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• W2088254280 abstract "Calcium-independent phospholipase A2 β (iPLA2 β) participates in numerous diverse cellular processes, such as arachidonic acid release, insulin secretion, calcium signaling, and apoptosis. Herein, we demonstrate the highly selective iPLA2β-catalyzed hydrolysis of saturated long-chain fatty acyl-CoAs (palmitoyl-CoA ≈ myristoyl-CoA ≫ stearoyl-CoA ≫ oleoyl-CoA ≈ arachidonoyl-CoA) present either as monomers in solution or guests in host membrane bilayers. Site-directed mutagenesis of the iPLA2β catalytic serine (S465A) completely abolished acyl-CoA thioesterase activity, demonstrating that Ser-465 catalyzes both phospholipid and acyl-CoA hydrolysis. Remarkably, incubation of iPLA2β with oleoyl-CoA, but not other long-chain acyl-CoAs, resulted in robust stoichiometric covalent acylation of the enzyme. Moreover, S465A mutagenesis or pretreatment of wild-type iPLA2β with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one unexpectedly increased acylation of the enzyme, indicating the presence of a second reactive nucleophilic residue that participates in the formation of the fatty acyl-iPLA2β adduct. Radiolabeling of intact Sf9 cells expressing iPLA2β with [3H]oleic acid demonstrated oleoylation of the membrane-associated enzyme. Partial trypsinolysis of oleoylated iPLA2β and matrix-assisted laser desorption ionization mass spectrometry analysis localized the acylation site to a hydrophobic 25-kDa fragment (residues ∼400–600) spanning the active site to the calmodulin binding domain. Intriguingly, calmodulin-Ca2+ blocked acylation of iPLA2β by oleoyl-CoA. Remarkably, the addition of low micromolar concentrations (5 μm) of oleoyl-CoA resulted in reversal of calmodulin-mediated inhibition of iPLA2 β phospholipase A2 activity. These results collectively identify the molecular species-specific acyl-CoA thioesterase activity of iPLA2 β, demonstrate the presence of a second active site that mediates iPLA2 β autoacylation, and identify long-chain acyl-CoAs as potential candidates mediating calcium influx factor activity. Calcium-independent phospholipase A2 β (iPLA2 β) participates in numerous diverse cellular processes, such as arachidonic acid release, insulin secretion, calcium signaling, and apoptosis. Herein, we demonstrate the highly selective iPLA2β-catalyzed hydrolysis of saturated long-chain fatty acyl-CoAs (palmitoyl-CoA ≈ myristoyl-CoA ≫ stearoyl-CoA ≫ oleoyl-CoA ≈ arachidonoyl-CoA) present either as monomers in solution or guests in host membrane bilayers. Site-directed mutagenesis of the iPLA2β catalytic serine (S465A) completely abolished acyl-CoA thioesterase activity, demonstrating that Ser-465 catalyzes both phospholipid and acyl-CoA hydrolysis. Remarkably, incubation of iPLA2β with oleoyl-CoA, but not other long-chain acyl-CoAs, resulted in robust stoichiometric covalent acylation of the enzyme. Moreover, S465A mutagenesis or pretreatment of wild-type iPLA2β with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one unexpectedly increased acylation of the enzyme, indicating the presence of a second reactive nucleophilic residue that participates in the formation of the fatty acyl-iPLA2β adduct. Radiolabeling of intact Sf9 cells expressing iPLA2β with [3H]oleic acid demonstrated oleoylation of the membrane-associated enzyme. Partial trypsinolysis of oleoylated iPLA2β and matrix-assisted laser desorption ionization mass spectrometry analysis localized the acylation site to a hydrophobic 25-kDa fragment (residues ∼400–600) spanning the active site to the calmodulin binding domain. Intriguingly, calmodulin-Ca2+ blocked acylation of iPLA2β by oleoyl-CoA. Remarkably, the addition