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acyl-[acyl-carrier-protein] synthetase from Escherichia coli
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[13] 2 - A c y l g l y c e r o p h o s p h o e t h a n o l a m i n e A c y l t r a n s f e r a s e / A c y l - [ A c y l - C a r r i e r - P r o t e i n ] S y n t h e t a s e f r o m E s c h e r i c h i a coli By SUZANNE JACKOWSKI, LI HSU, and CHARLES O. ROCK
Introduction 2-Acylglycerophosphoethanolamine (2-acyl-GPE) acyltransferase is a membrane-bound enzyme that either activates fatty acids for acyl transfer in the presence of ATP and Mg2÷ or transfers fatty acids from acyl-acyl carrier protein (ACP) to the 1-position of lysophospholipids. 1-3 2-AcylGPE acyltransferase is a heterodimer composed of a hydrophobic membrane-bound subunit and ACP. 4 The catalytic cycle of the acyltransferase/ synthetase is shown in Fig. 1. The first step is the ligation of a fatty acid to the 4'-phosphopantetheine sulfhydryl group of the ACP subunit. This reaction requires ATP and Mg2÷, and AMP and pyrophosphate are the products) The bound acyl-ACP remains associated with the complex in vivo, since exogenous fatty acids activated by this enzyme are not made available to other intracellular enzymes that utilize acyl-ACP. 3'4 However, in vitro, the presence of high salt concentrations in the assay mixture lowers the affinity of the acyltransferase subunit for ACP, leading to the dissociation and accumulation of acyl-ACP.4 This property forms the basis of the acyI-ACP synthetase activity measurement, 6 and it is clear that the acyltransferase and synthetase are dual catalytic activities of the same protein. The second step in the acyltransferase reaction is the transfer of the acyl moiety from ACP to the 2-acyl-GPE substrate to form phosphatidylethanolamine (PtdEtn). The 2-acyl-GPE acyltransferase is responsible for a number of physiological processes. The primary function of the enzyme system is to acylate 2-acyl-GPE that arises from the transfer of 1-position fatty acids to outer membrane lipoproteins or from the degradation of membrane phospholipids by phospholipase A1.3,7 The acyltransferase is also responsible for the 1 S. S. Taylor and E. C. Heath, J. Biol. Chem. 244, 6605 (1969). z H. Homma, M. Nishijima, T. Kobayashi, H. Okuyama, and S. Nojima, Biochim. Biophys. Acta 508, 165 (1981). 3 C. O. Rock, J. Biol. Chem. 259, 6188 (1984). 4 C. L. Cooper, L. Hsu, S. Jackowski, and C. O. Rock, J. Biol. Chem. 264, 7384 (1989). s T. K. Ray and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. U.S.A. 73, 4374 (1976). 6 C. O. Rock and J. E. Cronan, Jr., J. Biol. Chem. 254, 7116 (1979). 7 S. Jackowski and C. O. Rock, J. Biol. Chem. 261, 11328 (1986).
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, inc. All rights of reproduction in any form reserved.
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PtdE~AcyI-GPE Enz+ / ~ ACPSH "---7
ATP + Fatty Acid
Enz ,/ Enz+ Acy-ACP~7 AcyI-ACP
AMP +
PPi
FIG. 1. Catalytic cycle of 2-acyi-GPE acyltransferase/acyl-ACPsynthetase.
