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• W2068856232 abstract "Membranes of mammalian cells contain lysophosphatidic acid acyltransferase (LPAAT) activities that catalyze the acylation of sn-1-acyl lysophosphatidic acid (lysoPA) to form phosphatidic acid. As the biological roles and biochemical properties of the six known LPAAT isoforms have yet to be fully elucidated, we have characterized human LPAAT-β activity using two different assays. In a membrane-based assay, LPAAT-β used lysoPA and lysophosphatidylmethanol (lysoPM) but not other lysophosphoglycerides as an acyl acceptor, and it preferentially transferred 18:1, 18:0, and 16:0 acyl groups over 12:0, 14:0, 20:0, and 20:4 acyl groups. The fact that lysoPM could traverse cell membranes permitted additional characterization of LPAAT-β activity in cells: PC-3 and DU145 cells converted exogenously added lysoPM and 14C-labeled 18:1 into 14C-labeled phosphatidylmethanol (PM). The rate of PM formation was higher in cells that overexpressed LPAAT-β and was inhibited by the LPAAT-β inhibitor CT-32501. In contrast, if lysoPM and 14C-labeled 20:4 were added to PC-3 or DU145 cells, 14C-labeled PM was also formed, but the rate was neither higher in cells that overexpressed LPAAT-β nor inhibited by CT-32501. We propose that LPAAT-β catalyzes the intracellular transfer of 18:1, 18:0, and 16:0 acyl groups but not 20:4 groups to lysoPA. Membranes of mammalian cells contain lysophosphatidic acid acyltransferase (LPAAT) activities that catalyze the acylation of sn-1-acyl lysophosphatidic acid (lysoPA) to form phosphatidic acid. As the biological roles and biochemical properties of the six known LPAAT isoforms have yet to be fully elucidated, we have characterized human LPAAT-β activity using two different assays. In a membrane-based assay, LPAAT-β used lysoPA and lysophosphatidylmethanol (lysoPM) but not other lysophosphoglycerides as an acyl acceptor, and it preferentially transferred 18:1, 18:0, and 16:0 acyl groups over 12:0, 14:0, 20:0, and 20:4 acyl groups. The fact that lysoPM could traverse cell membranes permitted additional characterization of LPAAT-β activity in cells: PC-3 and DU145 cells converted exogenously added lysoPM and 14C-labeled 18:1 into 14C-labeled phosphatidylmethanol (PM). The rate of PM formation was higher in cells that overexpressed LPAAT-β and was inhibited by the LPAAT-β inhibitor CT-32501. In contrast, if lysoPM and 14C-labeled 20:4 were added to PC-3 or DU145 cells, 14C-labeled PM was also formed, but the rate was neither higher in cells that overexpressed LPAAT-β nor inhibited by CT-32501. We propose that LPAAT-β catalyzes the intracellular transfer of 18:1, 18:0, and 16:0 acyl groups but not 20:4 groups to lysoPA. Membrane-associated lysophosphatidic acid acyltransferase (LPAAT) activities, identified in bacteria, yeast, plant, and animal cells, catalyze the transfer of acyl groups from acyl-CoA to lysophosphatidic acid (lysoPA) to form phosphatidic acid (PA) (1Dircks L. Sul H.S. Acyltransferases of de novo glycerophospholipid biosynthesis.Prog. Lipid Res. 1999; 38: 461-479Google Scholar, 2Athenstaedt K. Daum G. Phosphatidic acid, a key intermediate in lipid metabolism.Eur. J. Biochem. 1999; 266: 1-16Google Scholar, 3Yamashita A. Sugiura T. Waku K. Acyltransferases and transacylases involved in fatty acid remodeling of phospholipids and metabolism of bioactive lipids in mammalian cells.J. Biochem. (Tokyo). 