used were as follows: A, 1 mm phosphlipid/6 mm taurocholate mixed micelles; B, 1 mm acylglycerol/6 mm taurocholate mixed micelles. Bis-PA, bisphosphatidic acid.View Large Image Figure ViewerDownload (PPT) PA-PLA2/MGL exhibited monoacylglycerol lipase activity with modest preference for polyunsaturated acyl groups at pH 8.5, with the specificity order linolenoyl > arachidonoyl > linoleoyl > oleoyl > stearoyl, whereas its di- and tri-acylglycerol lipase activities were very low (Fig. 3 B). This order was the case with an assay using mixtures of stearoyl-, oleoyl-, linoleoyl-, linolenoyl-, and arachidonoylglycerols (1 mm each) in the presence of 6 mm taurocholate (pH 8.5), as substrates to ensure the similar surface quality for each substrate. Cholesterol oleate, which was emulsified by three different methods as described under "Experimental Procedures" was not a substrate for PA-PLA2/MGL. CoA-independent transacylase activities were not detectable. Serine-modifying irreversible inhibitors, APMSF and MAFP, inhibited PLA2 and lipase activities to a similar extent (TableII). PA-PLA2/MGL also exhibited esterase activity toward p-nitrophenyl esters of short and long chain fatty acids. The specific activity for the valerate ester (580 μmol/min/mg) was significantly greater than the myristate ester (12 μmol/min/mg).Table IIInhibition of PA-PLA2 /MGL by serine-modifying irreversible inhibitors, APMSF, and MAFPInhibitorsResidual activi
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