acyl-CoA-independent uptake and incorporation of exogenous fatty acids and lysophospholipids into the membrane. 8'9
Assay Method Principle. The assay method for 2-acyl-GPE acyltransferase is based on measuring the formation of [14C]PtdEtn from 2-acyl-GPE and [14C]palmitic acid in the presence of ATP, Mg 2+ , and ACP. The reaction is terminated by applying an aliquot of the incubation mixture to the preadsorbant layer of a thin-layer chromatography plate, and, following development to separate unreacted fatty acid from PtdEtn, the amount of [14C]PtdEtn formed is quantitated by scintillation counting. The acyltransferase/synthetase may also be detected using the acyl-ACP synthetase assay. 5'6'I° The acyl-ACP synthetase assay technique is easier to perform and faster than the acyltransferase assay described in this chapter, but it measures a nonphysiological, low specific activity side reaction of the acyltransferase rather than its normal catalytic activity. 4 Reagents. [14C]Palmitic acid (specific activity - 5 5 Ci/mol) from any supplier is suitable. Purified (oxidant-free) Triton X-100, ATP, octyl-/3-Dglucoside, and Rh&opus arrhizus lipase are purchased from Boehringer Mannheim (Indianapolis, IN). Escherichia coli PtdEtn is purchased from Serdary Research Laboratories (London, ON, Canada). ACP is purified
8 c. o. Rock and S. Jackowski, J. Biol. Chem. 26@~127200985). 9 L. Hsu, S. Jackowski, and C. O. Rock, J. Bacteriol. 171, 1203(1989). 10See this series, Vol. 71 [21].
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TABLE I REAGENTS AND STOCK SOLUTIONS FOR 2-ACYLGLYCEROPHOSPHOETHANOLAMINE ACYLTRANSFERASE ASSAY Reagent
Microliters/assay
Final concentration
Tris-HC1, 1 M, pH 8.0 ATP, 0.1 M, pH 7.0 MgCI2, 0.1 M ACP, 100 p,M Dithiothreitol, 40 mM 2-AcyI-GPE, a 1 mM [14C]Palmitic acid, a 1 mM Protein in 2% Triton X-100
4 2 2 4 2 4 2 20
0.1 M 5 mM 5 mM 10 p.M 2 mM 100/zM 50/xM 0-10 mU
a These solutions are prepared in 2% Triton X-100.
as described in an earlier volume in this series. 11 The stock solutions required for the assay are listed in Table I and can be stored at - 20° for months. The exception is dithiothreitol, which should be prepared daily. Preparation of2-Acyl-GPE Substrate. 2-AcyI-GPE is prepared by the digestion of E. coli PtdEtn essentially as described by Homma and Nojima. 12 PtdEtn (4 /zmol) is added to a screw-top tube and the solvent evaporated. Next, 1.6 ml of 50 mM Bis-Tris, pH 5.6, is added, and the PtdEtn is suspended by two 10-sec rounds of sonication. Then, 0.2 ml of 100 mM CaCI 2 , 0.2 ml of 2 mg/ml R. arrhizus lipase, and 0.2 ml of diethyl ether are added. The mixture is incubated at 25 ° for 3 hr with shaking. Methanol (2 ml) is added to stop the reaction and the mixture extracted twice with 4 ml of hexane to remove the fatty acid. Next, 2.2 ml of chloroform, 0.5 ml methanol, and 0.2 ml of 0.5 M citric acid are added. The 2-acyl-GPE partitions into the bottom phase, which is collected, the solvent removed, and the 2-acyl-GPE suspended in chloroform-methanol (I : 1, v/v). 2-Acyl-GPE concentrations are determined by the spectrophotometric assay of Stewart ~3 using E. coli PtdEtn as a standard. 2-AcylGPE is aliquoted and stored under nitrogen at - 2 0 °. Stocks of 2-acylGPE should not be stored for longer than 1 week because by this time a significant fraction of the acyl groups have migrated to the 1-position and 2-acyl-GPE acyltransferase will not acylate 1-acyl-GPE. Method. The number of assays to be performed is determined, and the appropriate volumes of the assay regents (Table I) are combined. To each 11 See this series, Vol. 71 [41]; see also C. O. Rock and J. E. Cronan, Jr., Anal. Biochem. 102, 362 (1980). 12 H. Homma and S. Nojima, J. Biochem. (Tokyo) 91, 1103 (1982). 13 j. C. M. Stewart, Anal. Biochem. 104, 10 (1980).