1997; 122: 1-16Google Scholar). PA is a component of cell membranes and a key intermediate in the de novo synthesis of phosphoglycerides, the major components of cell membranes, and triacylglycerol, the major form of energy storage in plants and animals. Models that suggest that PA can activate numerous cell functions, including proliferation, cytoskeletal organization, vesicle trafficking and the oxidative burst in neutrophils, have also gained wide acceptance. Six human LPAAT isoforms (α–ζ), also known as 1-acyl-sn-glycerol-3-phosphate acyltransferase 1–6, have been cloned. Only the α and β isoforms, which share 34% sequence identity, have been shown to have significant LPAAT activity and to complement the growth defect in plsC, an Escherichia coli strain mutated in its sole LPAAT gene (4Leung D.W. The structure and functions of human lysophosphatidic acid acyltransferases.Front. Biosci. 2001; 6: D944-D953Google Scholar). These isoforms also contain two to four predicted transmembrane domains and two highly conserved motifs, H(X)4D and EGTR, which are essential for the catalytic activity of a family of acyltransferases (5Lewin T.M. Wang P. Coleman R.A. Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction.Biochemistry. 1999; 38: 5764-5771Google Scholar, 6Haque W. Garg A. Agarwal A.K. Enzymatic activity of naturally occurring 1-acylglycerol-3-phosphate-O-acyltransferase 2 mutants associated with congenital generalized lipodystrophy.Biochem. Biophys. Res. Commun. 2005; 327: 446-453Google Scholar, 7Dircks L.K. Ke J. Sul H.S. A conserved seven amino acid stretch important for murine mitochondrial glycerol-3-phosphate acyltransferase activity. Significance of arginine 318 in catalysis.J. Biol. Chem. 1999; 274: 34728-34734Google Scholar). Northern blot analysis of human LPAAT-α suggests that it has broad tissue distribution, whereas that of human LPAAT-β is more restricted, primarily to heart, liver, adipose, and pancreas (8West J. Tompkins C.K. Balantac N. Nudelman E. Meengs B. White T. Bursten S. Coleman J. Kumar A. Singer J.W. et al.Cloning and expression of two human lysophosphatidic acid acyltransferase cDNAs that enhance cytokine-induced signaling responses in cells.DNA Cell Biol. 1997; 16: 691-701Google Scholar, 9Stamps A.C. Elmore M.A. Hill M.E. Kelly K. Makda A.A. Finnen M.J. A human cDNA sequence with homology to non-mammalian lysophosphatidic acid acyltransferases.Biochem. J. 1997; 326: 455-461Google Scholar, 10Eberhardt C. Gray P.W. Tjoelker L.W. Human lysophosphatidic acid acyltransferase. cDNA cloning, expression, and localization to chromosome 9q34.3.J. Biol. Chem. 1997; 272: 20299-20305Google Scholar, 11Agarwal A.K. Arioglu E. De Almeida S. Akkoc N. Taylor S.I. Bowcock A.M. Barnes R.I. Garg A. AGPAT2 is mutated in congenital generalized lipodystrophy linked to chromosome 9q34.Nat. Genet. 2002; 31: 21-23Google Scholar). The inherited loss of function of LPAAT-β has also been linked to a rare form of congenital, generalized lipodystrophy, characterized by a nearly complete absence of adipose tissue from birth (12Garg A. Lipodystrophies.Am. J. Med. 2000; 108: 143-152Google Scholar, 13Magre J. Delepine M. Khallouf E. Identification of the gene altered in Berardinelli-Seip congenital lipodystrophy on chromosome 11q13.Nat. Genet. 2001; 28: 365-370Google Scholar). Although membrane-associated LPAAT activities were first described by Kornberg and Pricer (14Kornberg A. Pricer Jr., W.E. Enzymatic esterification of alpha-glycerophosphate by long chain fatty acids.J. Biol. Chem. 1953; 204: 345-357Google Scholar) in 1953, progress has been slow to understand their cellular function(s) and to separately define each isozyme's enzymatic properties. Significant steps could be made if these enzymes were solubilized from the membranes and purified to homogeneity. Unfortunately, attempts to solubilize LPAATs into detergent micelles have resulted in the immediate and complete loss of enzymatic activity. Thus, most information