of low micromolar concentrations (5 μm) of oleoyl-CoA resulted in reversal of calmodulin-mediated inhibition of iPLA2 β phospholipase A2 activity. These results collectively identify the molecular species-specific acyl-CoA thioesterase activity of iPLA2 β, demonstrate the presence of a second active site that mediates iPLA2 β autoacylation, and identify long-chain acyl-CoAs as potential candidates mediating calcium influx factor activity. Phospholipases A2 (PLA2s) 2The abbreviations used are: PLA2, phospholipase A2; BEL, (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one; BODIPY-PC, 2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3α, 4α-diaza-s-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine; CaM, calmodulin; CIF, calcium influx factor; DOPS, 1,2-dioleoyl-sn-glycero-3-phospho-l-serine; iPLA2, calcium-independent phospholipase A2; LPC, lysophosphatidylcholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; ESI, electrospray ionization; PC, phosphocholine; MALDI, matrix-assisted laser desorption ionization; TOF, time of flight; MS, mass spectral.2The abbreviations used are: PLA2, phospholipase A2; BEL, (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one; BODIPY-PC, 2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3α, 4α-diaza-s-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine; CaM, calmodulin; CIF, calcium influx factor; DOPS, 1,2-dioleoyl-sn-glycero-3-phospho-l-serine; iPLA2, calcium-independent phospholipase A2; LPC, lysophosphatidylcholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; ESI, electrospray ionization; PC, phosphocholine; MALDI, matrix-assisted laser desorption ionization; TOF, time of flight; MS, mass spectral. catalyze the hydrolysis of ester-linked fatty acids from glycerophospholipids, thereby regulating cellular signaling pathways through the generation of lysophospholipids, free fatty acids (e.g. arachidonic acid), and their downstream metabolites. In eukaryotes, PLA2s are broadly categorized into three families: secretory, cytosolic, and calcium-independent phospholipases A2 (iPLA2) (1Kudo I. Murakami M. Prostaglandins Other Lipid Mediat. 2002; 68: 3-58Crossref PubMed Scopus (653) Google Scholar). Secretory PLA2s are low molecular mass (∼12–15 kDa) extracellular enzymes that require high micromolar to millimolar concentrations of Ca2+ for catalysis (2Scott D.L. Sigler P.B. Adv. Protein Chem. 1994; 45: 53-88Crossref PubMed Google Scholar, 3Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Six cytosolic phospholipases A2 (α, β,γ, δ, ε, and ζ) have been characterized at present, five of which (α, β, δ, ε, and ζ) contain C2 domains that require submicromolar Ca2+ for membrane association (4Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar, 5Underwood K.W. Song C. Kriz R.W. Chang X.J. Knopf J.L. Lin L.L. J. Biol. Chem. 1998; 273: 21926-21932Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 6Pickard R.T. Strifler B.A. Kramer R.M. Sharp J.D. J. Biol. Chem. 1999; 274: 8823-8831Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 7Chiba H. Michibata H. Wakimoto K. Seishima M. Kawasaki S. Okubo K. Mitsui H. Torii H. Imai Y. J. Biol. Chem. 2004; 279: 12890-12897Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Calcium-independent PLA2s are intracellular, do not require calcium ion for membrane association or catalysis, and currently comprise seven family members (α, β, γ, δ, ε, ζ, η) (8Andrews D.L. Beames B. Summers M.D. Park W.D. Biochem. J. 1988; 252: 199-206Crossref PubMed Scopus (206) Google Scholar, 9Wolf M.J. Gross R.W. J. Biol. Chem. 1996; 271: 30879-30885Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 10Tang 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 (273) Google