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10 × 75 mm glass assay tube, 20/zl of the reaction mixture is added, and the reaction is initiated by the addition of 20/zl of the protein solution in 2% Triton X-100. The tubes are briefly vortex mixed and placed in a 37 ° water bath for 10 min. Incubations are terminated by the addition of 0.2 ml of ethanol. The mixture is evaporated to dryness under a stream of nitrogen, resuspended in chloroform-methanol (1 : 1, v/v), and the entire sample applied to the preadsorbant layer of a silica gel G plate. The thin-layer plate is developed with chloroform-methanol-acetic acid (85 : 15 : 10, v/v), and the areas corresponding to PtdEtn and fatty acid are located by comparison with standards chromatographed in one lane of the same plate. These two areas are removed, and the amount of radioactivity incorporated into PtdEtn is determined by scintillation counting. Counting the fatty acid area is a useful control to confirm that the amount of radioactivity recovered from the thin-layer plate corresponds to the amount of carbon-14 initially added to the assay. Results are expressed as nanomoles per minute per milligram protein. Purification
Initial Purification Steps. The initial steps in the purification of 2-acylGPE acyltransferase/acyl-ACP synthetase are identical to the procedure outlined by Rock and Cronan for the purification of acyl-ACP synthetase activity. 6'1°All procedures are performed at 4° unless otherwise indicated. The cells are disrupted by passage through a French pressure cell at 16,000 psi. The homogenate is then centrifuged at 15,000 g for 20 min to remove debris and unbroken cells, and the pellet is discarded. The supernatant is adjusted to 10 mM in MgCI2 by the addition of the appropriate volume of 1 M MgCI 2. The suspension then is sedimented at 80,000 g for 90 min, and the supernatant is discarded. The resulting pellets are homogenized thoroughly in 150 ml of 50 mM Tris-HC1, pH 8.0, and then 150 ml of 50 mM Tris-HC1, pH 8.0, containing I M NaC1, and 20 mM MgC12 is added to the membrane suspension. The solution is gently stirred for 15 min, and the membranes are sedimented at 80,000 g for 90 min. The membrane pellets from the salt wash are homogenized thoroughly in 100 ml of 50 mM Tris-HCl, pH 8.0, and to this suspension I00 ml of 50 mM Tris-HCl, pH 8.0, containing 4% Triton X-100 and 20 mM MgCI 2 is added, and the solution is gently stirred for 30 min. The suspension then is centrifuged at 80,000 g for 90 min to remove unsolubilized material, and the Triton X-100 supernatant is saved. The Triton X-100 extract (200 ml) is then adjusted to 5 mM ATP by the addition of 20 ml of 0.1 M ATP in 50 mM Tris-HC1, pH 8.0. The protein concentration should be between 2 and 5 mg-ml. The Triton X-100 extract
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is then placed in a 37° water bath, stirred until the temperature reaches 33°, and then placed in a 55 ° water bath and stirred on ice and centrifuged at 15,000 g for 20 min to remove denatured proteins. Blue-Sepharose Chromatography. A column (1.2 x 35 cm) is packed with Blue-Sepharose CL-6B and equilibrated with 50 mM Tris-HCl, pH 8.0, containing 2% Triton X-100 at 4°. The supernatant from the heat step is applied to the column. The column is then rinsed with 6 column volumes of 50 mM Tris-HCl, pH 8.0, containing 2% Triton X-100, followed by 6 column volumes of the same buffer containing 0.6 M NaCI. The majority of the protein adsorbed to the Blue-Sepharose column is eluted with 0.6 M NaCI, and acyltransferase/synthetase activity is desorbed from the column by elution with 0.5 M KSCN in 50 mM Tris-HC1, pH 8.0, 2% Triton X-100. ATP-Charged DEAE-Cellulose Chromatography. The final step in the procedure is chromatography on ATP-charged DEAE-cellulose. A 17-ml column of Whatman (Clifton, N