on LPAAT enzymatic properties comes from experiments using cell lysates or membrane preparations containing a recombinant LPAAT isozyme. When the potential acyl donors 14:0-CoA, 16:0-CoA, 18:0-CoA, and 20:4-CoA were tested separately against lysates from COS 7 cells containing human recombinant LPAAT-β, all acyl donors were active. When the potential acyl acceptors sn-1-18:1 lysoPA, sn-1-acyl lysophosphatidylcholine (lysoPC), and lysoplatelet-activating factor (sn-1-alkyl lysoPC) were tested separately, only lysoPA was active (10Eberhardt C. Gray P.W. Tjoelker L.W. Human lysophosphatidic acid acyltransferase. cDNA cloning, expression, and localization to chromosome 9q34.3.J. Biol. Chem. 1997; 272: 20299-20305Google Scholar). A separate study showed that 18:1-CoA was also an acceptable acyl donor (8West J. Tompkins C.K. Balantac N. Nudelman E. Meengs B. White T. Bursten S. Coleman J. Kumar A. Singer J.W. et al.Cloning and expression of two human lysophosphatidic acid acyltransferase cDNAs that enhance cytokine-induced signaling responses in cells.DNA Cell Biol. 1997; 16: 691-701Google Scholar). Studies of human or murine LPAAT-α expressed in JC201, COS 7, or Sf21 cells have shown that it has broad preference for acyl-CoAs and can use lysoPA but not lysoPC, lysophosphatidylethanolamine, lysophosphatidylinositol, or lysoplatelet-activating factor as an acyl acceptor (9Stamps A.C. Elmore M.A. Hill M.E. Kelly K. Makda A.A. Finnen M.J. A human cDNA sequence with homology to non-mammalian lysophosphatidic acid acyltransferases.Biochem. J. 1997; 326: 455-461Google Scholar, 15Yamashita A. Kawagishi N. Miyashita T. Nagatsuka T. Sugiura T. Kume K. Shimizu T. Waku K. ATP-independent fatty acyl-coenzyme A synthesis from phospholipid: coenzyme A-dependent transacylation activity toward lysophosphatidic acid catalyzed by acyl-coenzyme A:lysophosphatidic acid acyltransferase.J. Biol. Chem. 2001; 276: 26745-26752Google Scholar, 16Aguado B. Campbell R.D. Characterization of a human lysophosphatidic acid acyltransferase that is encoded by a gene located in the class III region of the human major histocompatibility complex.J. Biol. Chem. 1998; 273: 4096-4105Google Scholar). In spite of these efforts, the LPAATs' enzymatic properties need a more detailed characterization to fully understand the effect that LPAAT catalysis may have on biological processes requiring PA. To this end, we studied the substrate specificity of LPAAT-β in more detail using Sf9 membrane preparations containing recombinant LPAAT-β and compared it with that of native Sf9 membranes and Sf9 membrane preparations containing recombinant LPAAT-α. We also developed a novel assay to estimate LPAAT-β activity in whole cells. This assay measured the intracellular formation of phosphatidylmethanol (PM) and took advantage of two reagents, lysophosphatidylmethanol (lysoPM) and LPAAT-β chemical inhibitors. LysoPM was shown to be an acyl acceptor for LPAAT-β but not LPAAT-α, and unlike lysoPA, it readily traversed cell membranes. The LPAAT-β chemical inhibitors show submicromolar potency against LPAAT-β but do not inhibit LPAAT-α even at 40 μM and were used to help differentiate the activity of the two isozymes. We used this second assay to test whether the LPAAT-β acyl chain transfer specificity observed in the membrane assay was reciprocated in cells. 14C-labeled 18:1, 14C-labeled 20:4, 14C-labeled 18:1-CoA, 3H-labeled sn-1-18:1 lysoPA, and γ-33P-labeled ATP were from Perkin-Elmer; 33P-labeled sn-1-18:1 lysoPA was prepared using E. coli diacylglycerol kinase (Calbiochem) to phosphorylate 1-monoolein (Doosan Serdery Research Laboratories) with γ-33P-labeled ATP as described (17Hollenback D. Glomset J.A. On the relation between a stearoyl-specific transacylase from bovine testis membranes and a copurifying acyltransferase.Biochemistry. 