Scholar, 11Mancuso D.J. Jenkins C.M. Gross R.W. J. Biol. Chem. 2000; 275: 9937-9945Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), all of which contain conserved nucleotide-binding (GXGXXG) and lipase (GXSXG) sequence motifs. Studies of calcium-independent phospholipase A2β have revealed its importance in agonist-induced arachidonic acid release (12Lehman J.J. Brown K.A. Ramanadham S. Turk J. Gross R.W. J. Biol. Chem. 1993; 268: 20713-20716Abstract Full Text PDF PubMed Google Scholar, 13Wolf M.J. Wang J. Turk J. Gross R.W. J. Biol. Chem. 1997; 272: 1522-1526Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), apoptosis (14Atsumi G. Tajima M. Hadano A. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 13870-13877Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 15Ramanadham S. Hsu F.F. Zhang S. Jin C. Bohrer A. Song H. Bao S. Ma Z. Turk J. Biochemistry. 2004; 43: 918-930Crossref PubMed Scopus (88) Google Scholar), lymphocyte proliferation (16Roshak A.K. Capper E.A. Stevenson C. Eichman C. Marshall L.A. J. Biol. Chem. 2000; 275: 35692-35698Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), fat cell differentiation (17Su X. Mancuso D.J. Bickel P.E. Jenkins C.M. Gross R.W. J. Biol. Chem. 2004; 279: 21740-21748Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), insulin secretion (18Ramanadham S. Gross R.W. Han X. Turk J. Biochemistry. 1993; 32: 337-346Crossref PubMed Scopus (123) Google Scholar), and lysolipid production mediating capacitive calcium influx (19Smani T. Zakharov S.I. Leno E. Csutora P. Trepakova E.S. Bolotina V.M. J. Biol. Chem. 2003; 278: 11909-11915Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 20Smani T. Zakharov S.I. Csutora P. Leno E. Trepakova E.S. Bolotina V.M. Nat. Cell Biol. 2004; 6: 113-120Crossref PubMed Scopus (234) Google Scholar). In addition, recent experiments utilizing transgenic mice selectively overexpressing iPLA2β in myocardium have provided evidence that cardiac ischemia activates iPLA2β, precipitating ventricular tachyarrythmias, which can be suppressed by pretreatment with the mechanism-based iPLA2 inhibitor, (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (21Mancuso D.J. Abendschein D.R. Jenkins C.M. Han X. Saffitz J.E. Schuessler R.B. Gross R.W. J. Biol. Chem. 2003; 278: 22231-22236Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Previously, we have demonstrated that iPLA2β activity is regulated through calmodulin-mediated inhibition of phospholipase A2 activity in the presence of physiologic concentrations of calcium (∼200 nm) (22Wolf M.J. Gross R.W. J. Biol. Chem. 1996; 271: 20989-20992Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Subsequent experiments identified the calmodulin binding domain of iPLA2β, containing “1-9-14” and IQ sequence motifs, located ∼160 and 240 amino acid residues, respectively, from the active site (23Jenkins C.M. Wolf M.J. Mancuso D.J. Gross R.W. J. Biol. Chem. 2001; 276: 7129-7135Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). During cellular stimulation and the depletion of intracellular Ca2+ stores, activation of iPLA2β has been proposed to occur through disassociation of the iPLA2β·CaM complex (13Wolf M.J. Wang J. Turk J. Gross R.W. J. Biol. Chem. 1997; 272: 1522-1526Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 20Smani T. Zakharov S.I. Csutora P. Leno E. Trepakova E.S. Bolotina V.M. Nat. Cell Biol. 2004; 6: 113-120Crossref PubMed Scopus (234) Google Scholar), potentially through the actions of a low molecular weight cellular component known as calcium influx factor (CIF) (20Smani T. Zakharov S.I. Csutora P. Leno E. Trepakova E.S. Bolotina V.M. Nat. Cell Biol. 2004; 6: 113-120Crossref PubMed Scopus (234) Google Scholar, 24Bolotina V.M. Sci. STKE 2004. 