J) DE-52 is packed and equilibrated in 50 mM Tris-HCl, pH 8.0, 2% Triton X-100. The column is then washed with 50 ml of 5 mM ATP in the same buffer and excess nucleotide removed by washing with buffer without ATP. The dialyzed KSCN eluate from the Blue-Sepharose step is loaded on the ATP-charged Whatman DE-52 column and the column washed with the column buffer. In the case where the samples are to be analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis, the column is exchanged in 50 mM Tris-HCl, pH 8.0, 30 mM octyl-fl-D-glucoside. Activity is eluted with 0.5 M NaC1 in either octyl/3-o-glucoside or Triton X-100 buffer. Comments on Purification. The results of the purification procedure are presented in Table II. Throughout the purification procedure, the ratio of 2-acyl-GPE acyltransferase to acyl-ACP synthetase activity remains TABLE II PURIFICATION OF 2-ACYLGLYCEROPHOSPHOETHANOLAMINE ACYLTRANSFERASE[ ACYL-[AcYL-CARRIER-PROTEIN] SYNTHETASE
Purification step
2-Acyl-GPE acyltransferase (nmol/min/mg)
Acyl-ACP synthetase (nmol/min/mg)
Ratio
Recovery (%)
Membrane s Washed membranes Triton extract Heat supernatant Blue- Sepharose DEAE-ceUulose
0.300 0.437 0.958 1.73 50.6 102.4
0.092 0.117 0.273 0.490 16.24 28.4
3.26 3.74 3.51 3.53 3.12 3.61
100 67.8 48.5 45.8 9.9 7.7
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constant. The specific activities of the preparations for 2-acyl-GPE acyltransferase are consistently 10-fold higher than the specific activity of acylACP synthetase. At stages of purification prior to the Blue-Sepharose chromatography, 2-acyl-GPE acyltransferase activity is not dependent on the addition of ACP to the assay. However, the conditions of the BlueSepharose step dissociate the ACP subunit from the acyltransferase, and, after this step, acyltransferase activity becomes completely dependent on the addition of ACP to the assay. This purification procedures results in the 3000-fold purification of 2-acyl-GPE acyltransferase/acyl-ACP synthetase in 7.7% yield. SDS gel electrophoresis of the final product shows a single band with an apparent molecular weight of 27,000. Properties 2-Acyl-GPE acyltransferase/acyl-ACP synthetase is an inner membrane enzyme that is not subject to catabolite repression, and it is genetically and biochemically distinct from acyl-CoA synthetase and glycerolphosphate and 1-acylglycerol-phosphate acyltransferases. The enzyme specifically catalyzes the transfer of fatty acids to the 1-position; l-acylGPE is not a substrate. The enzyme acylates 2-acylglycerophosphocholine as readily as 2-acyl-GPE but does not acylate 2-acylglycerophosphoglycerol. The enzyme prefers saturated fatty acids (palmitate and myristate) as substrates and is less active on shorter (decanoate) and unsaturated (oleate) fatty acids. Poor substrates have both a higher Km and lower Vmax . The acyltransferase binds ACP with high affinity (Kd --60 nM) and catalyzes the acyltransferase reaction without the dissociation of ACP or acyl-ACP from the enzyme. ACP is dissociated from the protein complex by exposure to high salt. This property accounts for the accumulation of acyl-ACP in assays performed in the presence of 0.4 M LiCI and explains the acyl-ACP synthetase activity expressed by the acyltransferase. There is little doubt that the protein functions only as an acyltranferase in v i v o and does not catalyze the formation ofacyl-ACP that can be made available to other proteins. Genetics and Molecular Biology A replica print procedure was used to isolate mutants that lacked both 2-acyl-GPE acyltransferase and acyl-ACP synthetase activities. This mutation maps to the 61-rain region of the E . c o l i chromosome, and the allele is designated a a s . ~4 The a a s mutants were then employed to isolate 14 L. Hsu, S. Jackowski, and C. O. Rock, J. Biol. Chem. 266, 13783 (1991).