1998; 37: 363-376Google Scholar); all other lipids were from Avanti Polar Lipids. Silica Gel 60 HP-TLC plates were from Analtech. Organic solvents were American Chemical Society grade or better from J. T. Baker or EM Science. The synthesis of compounds CT-32228 and CT-32212 (18Bhatt R. Gong B. Hong F. Jenkins S.A. Klein J.P. Kumar A.M. Tulinsky J. PCT Int. Appl. WO 03/037346. 2003; Google Scholar) and of compound CT-32501 (19Bhatt R. Gong B. Hong F. Jenkins S.A. Klein J.P. Kohm C.T. Tulinsky J. US Patent Appl. 20040204386. 2004; Google Scholar) were as described. Anti-LPAAT-α and anti-LPAAT-β antibodies have been described (20Bonham L. Leung D.W. White T. Hollenback D. Klein P. Tulinsky J. Coon M. de Vries P. Singer J.W. Lysophosphatidic acid acyltransferase-beta: a novel target for induction of tumour cell apoptosis.Expert Opin. Ther. Targets. 2003; 7: 643-661Google Scholar). All other chemicals were of reagent grade from Sigma. sn-1-18:1 lysoPM was synthesized from sn-1-18:1 lysoPA essentially as described (21Jain M.K. Gelb M.H. Phospholipase A2-catalyzed hydrolysis of vesicles: uses of interfacial catalysis in the scooting mode.Methods Enzymol. 1991; 197: 112-125Google Scholar). One gram of the protonated form of sn-1-18:1 lysoPA was dissolved in 50 ml of chloroform and mixed with an ethereal solution of diazomethane until a yellow color persisted. The solvent was evaporated, and the dimethylated lysoPA was mixed with 2.0 g of NaI in anhydrous 2-butanone and then refluxed for 90 min. The solvent was removed, and the lysoPM was purified by flash chromatography as the sodium salt by eluting with chloroform-methanol-acetic acid-water (50:30:15:5). The solvent was evaporated, and the lysoPM was reconstituted in chloroform. This yielded 800 mg of lysoPM, whose structure and purity (>95%) were confirmed by TLC, LC-MS, and NMR. Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. PC-3, a human prostate tumor cell line (American Type Culture Collection; CRL-1435), was cultured in Ham's F12K medium supplemented with 2 mM l-glutamine, 1.5 g/l sodium bicarbonate, and 10% fetal bovine serum. DU145, a human prostate tumor cell line (American Type Culture Collection; HTB-81), was cultured in Eagle's minimum essential medium supplemented with 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, and 10% fetal bovine serum. For the overexpression of LPAATs in these cells, human LPAAT-β cDNA and human LPAAT-α cDNA were cloned separately into a modified version of the LXSN retroviral vector (Clontech) (22Miller A.D. Rosman G.J. Improved retroviral vectors for gene transfer and expression.Biotechniques. 1989; 7 (989): 980-986Google Scholar). The Moloney murine leukemia virus long terminal repeat drives LPAAT expression, and the SV40 promoter drives the selectable marker (neo) expression. As a further modification of the vector, a recognition site for cre recombinase (23Sauer B. Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae.Mol. Cell. Biol. 1987; 7: 2087-2096Google Scholar) was introduced into the long terminal repeat regions. Control vectors containing alkaline phosphatase cDNA, green fluorescent protein (GFP) cDNA, or vector alone were also produced using the same method. The retroviral packaging cell lines for LPAAT-β, LPAAT-α, and control vectors were produced and characterized as described (24Miller A.D. Miller D.G. Garcia J.V. Lynch C.M. Use of retroviral vectors for gene transfer and expression.Methods Enzymol. 