2004; : PE34Google Scholar). Although CIF was first described and partially characterized more than 10 years ago as a diffusible messenger released upon intracellular Ca2+ store depletion, which stimulated Ca2+ influx through the plasma membrane (25Randriamampita C. Tsien R.Y. Nature. 1993; 364: 809-814Crossref PubMed Scopus (783) Google Scholar, 26Randriamampita C. Tsien R.Y. J. Biol. Chem. 1995; 270: 29-32Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), the precise molecular identity of CIF is unknown. Multiple fatty acyl-CoA thioesterases have been purified from mammalian cytosol, peroxisomes, and mitochondria and have been cloned and characterized with respect to substrate selectivity, enzyme kinetics, and sensitivity to various inhibitors (27Hunt M.C. Alexson S.E. Prog. Lipid Res. 2002; 41: 99-130Crossref PubMed Scopus (212) Google Scholar). In general, fatty acyl-CoA thioesterases have been classified as those that are induced by peroxisome proliferators (Type-I or Type-II thioesterases) and those that do not share significant sequence homology with these isoforms (27Hunt M.C. Alexson S.E. Prog. Lipid Res. 2002; 41: 99-130Crossref PubMed Scopus (212) Google Scholar). Several other mammalian enzymes, such as lysophospholipases (28Gross R.W. Biochemistry. 1983; 22: 5641-5646Crossref PubMed Scopus (25) Google Scholar), secretory phospholipase A2 (29Nocito M. Roy G. Villar L.M. Palacios C. Serrano A. Alvarez-Cermeno J.C. Gonzalez-Porque P. Biochim. Biophys. Acta. 1996; 1299: 17-22Crossref PubMed Scopus (6) Google Scholar), and palmitoyl-protein thioesterases (30Camp L.A. Verkruyse L.A. Afendis S.J. Slaughter C.A. Hofmann S.L. J. Biol. Chem. 1994; 269: 23212-23219Abstract Full Text PDF PubMed Google Scholar, 31Soyombo A.A. Hofmann S.L. J. Biol. Chem. 1997; 272: 27456-27463Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 32Duncan J.A. Gilman A.G. J. Biol. Chem. 1998; 273: 15830-15837Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), have also been shown to exhibit acyl-CoA hydrolase activity. Interestingly, hepatocyte nuclear factor-4α has been recently demonstrated to hydrolyze fatty acyl-CoAs, followed by binding of the fatty acid product to hepatocyte nuclear factor-4α, thereby allowing cross-talk between the acyl-CoA and free fatty acid binding domains (33Hertz R. Kalderon B. Byk T. Berman I. Za'tara G. Mayer R. Bar-Tana J. J. Biol. Chem. 2005; 280: 24451-24461Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In addition to its regulation by calmodulin, iPLA2β binds ATP through a conserved nucleotide binding motif (GXGXXG), resulting in both enzyme stabilization and activation (34Hazen S.L. Gross R.W. J. Biol. Chem. 1991; 266: 14526-14534Abstract Full Text PDF PubMed Google Scholar, 35Hazen S.L. Gross R.W. Biochem. J. 1991; 280: 581-587Crossref PubMed Scopus (51) Google Scholar). From this perspective, we considered the possibility that iPLA2β might also bind and hydrolyze long-chain acyl-CoAs, given the structural similarity of the 3′-phosphoadenosine moiety of CoA to ATP. In this paper, we demonstrate that iPLA2β catalyzes the hydrolysis of saturated long-chain acyl-CoAs present as either monomers or as guests in host membrane vesicles at physiologic concentrations (1–5 mol %). Moreover, highly selective autoacylation of iPLA2β by oleoyl-CoA occurred at a second nucleophilic site(s) within the catalytic domain, which is protected from oleoylation by Ca2+-activated calmodulin. Finally, oleoyl-CoA was found to attenuate calmodulin-mediated inhibition of iPLA2β phospholipase A2 activity. In summary, these results describe the previously unrecognized fatty acyl-CoA thioesterase activity and fatty acyl-CoA dependent covalent acylation of iPLA2β, thereby revealing additional levels of complexity in this multifunctional signaling enzyme. Materials—[1-14C]palmitoyl-CoA, [1-14C]oleoyl-CoA, and 1-palmitoyl-2-[1-14C]oleoyl-sn-glycero-3-phosphocholine were obtained from PerkinElmer Life Sciences. [1-14C]Arachidonoyl-CoA, [1-14C]myristoyl-CoA, [1-14C]stearoyl-CoA, and [methyl-14C]human albumin were obtained from American Radiolabeled Chemicals. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] (DOPS) were purchased from Avanti Polar Lipids. Sf9 cell culture reagents and 2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3α,4α-diaza-s-indacene-3-propionyl)amino) undecyl)-sn-glycero-3-phosphocholine (BODIPY-PC) were purchased from Invitrogen. High purity bovine calmodulin was obtained from Calbiochem. Most other materials were obtained from either Sigma or Fisher. BEL was purchased from Cayman Chemical and separated into individual enantiomers as described previously (36Jenkins C.M. Han X. Mancuso D.J. Gross R.W. J. Biol. Chem. 2002; 277: 32807-32814Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Construction of Recombinant Baculoviruses Encoding His-tagged Wild-type and S465A Mutant iPLA2β Proteins—cDNA encoding wild-type C. griseus (Chinese hamster) iPLA2β (9Wolf M.J. Gross R.W. J. Biol. Chem. 1996; 271: 30879-30885Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) was utilized as template for PCR amplification of the sequence to introduce six in-frame His codons followed by a stop codon and XhoI restriction site (3′ primer) for subcloning into pFASTBac1. The 5′ primer contained a SalI site and Kozak sequence (GCCACC) 5′ of the ATG start codon. The His-tagged S465A iPLA2β construct was created by PCR amplification of the mutant cDNA (9Wolf M.J. Gross R.W. J. Biol. Chem. 1996; 271: 30879-30885Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) utilizing a 5′ primer containing an EcoRI restriction site and a3′ primer encoding a His6 tag followed by a SalI site for subcloning into pFASTBac1. The 2.4-kb products were each inserted into a bacmid shuttle vector and sequenced in both directions to confirm the integrity of the constructs. S. frugiperda (Sf9) cells in 35-mm plates containing supplemented Grace's medium were transfected and incubated at 27 °C for 72 h to produce high titer baculoviral stocks for infection of Sf9 cells. Expression and Affinity Purification of iPLA2βHis6 from Sf9 Cells—Sf9 cells were grown as 100 ml of suspended cultures (in 1-liter plastic bottles) in supplemented Grace's insect medium containing 10% heat-inactivated fetal bovine serum and 0.1% Pluronic F-68. Following infection of 3 × 100-ml cultures of Sf9 cells (1.5 × 106 cells/ml) with baculovirus encoding iPLA2βHis6 for 48 h, cells were harvested by centrifugation (250 × g for 10 min), washed once with Grace's insect medium without serum, and resuspended in 30 ml of 25 mm sodium phosphate, pH 7.8, 20% glycerol, 2 mm β-ME, 5 μg/ml aprotinin, 5 μg/ml leupeptin. After lysing the cells by sonication (30 1-s bursts), the homogenate was centrifuged at 100,000 × g for 1 h to obtain the cytosol, to which NaCl was added to a final concentration of 250 mm. The cytosol was then mixed by inversion with 3 ml of HIS-Select-Co2+ affinity resin (Sigma) for 1 h, and the cytosol-resin suspension was poured into an Amersham Biosciences 1 × 10-cm column. Following washing of the settled resin with 30 ml of Buffer A (25 mm sodium phosphate, pH 7.8, containing 500 mm NaCl, 20% glycerol, and 2 mm β-ME), bound protein was eluted from the column at a flow rate of 0.25 ml/min utilizing a 250 mm imidazole gradient in Buffer A (50-ml total volume) generated using an Amersham Biosciences fast protein liquid