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plasmids that carry the a a s gene. One such plasmid, pLCH3, contains the full a a s coding sequence, and strains that harbor this plasmid possess 10fold higher specific activities of both 2-acyl-GPE acyltransferase and acylACP synthetase) 4 Thus, strains harboring this plasmid are an excellent source for purifying the acyltransferase/synthetase either for biochemical characterization or in routine preparation of the enzyme for the synthesis of acyl-ACPs. Synthesis of AcyI-ACP Although the acyltransferase/synthetase functions only as an acyltransferase in vivo, its ability to synthesize acyl-ACP in vitro has made it a useful tool for the preparation of defined acyl-ACPs to be used as substrates for other enzymes. 15 Our laboratory still uses the techniques outlined in a previous volume in this series 15 to synthesize acyl-ACP using the acyltransferase/synthetase, but there is a useful modification introduced by others 16: that warrants consideration. These investigators 16,17have taken advantage of the tight binding of the acyltransferase to Blue-Sepharose and the fact that the synthetase remains active when coupled to the resin. 6,16: The enzymatically active column is used directly to catalyze the formation of acyl-ACP by the continuous cycling of a reaction mixture ( - 6 0 ml) over the column for 24 hr with a peristaltic pump at 4°. 17 The reaction mixture contains 0.6 mg/ml ACP, 0.1 M TrisHCI, pH 8.0, 0.4 M LiCI, 10 mM ATP, 10 mM MgCI2,2 mM dithiothreitol, 0.07% Triton X-100, and 100/xM of the desired fatty acid) 7 Yields of acyl-ACP up to 90% or greater have been reported, and the acyl-ACP synthetase/Blue Sepharose column remains stable for at least 4 months at 4oC. 17
Acknowledgments This work was supported by National Institutes of Health Grant GM 28035, Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities. We thank Pare Jackson for excellent technical assistance.
t5 See this series, Vol. 72 [27]; see also C. O. Rock and J. L. Garwin, J. Biol. Chem. 254, 7123 (1979). 16 p. R. Green, A. H. Merrill, Jr., and R. M. Bell, J. Biol. Chem. 256, 11151 (1981). 17 M. S. Anderson, C. E. Bulawa, and C. R. H. Raetz, J. Biol. Chem. 260, 15536 (1985).
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[13] 2 - A c y l g l y c e r o p h o s p h o e t h a n o l a m i n e A c y l t r a n s f e r a s e / A c y l - [ A c y l - C a r r i e r - P r o t e i n ] S y n t h e t a s e f r o m E s c h e r i c h i a coli By SUZANNE JACKOWSKI, LI HSU, and CHARLES O. ROCK
Introduction 2-Acylglycerophosphoethanolamine (2-acyl-GPE) acyltransferase is a membrane-bound enzyme that either activates fatty acids for acyl transfer in the presence of ATP and Mg2÷ or transfers fatty acids from acyl-acyl carrier protein (ACP) to the 1-position of lysophospholipids. 1-3 2-AcylGPE acyltransferase is a heterodimer composed of a hydrophobic membrane-bound subunit and ACP. 4 The catalytic cycle of the acyltransferase/ synthetase is shown in Fig. 1. The first step is the ligation of a fatty acid to the 4'-phosphopantetheine sulfhydryl group of the ACP subunit. This reaction requires ATP and Mg2÷, and AMP and pyrophosphate are the products) The bound acyl-ACP remains associated with the complex in vivo, since exogenous fatty acids activated by this enzyme are not made available to other intracellular enzymes that utilize acyl-ACP. 3'4 However, in vitro, the presence of high salt concentrations in the assay mixture lowers the affinity of the acyltransferase subunit for ACP, leading to the dissociation and accumulation of acyl-ACP.4 This property forms the basis of the acyI-ACP synthetase activity measurement, 6 and it is clear that the acyltransferase and synthetase are dual catalytic activities of the same protein. The second step in the acyltransferase reaction is the transfer of the acyl moiety from ACP to the 2-acyl-GPE substrate to form phosphatidylethanolamine (PtdEtn). The 2-acyl-GPE acyltransferase is responsible for a number of physiological processes. The primary function of the enzyme system is to acylate 2-acyl-GPE that arises from the transfer of 1-position fatty acids to outer membrane lipoproteins or from the degradation of membrane phospholipids by phospholipase A1.3,7 The acyltransferase is also responsible for the 1 S. S. Taylor and E. C. Heath, J. Biol. Chem. 244, 6605 (1969). z H. Homma, M. Nishijima, T. Kobayashi, H. Okuyama, and S. Nojima, Biochim. Biophys. Acta 508, 165 (1981). 3 C. O. Rock, J. Biol. Chem. 259, 6188 (1984). 4 C. L. Cooper, L. Hsu, S. Jackowski, and C. O. Rock, J. Biol. Chem. 264, 7384 (1989). s T. K. Ray and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. U.S.A. 73, 4374 (1976). 6 C. O. Rock and J. E. Cronan, Jr., J. Biol. Chem. 254, 7116 (1979). 7 S. Jackowski and C. O. Rock, J. Biol. Chem. 261, 11328 (1986).