1993; 217: 581-599Google Scholar). Clones producing high-titer virus (0.5–1 × 106 G418-resistant colony-forming units/ml) were used for the transduction of PC-3 and DU145 cells in their respective media; clonal cell lines were subsequently generated. None of the transduced cells exhibited any gross morphological or proliferative alterations. The magnitude of overexpression of each recombinant LPAAT was determined by assaying total endogenous LPAAT activity in cell lysates essentially as described (25Hideshima T. Chauhan D. Hayashi T. Podar K. Akiyama M. Mitsiades C. Itsiades N.M. Gong B. Bonham L. de Vries P. et al.Antitumor activity of lysophosphatidic acid acyltransferase-beta inhibitors, a novel class of agents, in multiple myeloma.Cancer Res. 2003; 63: 8428-8436Google Scholar). Briefly, transduced PC-3 and DU145 cells were harvested by trypsinization, suspended in 400 μl of ice-cold PBS, and probe-sonicated with a Branson Sonifier at output = 2, duty cycle = 2 for 10 pulses. The samples were centrifuged at 1,500 g for 2 min at 4°C to obtain the supernatants, which were quantified for protein. The LPAAT activity in these supernatants was estimated during a 7 min incubation at 37°C in 12 × 75 mm silanized, borosilicate glass test tubes containing 50 μl of 50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mg/ml fatty acid-free BSA, 0.2 mM 14C-labeled 18:1-CoA (∼40,000 cpm/assay), 0.2 mM sn-1-18:1 lysoPA, and 0.005–0.03 mg/ml protein; the reaction was quenched with organic solvent. The 14C-labeled PA was resolved by TLC on Silica Gel 60 plates, and the density of radioactive bands was quantitated by exposing the plate to a phosphor screen overnight and scanning the phosphor screen with a Storm 840 PhosphorImager (GE Healthcare Life Sciences) as described (26White T. Bursten S. Federighi D. Lewis R.A. Nudelman E. High-resolution separation and quantification of neutral lipid and phospholipid species in mammalian cells and sera by multi-one-dimensional thin-layer chromatography.Anal. Biochem. 1998; 258: 109-117Google Scholar). The increase in total LPAAT activity achieved in PC-3 cells was typically 5- to 20-fold for LPAAT-α and 20- to 45-fold for LPAAT-β. One clone from PC-3 cells containing recombinant LPAAT-β was used frequently in this study and is referred to as PC-3/β1 (or β1 in Fig. 3D). The increase in total LPAAT activity achieved in DU145 cells typically was 2- to 5-fold for LPAAT-α and 4- to 10-fold for LPAAT-β.Fig. 3.Acylation of sn-1-18:1 lysoPM with 14C-labeled 18:1 in PC-3 cells: effects of time, lysoPM concentration, and total LPAAT activity. A: Separate wells containing wild-type (wt) PC-3 cells or PC-3 cells transduced with LPAAT-β (β1), LPAAT-α (α1), or green fluorescent protein (GFP) cDNAs were incubated with 14C-labeled 18:1 (220,000 cpm) and the indicated concentrations of sn-1-18:1 lysoPM and CT-32228, as described in Experimental Procedures. After lipid extraction, TLC, and exposure of the plate to a phosphor screen (see Experimental Procedures), the screen was scanned with a Storm PhosphorImager in phosphor mode. The migration of lipid standards is indicated: NL, neutral lipids; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI/PS, phosphatidylinositol/phosphatidylserine; ori, origin. B: Effect of lysoPM concentration on PM formation. PC-3 (squares) and PC-3/β1 (circles) cells were incubated (40 min, 37°C) in the presence of 0.2 μCi of 14C-labeled 18:1 and the indicated concentrations of sn-1-18:1 lysoPM. Samples were processed as described for A, and 14C-labeled PM was quantitated and normalized to the total radioactivity detected in the lane. Results are representative of two experiments performed in triplicate. Error bars in all panels indicate SD. C: Rate of PM formation. PC-3 (squares) and PC-3/β1 (circles) cells were incubated in the presence