chromatography system. Column fractions were assayed for iPLA2 activity as described below, pooled, and dialyzed overnight against Buffer B (25 mm imidazole, pH 7.8, containing 20% glycerol, 1 mm dithiothreitol, and 1 mm EGTA). The dialyzed sample was applied to a 2.5-ml column of ATP-agarose equilibrated with Buffer B and washed with Buffer B containing 1 mm AMP and 50 mm NaCl. Bound iPLA2βHis6 was eluted with Buffer B containing 2 mm ATP and 100 mm NaCl, dialyzed against Buffer B (EGTA concentration was reduced to 0.1 mm) containing 100 mm NaCl, flash frozen in liquid nitrogen, and stored at –80 °C. Approximately 1 mg (65% yield) of iPLA2βHis6 with a specific activity of 0.5 μmol of oleic acid/min/mg, utilizing 5 μm [14C]POPC as substrate, was typically recovered from 300 ml of cultured Sf9 cells by this procedure. Phospholipase A2 and Acyl-CoA Hydrolase Enzymatic Assays—Purified recombinant iPLA2βHis6 (0.1–1 μg) was incubated with radiolabeled phospholipid or acyl-CoA in 25 mm Tris-HCl, pH 7.2, containing 1 mm EGTA (200 μl final volume) for 1–2 min at 37 °C. In experiments using acyl-CoAs as guests in host phospholipid bilayers, radiolabeled acyl-CoAs were incorporated into POPC/DOPS (90:10 mol %) vesicles (100 μm) before addition to the reaction mix. Long-chain acyl-CoAs have been previously demonstrated to integrate into lipid bilayers within seconds (37Cohen Simonsen A. Bernchou Jensen U. Faergeman N.J. Knudsen J. Mouritsen O.G. FEBS Lett. 2003; 552: 253-258Crossref PubMed Scopus (25) Google Scholar). Reactions were terminated by extraction of the released radiolabeled fatty acids into 100 μl of butanol, separation of fatty acids from unreacted substrate by thin layer chromatography, and quantitation by scintillation spectroscopy as previous described (38Hazen S.L. Stuppy R.J. Gross R.W. J. Biol. Chem. 1990; 265: 10622-10630Abstract Full Text PDF PubMed Google Scholar). For experiments examining the effects of acyl-CoAs on calmodulin-mediated inhibition of iPLA2β, phospholipase A2 activity was continuously measured utilizing a SPECTRAmax GEMINI XS Dual-Scanning Microplate Spectrofluorometer (Molecular Devices). BODIPY-PC substrate (1.17 mm in Me2SO, 5 μm final concentration) was co-sonicated (10 min at 40% power, 50% duty cycle) with POPC (95 μm final concentration) in 25 mm HEPES, pH 7.2. Oleoyl-CoA and CaCl2 were added at the indicated concentration to the lipid vesicles before addition to iPLA2β with or without CaM (preincubated on ice for 10 min) present in individual wells of a black 96-well microtiter plate. Fluorescence readings were acquired at 15-s intervals for 2 min at 37 °C utilizing excitation/emission wavelengths of 495/515 nm, respectively. Electrospray Ionization (ESI)/Mass Spectral (MS) Analyses—Purified iPLA2β (0.25 μg) was incubated with POPC (95 μm) vesicles containing BODIPY-PC (5 μm) for 5 min at 37 °C as described above. The reactions were stopped by the addition of 4 ml of chloroform/methanol (1:1, v/v) containing internal standards (i.e. 14:1–14:1 PC and 17:0 LPC), and the lipid species were extracted into the chloroform layer as described previously (39Han X. Gross R.W. Mass Spectrom. Rev. 2005; 24: 367-412Crossref PubMed Scopus (890) Google Scholar). Extracted lipid samples were filtered with Millex-FG 0.20-μm filters (Millipore, Bedford, MA) and were routinely stored in 200 μl of chloroform/methanol (1/1, v/v) under nitrogen at –20 °C. ESI/MS analysis was performed using a Thermo Finnigan TSQ Quantum Plus spectrometer (San Jose, CA) equipped with an electrospray ion source as previously described in detail (40Han X. Yang J. Cheng H. Ye H. Gross R.W. Anal. Biochem. 2004; 330: 317-331Crossref PubMed Scopus (176) Google Scholar, 41Han X. Cheng H. Mancuso D.J. Gross R.W. Biochemistry. 