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, inc. All rights of reproduction in any form reserved.
112
ACYLTRANSFERASES
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PtdE~AcyI-GPE Enz+ / ~ ACPSH "---7
ATP + Fatty Acid
Enz ,/ Enz+ Acy-ACP~7 AcyI-ACP
AMP +
PPi
FIG. 1. Catalytic cycle of 2-acyi-GPE acyltransferase/acyl-ACPsynthetase.
acyl-CoA-independent uptake and incorporation of exogenous fatty acids and lysophospholipids into the membrane. 8'9
Assay Method Principle. The assay method for 2-acyl-GPE acyltransferase is based on measuring the formation of [14C]PtdEtn from 2-acyl-GPE and [14C]palmitic acid in the presence of ATP, Mg 2+ , and ACP. The reaction is terminated by applying an aliquot of the incubation mixture to the preadsorbant layer of a thin-layer chromatography plate, and, following development to separate unreacted fatty acid from PtdEtn, the amount of [14C]PtdEtn formed is quantitated by scintillation counting. The acyltransferase/synthetase may also be detected using the acyl-ACP synthetase assay. 5'6'I° The acyl-ACP synthetase assay technique is easier to perform and faster than the acyltransferase assay described in this chapter, but it measures a nonphysiological, low specific activity side reaction of the acyltransferase rather than its normal catalytic activity. 4 Reagents. [14C]Palmitic acid (specific activity - 5 5 Ci/mol) from any supplier is suitable. Purified (oxidant-free) Triton X-100, ATP, octyl-/3-Dglucoside, and Rh&opus arrhizus lipase are purchased from Boehringer Mannheim (Indianapolis, IN). Escherichia coli PtdEtn is purchased from Serdary Research Laboratories (London, ON, Canada). ACP is purified
8 c. o. Rock and S. Jackowski, J. Biol. Chem. 26@~127200985). 9 L. Hsu, S. Jackowski, and C. O. Rock, J. Bacteriol. 171, 1203(1989). 10See this series, Vol. 71 [21].
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TABLE I REAGENTS AND STOCK SOLUTIONS FOR 2-ACYLGLYCEROPHOSPHOETHANOLAMINE ACYLTRANSFERASE ASSAY Reagent
Microliters/assay
Final concentration
Tris-HC1, 1 M, pH 8.0 ATP, 0.1 M, pH 7.0 MgCI2, 0.1 M ACP, 100 p,M Dithiothreitol, 40 mM 2-AcyI-GPE, a 1 mM [14C]Palmitic acid, a 1 mM Protein in 2% Triton X-100
4 2 2 4 2 4 2 20
0.1 M 5 mM 5 mM 10 p.M 2 mM 100/zM 50/xM 0-10 mU
a These solutions are prepared in 2% Triton X-100.