of 50 μM sn-1-18:1 lysoPM and 0.2 μCi of 14C-labeled 18:1 for the indicated times at 37°C. Samples were processed and radioactivity quantitated as described for B, except that the data for this panel are expressed in cpm PM, because PM formation expressed as the percentage of the lane did not change over time. Results are representative of two experiments performed in triplicate. D: Total endogenous LPAAT activity in PC-3 wild-type (WT) cells and PC-3 cells transduced with retroviral vectors containing no transcript (X1 and X2), GFP (GFP1, GFP2, GFP3), LPAAT-α (α1, α2, α3), or LPAAT-β (β1, β2, β3) cDNA was measured with the LPAAT activity assay in cell lysates (see Experimental Procedures). For reference, total LPAAT activity in the lysates from wild-type cells was 18 ± 5 (n = 8) nmol PA formed/min/mg protein. E: Effect of the overexpression of LPAAT-β or LPAAT-α on 14C-labeled PM formation in PC-3 cells. PC-3 wild-type cells or cells transduced as described for D were incubated separately (40 min, 37°C) in the presence of 0.4 μCi of 14C-labeled 18:1 and 50 μM sn-1-18:1 lysoPM (black bars). In some cases, 10 μM of the LPAAT-β inhibitor CT-32228 was added to the cells 12 min before the addition of the 14C-labeled 18:1 and the lysoPM (white bars). Approximately 4% of the total radioactivity was incorporated into the lipid fraction for each of the clones. Results are representative of two experiments performed in duplicate. Note that β1 described in A, D, and E refers to the PC-3/β1 clonal cell line.View Large Image Figure ViewerDownload (PPT)Fig. 3.Acylation of sn-1-18:1 lysoPM with 14C-labeled 18:1 in PC-3 cells: effects of time, lysoPM concentration, and total LPAAT activity. A: Separate wells containing wild-type (wt) PC-3 cells or PC-3 cells transduced with LPAAT-β (β1), LPAAT-α (α1), or green fluorescent protein (GFP) cDNAs were incubated with 14C-labeled 18:1 (220,000 cpm) and the indicated concentrations of sn-1-18:1 lysoPM and CT-32228, as described in Experimental Procedures. After lipid extraction, TLC, and exposure of the plate to a phosphor screen (see Experimental Procedures), the screen was scanned with a Storm PhosphorImager in phosphor mode. The migration of lipid standards is indicated: NL, neutral lipids; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI/PS, phosphatidylinositol/phosphatidylserine; ori, origin. B: Effect of lysoPM concentration on PM formation. PC-3 (squares) and PC-3/β1 (circles) cells were incubated (40 min, 37°C) in the presence of 0.2 μCi of 14C-labeled 18:1 and the indicated concentrations of sn-1-18:1 lysoPM. Samples were processed as described for A, and 14C-labeled PM was quantitated and normalized to the total radioactivity detected in the lane. Results are representative of two experiments performed in triplicate. Error bars in all panels indicate SD. C: Rate of PM formation. PC-3 (squares) and PC-3/β1 (circles) cells were incubated in the presence of 50 μM sn-1-18:1 lysoPM and 0.2 μCi of 14C-labeled 18:1 for the indicated times at 37°C. Samples were processed and radioactivity quantitated as described for B, except that the data for this panel are expressed in cpm PM, because PM formation expressed as the percentage of the lane did not change over time. Results are representative of two experiments performed in triplicate. D: Total endogenous LPAAT activity in PC-3 wild-type (WT) cells and PC-3 cells transduced with retroviral vectors containing no transcript (X1 and X2), GFP (GFP1, GFP2, GFP3), LPAAT-α (α1, α2, α3), or LPAAT-β (β1, β2, β3) cDNA was measured with the LPAAT activity assay in cell lysates (see Experimental Procedures). For reference, total LPAAT activity in the lysates from wild-type cells