2004; 43: 15584-15594Crossref PubMed Scopus (63) Google Scholar). Samples were diluted with chloroform/methanol (1:1, v/v) (∼5 pmol/μl) prior to direct infusion into the ESI ion source at a flow rate of 4 μl/min with a 2-min period of signal averaging. Tandem mass scanning of neutral loss of 59 atomic mass units (loss of trimethylamine) was performed at a collision energy of 24 eV and a collision gas pressure of 1.0 millitorr. Covalent Modification of iPLA2β with 14C-labeled Long-chain Acyl-CoAs—Purified recombinant iPLA2βHis6 was incubated with POPC vesicles (90 μm) containing 10 mol % [1-14C]acyl-CoA for 1 h at 37 °C. In some experiments, iPLA2βHis6 was preincubated with BEL (3 min at 23 °C), N-ethylmaleimide (5 min at 30 °C) or iodoacetamide (5 min at 30 °C) prior to the addition of radiolabeled acyl-CoA. Chloroform/methanol precipitation of some samples was performed as described (42Wessel D. Flugge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3108) Google Scholar), utilizing 15 μg of bovine serum albumin as carrier. In experiments to examine the nature of the covalent linkage between oleic acid and iPLA2β, acid (HCl), base (NaOH), and hydroxylamine were added to the indicated concentrations, and the samples were incubated at 30 °C for 1 h. Bovine serum albumin (15 μg) and SDS-PAGE loading buffer were then added to each sample prior to dialysis against 50 mm Tris-HCl, pH 6.8, containing 10% glycerol and 1% SDS for 4 h. Samples were electrophoresed by SDS-PAGE, fixed (40% methanol containing 10% acetic acid), stained with Coomassie Blue R-250, incubated in Amplify fluorographic reagent, dried, and exposed to Eastman Kodak Co. Biomax MR film for 2–5 days at –80 °C. Partial Trypsinolysis of Oleoylated iPLA2β—Purified iPLA2βHis6 (10 μm) was incubated with 50 μm [1-14C]oleoyl-CoA or unlabeled oleoyl-CoA in 25 mm imidazole, pH 7.8, containing 50 mm NaCl, 0.1 mm EGTA, 1 mm dithiothreitol, and 20% glycerol for 1 h at 37°C. Excess [1-14C]oleoyl-CoA was removed by using a Micro Bio-Spin (Bio-Rad) column equilibrated with the above buffer. Recovered iPLA2β was partially digested with trypsin (1:25, w/w) for 1–30 min at 37 °C. Tryptic peptides were separated by SDS-PAGE, fixed in 40% methanol, 10% glacial acetic acid, stained with Coomassie Blue, and destained in the fixation solution. Gels containing the radiolabeled peptide fragments were soaked in Amplify fluorogenic reagent (Amersham Biosciences), dried, and exposed to film. In parallel samples utilizing unlabeled oleoyl-CoA, the band corresponding to the ∼25-kDa radiolabeled fragment was excised, cut into ∼1 × 1-mm pieces, and destained further by washing with 50% acetonitrile at 37 °C. The gel pieces were then dried in a SpeedVac, resuspended in 50 mm ammonium bicarbonate (100 μl) containing 0.5 μg of sequencing grade modified trypsin (Promega), and incubated for 12 h at 37 °C. After aliquoting the supernatant solution to a separate tube, residual peptides in the gel pieces were extracted into 50% acetonitrile, 20% isopropyl alcohol, 0.1% trifluoroacetic acid, combined with the supernatant solution, and concentrated utilizing a SpeedVac. MALDI-TOF Analysis of iPLA2β Tryptic Fragments—Concentrated peptide samples were diluted with 0.5% trifluoroacetic acid, absorbed to a C18 Zip-Tip (Millipore), and desorbed with a solution composed of 50% acetonitrile, 20% isopropyl alcohol, 0.1% trifluoroacetic acid, and containing in addition 5 mg/ml α-cyano-4-hydroxycinnamic acid. Samples were applied to 192-spot sample plates (ABI) and allowed to air-dry. MS analyses were performed utilizing an Applied Biosystems 4700 Proteomics Analyzer (Framingham, MA), which possesses