as described in an earlier volume in this series. 11 The stock solutions required for the assay are listed in Table I and can be stored at - 20° for months. The exception is dithiothreitol, which should be prepared daily. Preparation of2-Acyl-GPE Substrate. 2-AcyI-GPE is prepared by the digestion of E. coli PtdEtn essentially as described by Homma and Nojima. 12 PtdEtn (4 /zmol) is added to a screw-top tube and the solvent evaporated. Next, 1.6 ml of 50 mM Bis-Tris, pH 5.6, is added, and the PtdEtn is suspended by two 10-sec rounds of sonication. Then, 0.2 ml of 100 mM CaCI 2 , 0.2 ml of 2 mg/ml R. arrhizus lipase, and 0.2 ml of diethyl ether are added. The mixture is incubated at 25 ° for 3 hr with shaking. Methanol (2 ml) is added to stop the reaction and the mixture extracted twice with 4 ml of hexane to remove the fatty acid. Next, 2.2 ml of chloroform, 0.5 ml methanol, and 0.2 ml of 0.5 M citric acid are added. The 2-acyl-GPE partitions into the bottom phase, which is collected, the solvent removed, and the 2-acyl-GPE suspended in chloroform-methanol (I : 1, v/v). 2-Acyl-GPE concentrations are determined by the spectrophotometric assay of Stewart ~3 using E. coli PtdEtn as a standard. 2-AcylGPE is aliquoted and stored under nitrogen at - 2 0 °. Stocks of 2-acylGPE should not be stored for longer than 1 week because by this time a significant fraction of the acyl groups have migrated to the 1-position and 2-acyl-GPE acyltransferase will not acylate 1-acyl-GPE. Method. The number of assays to be performed is determined, and the appropriate volumes of the assay regents (Table I) are combined. To each 11 See this series, Vol. 71 [41]; see also C. O. Rock and J. E. Cronan, Jr., Anal. Biochem. 102, 362 (1980). 12 H. Homma and S. Nojima, J. Biochem. (Tokyo) 91, 1103 (1982). 13 j. C. M. Stewart, Anal. Biochem. 104, 10 (1980).
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10 × 75 mm glass assay tube, 20/zl of the reaction mixture is added, and the reaction is initiated by the addition of 20/zl of the protein solution in 2% Triton X-100. The tubes are briefly vortex mixed and placed in a 37 ° water bath for 10 min. Incubations are terminated by the addition of 0.2 ml of ethanol. The mixture is evaporated to dryness under a stream of nitrogen, resuspended in chloroform-methanol (1 : 1, v/v), and the entire sample applied to the preadsorbant layer of a silica gel G plate. The thin-layer plate is developed with chloroform-methanol-acetic acid (85 : 15 : 10, v/v), and the areas corresponding to PtdEtn and fatty acid are located by comparison with standards chromatographed in one lane of the same plate. These two areas are removed, and the amount of radioactivity incorporated into PtdEtn is determined by scintillation counting. Counting the fatty acid area is a useful control to confirm that the amount of radioactivity recovered from the thin-layer plate corresponds to the amount of carbon-14 initially added to the assay. Results are expressed as nanomoles per minute per milligram protein. Purification
Initial Purification Steps. The initial steps in the purification of 2-acylGPE acyltransferase/acyl-ACP synthetase are identical to the procedure outlined by Rock and Cronan for the purification of acyl-ACP synthetase activity. 6'1°All procedures are performed at 4° unless otherwise indicated. The cells are disrupted by passage through a French pressure cell at 16,000 psi. The homogenate is then centrifuged at 15,000 g for 20 min to remove debris and unbroken cells, and the pellet is discarded. The supernatant is adjusted to 10 mM in MgCI2 by the addition of the appropriate volume of 1 M MgCI 2. The suspension then is sedimented at 80,000 g for 90 min, and the supernatant is discarded. The resulting pellets are homogenized thoroughly in 150 ml of 50 mM Tris-HC1, pH 8.0, and then 150 ml of 50 mM Tris-HC1, pH 8.0, containing I M NaC1, and 20 mM MgC12 is added to the membrane suspension. The solution is gently stirred for 15 min, and