was 18 ± 5 (n = 8) nmol PA formed/min/mg protein. E: Effect of the overexpression of LPAAT-β or LPAAT-α on 14C-labeled PM formation in PC-3 cells. PC-3 wild-type cells or cells transduced as described for D were incubated separately (40 min, 37°C) in the presence of 0.4 μCi of 14C-labeled 18:1 and 50 μM sn-1-18:1 lysoPM (black bars). In some cases, 10 μM of the LPAAT-β inhibitor CT-32228 was added to the cells 12 min before the addition of the 14C-labeled 18:1 and the lysoPM (white bars). Approximately 4% of the total radioactivity was incorporated into the lipid fraction for each of the clones. Results are representative of two experiments performed in duplicate. Note that β1 described in A, D, and E refers to the PC-3/β1 clonal cell line.View Large Image Figure ViewerDownload (PPT) PM synthesis was estimated in cultured cells by quantitating the conversion of exogenously added lysoPM and 14C-labeled fatty acid to 14C-labeled PM during a 35 min incubation at 37°C. PC-3 and DU145 cells, containing a recombinant protein as indicated, were separately plated onto 96-well plates in 500 μl of the appropriate medium (see above) and grown to 70–80% confluence. On the day of the assay, the medium volume was adjusted to 96 μl. The lysoPM and 14C-labeled fatty acid stocks were prepared separately by drying each solvent under a stream of nitrogen and resuspending the lysoPM in PBS and the 14C-labeled fatty acid in PBS + 20 mg/ml fatty acid-free BSA. The assay was performed by incubating 2 μl of the lysoPM stock with the cells for 5 min, adding 2 μl of the 14C-fatty acid stock, and incubating for an additional 30 min. The final concentrations were 50 μM sn-1-18:1 lysoPM and 0.2 μCi of 14C-labeled 18:1 in a total volume of 100 μl, unless stated otherwise. Background PM levels were determined by adding PBS without lysoPM. The assay was terminated by removing the incubation mixture, washing the cells three times with ice-cold PBS, discarding the final wash, and then adding 0.25 ml of methanol. The plates were gently swirled, and the methanol was transferred to glass test tubes. This method extracted >98% of the PM from the wells (data not shown). Carrier 18:1-18:1 PM (4 μg), 1.0 ml of chloroform, 0.25 ml of methanol, and 0.25 ml of 0.2 M H3PO4 were added to each tube. The tubes were vortexed for 10 s and centrifuged for 2 min at 500 g to clarify the phases; the upper phase was discarded. The lower phase was dried under a stream of nitrogen, and the sample was resuspended in 100 μl of chloroform-methanol (2:1). 14C-labeled PM was typically resolved by TLC in chloroform/methanol/NH4OH (65:30:4). To help confirm the identity of the PM, in some instances a two-solvent system was also used in which the TLC plates were first developed in chloroform/methanol/NH4OH (70:25:3), dried thoroughly, and then developed in chloroform-methanol-acetic acid-water (85:10:8:1). The density of radioactive bands was scanned and quantified with a Storm PhosphorImager as described above. Unless stated otherwise, 14C-labeled PM formation is expressed as a percentage of the lane: the pixel volume of the PM region ÷ the pixel volume of the entire lane × 100, where pixel volume is the signal intensity calculated with the Storm PhosphorImager and is directly proportional to the amount of radioactivity. This calculation provided a relative rate of esterification of radioactive fatty acid into lysoPM versus the entire lipid fraction and served as a loading control. For experiments using LPAAT-β chemical inhibitors, the inhibitors were diluted from a DMSO stock into PBS + 4 mg/ml BSA. The volume of medium was adjusted to 94 μl, and 2 μl of inhibitor stock or excipient alone was added to the cells 5 min