a 200-Hz Nd:YAG laser operating at 355 nm. The mass accuracy of the instrument was externally calibrated to the 4700 Proteomics analyzer calibration mixture of peptides. For MALDI-MS analysis, spectra were obtained by the accumulation of 2500 consecutive laser shots at a collision energy of 1 kV with air serving as the collision gas. Calculations of predicted peptide and peptide fragment masses were performed using programs developed at the University of California, San Francisco, Mass Spectrometry Facility (available on the World Wide Web at prospector.ucsf.edu). Metabolic Labeling of iPLA2β in Intact Sf 9 Cells—Cultures (100 ml) of Sf9 cells (1.5 × 106 cells/ml) were infected with control (pFB empty vector) or iPLA2βHis6-encoding baculovirus and incubated at 27 °C for 45 h. The cells were then pelleted by centrifugation (250 × g for 10 min), resuspended in 10 ml of Grace's insect medium (Sigma) containing 1% heat inactivated fetal bovine serum, 0.1% Pluronic F-68, and 1 mCi of [3H]oleic acid. Following incubation at 27 °C for 3 h, cells were harvested by centrifugation (250 × g for 10 min) and resuspended in 5 ml of 25 mm sodium phosphate, pH 7.8, containing 20% glycerol, 5 μg/ml aprotinin, and 5 μg/ml leupeptin. After sonication, cytosol and membrane fractions were separated by ultracentrifugation as described above. Other Procedures—SDS-PAGE was performed according to Laemmli (43Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). ECL Western analyses for iPLA2βHis6 were performed utilizing a monoclonal anti-His antibody (BD Biosciences) in conjunction with an anti-mouse IgG-horseradish peroxidase conjugate. Silver staining of SDS-polyacrylamide gels was performed as described (44Nesterenko M.V. Tilley M. Upton S.J. J. Biochem. Biophys. Methods. 1994; 28: 239-242Crossref PubMed Scopus (308) Google Scholar). Protein concentration was determined by a version of the Bradford protein assay (Bio-Rad) with bovine serum albumin as a standard. iPLA2β Hydrolyzes Palmitoyl-CoA and Is Inhibited at Submicellar Concentrations of Substrate—Due to the structural similarity between ATP and the 3′-phosphoadenosine moiety of CoA, we hypothesized that iPLA2β could bind to, and potentially hydrolyze, the thioester linkage of long-chain fatty acyl-CoAs. Accordingly, we overexpressed iPLA2βHis6 in Sf9 cells and purified the enzyme to apparent homogeneity (as determined by SDS-PAGE/silver staining) by sequential cobalt and ATP affinity chromatographies as described under “Experimental Procedures.” Initial assays with iPLA2β utilizing supramicellar concentrations of palmitoyl-CoA (100 μm) typically used for acyl-CoA thioesterases revealed very low rates of hydrolysis (Fig. 1A). Remarkably, robust palmitoyl-CoA thioesterase activity was demonstrated at low micromolar concentrations of substrate with a maximal rate of ∼250 nmol of palmitic acid/min/mg at 2.5 μm palmitoyl-CoA (Fig. 1A). Similar acyl-CoA-mediated substrate inhibition was observed in previous studies of a purified rabbit heart mitochondrial thioesterase (45Gross R.W. Biochim. Biophys. Acta. 1984; 802: 197-202Crossref PubMed Scopus (6) Google Scholar) and peroxisomal acyl-CoA thioesterase 2 (46Hunt M.C. Solaas K. Kase B.F. Alexson S.E. J. Biol. Chem. 2002; 277: 1128-" @default.
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• W2088254280 date "2006-06-01" @default.
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• W2088254280 title "Highly Selective Hydrolysis of Fatty Acyl-CoAs by Calcium-independent Phospholipase A2β" @default.
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• W2088254280 doi "https://doi.org/10.1074/jbc.m511623200" @default.
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