before the addition of the lysoPM stock. The final concentration of DMSO was 0.01% (v/v) and did not affect the outcome of the assay (data not shown). IC50 values were determined using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego CA; www.graphpad.com). Other changes in the assay are noted in the text and figure legends. For the construction of Baculovirus expression vectors, the full-length human LPAAT-α and LPAAT-β cDNAs were amplified by PCR from the DNA templates pCE9.LPAAT-α and pCE9. LPAAT-β (8West J. Tompkins C.K. Balantac N. Nudelman E. Meengs B. White T. Bursten S. Coleman J. Kumar A. Singer J.W. et al.Cloning and expression of two human lysophosphatidic acid acyltransferase cDNAs that enhance cytokine-induced signaling responses in cells.DNA Cell Biol. 1997; 16: 691-701Google Scholar) using the primers 5′-TGATATCCGAAGAAGATCTTATGGATTTGTGGCCAGGGGC-3′ (olpa1F) and 5′-CAGGCTCTAGATCACCCACCGCCCCCAGGCTTC-3′ (olpa1R) for LPAAT-α and the primers 5′-TGATATCCGAAGAAGATCTTATGGAGCTGTGGCCGTGTC-3″ (olpb1F) and 5′-CAGGCTCTAGACTACTGGGCCGGCTGCAC-3′ (olpb1R) for LPAAT-β. The ∼870 bp fragments generated were reamplified by PCR using the primers 5′-CCTACGTCGACATGGAACAAAAATTGATATCCGAAGAAGATC-3′ (olp2F) and olpa1R for LPAAT-α and the primers olp2F and olpb1R for LPAAT-β. The ∼890 bp fragments generated were then cleaved with SalI and XbaI for insertion into pFastBac™ HTc vector (Invitrogen) between the SalI and XbaI sites. The plasmids pFB.LPAAT-α and pFB.LPAAT-β were further modified by cleaving them with RsrII and SalI, converting the cohesive ends to blunt ends with Klenow DNA polymerase and deoxynucleoside triphosphate and recircularizing by ligase to remove the polyhistidine tag coding region. These plasmids were then transformed into E. coli DH10Bac™ (Invitrogen) for the generation of recombinant Bacmid DNAs for transfection into Sf9 insect cells for the production of recombinant Baculovirus stocks using the protocol described in the Bac-to-Bac® Baculovirus Expression System (Invitrogen), a eukaryotic expression system for generating recombinant Baculovirus through site-specific transposition in E. coli. Viral stocks harvested from the transfected cells were then used to infect fresh insect cells for the subsequent expression of LPAAT-α and LPAAT-β proteins. All steps were performed at 0–4°C. Sf9 cell pellets (∼108 cells) containing recombinant LPAAT were thawed and resuspended in 5 ml of buffer A [20 mM HEPES, pH 7.5, 1 mM DTT, 1 mM EDTA, 20% (w/v) glycerol, 1 mM benzamidine, 1 μg/ml soybean trypsin inhibitor, and 1 μg/ml pepstatin A] with 1 mM Pefabloc. The cells were lysed by sonication using a Branson Sonifier at output = 2, duty cycle = 2, 10 pulses each for 10 s. DTT was then added to 1 mM from a 1 M stock. The samples were centrifuged at 1,500 rpm for 5 min. The resulting supernatant was centrifuged at 100,000 g for 1 h. The resulting pellet, which contained the Sf9 membranes, was resuspended in buffer A + 1 mM DTT and stored at −70°C. LPAAT-β and LPAAT-α activities were stable in this form for >2 years. Enzyme assays were performed in 12 × 75 mm silanized, borosilicate glass test tubes at 37°C for 3–10 min. For the estimation of LPAAT activity in Sf9 membrane preparations, typical assay mixtures (300 μl) consisted of 251 μl of buffer B (50 mM HEPES, pH 7.5, 1 mM EDTA, and 100 mM NaCl), 25 μl of enzyme in buffer B + 10% glycerol, 12 μl of 14C-labeled 18:1-CoA (40,000–100,000 cpm), and 12 μl of sn-1-18:1 lysoPA. To prevent the nonspecific binding of substrates to the tubes, stock solutions of each substrate were prepared separately in DMSO. The final concentration of DMSO in all assays" @default.
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