the membranes are sedimented at 80,000 g for 90 min. The membrane pellets from the salt wash are homogenized thoroughly in 100 ml of 50 mM Tris-HCl, pH 8.0, and to this suspension I00 ml of 50 mM Tris-HCl, pH 8.0, containing 4% Triton X-100 and 20 mM MgCI 2 is added, and the solution is gently stirred for 30 min. The suspension then is centrifuged at 80,000 g for 90 min to remove unsolubilized material, and the Triton X-100 supernatant is saved. The Triton X-100 extract (200 ml) is then adjusted to 5 mM ATP by the addition of 20 ml of 0.1 M ATP in 50 mM Tris-HC1, pH 8.0. The protein concentration should be between 2 and 5 mg-ml. The Triton X-100 extract
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is then placed in a 37° water bath, stirred until the temperature reaches 33°, and then placed in a 55 ° water bath and stirred on ice and centrifuged at 15,000 g for 20 min to remove denatured proteins. Blue-Sepharose Chromatography. A column (1.2 x 35 cm) is packed with Blue-Sepharose CL-6B and equilibrated with 50 mM Tris-HCl, pH 8.0, containing 2% Triton X-100 at 4°. The supernatant from the heat step is applied to the column. The column is then rinsed with 6 column volumes of 50 mM Tris-HCl, pH 8.0, containing 2% Triton X-100, followed by 6 column volumes of the same buffer containing 0.6 M NaCI. The majority of the protein adsorbed to the Blue-Sepharose column is eluted with 0.6 M NaCI, and acyltransferase/synthetase activity is desorbed from the column by elution with 0.5 M KSCN in 50 mM Tris-HC1, pH 8.0, 2% Triton X-100. ATP-Charged DEAE-Cellulose Chromatography. The final step in the procedure is chromatography on ATP-charged DEAE-cellulose. A 17-ml column of Whatman (Clifton, N J) DE-52 is packed and equilibrated in 50 mM Tris-HCl, pH 8.0, 2% Triton X-100. The column is then washed with 50 ml of 5 mM ATP in the same buffer and excess nucleotide removed by washing with buffer without ATP. The dialyzed KSCN eluate from the Blue-Sepharose step is loaded on the ATP-charged Whatman DE-52 column and the column washed with the column buffer. In the case where the samples are to be analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis, the column is exchanged in 50 mM Tris-HCl, pH 8.0, 30 mM octyl-fl-D-glucoside. Activity is eluted with 0.5 M NaC1 in either octyl/3-o-glucoside or Triton X-100 buffer. Comments on Purification. The results of the purification procedure are presented in Table II. Throughout the purification procedure, the ratio of 2-acyl-GPE acyltransferase to acyl-ACP synthetase activity remains TABLE II PURIFICATION OF 2-ACYLGLYCEROPHOSPHOETHANOLAMINE ACYLTRANSFERASE[ ACYL-[AcYL-CARRIER-PROTEIN] SYNTHETASE
Purification step
2-Acyl-GPE acyltransferase (nmol/min/mg)
Acyl-ACP synthetase (nmol/min/mg)
Ratio
Recovery (%)
Membrane s Washed membranes Triton extract Heat supernatant Blue- Sepharose DEAE-ceUulose
0.300 0.437 0.958 1.73 50.6 102.4
0.092 0.117 0.273 0.490 16.24 28.4
3.26 3.74 3.51 3.53 3.12 3.61
100 67.8 48.5 45.8 9.9 7.7
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ACYLTRANSFERASES
[ 13]
constant. The specific activities of the preparations for 2-acyl-GPE acyltransferase are consistently 10-fold higher than the specific activity of acylACP synthetase. At stages of purification prior to the Blue-Sepharose chromatography, 2-acyl-GPE acyltransferase activity is not dependent on the addition of ACP to the assay. However, the conditions of the BlueSepharose step dissociate the ACP subunit from the acyltransferase, and, after this step, acyltransferase activity becomes completely dependent on the addition of ACP to the assay. This purification procedures results in the 3000-fold purification of 2-acyl-GPE acyltransferase/acyl-ACP synthetase in 7.7% yield. SDS gel electrophoresis of the final product shows a single band with an apparent molecular weight of 27,000. Properties 2-Acyl-GPE acyltransferase/acyl-ACP synthetase is an inner membrane enzyme that is not subject to catabolite repression, and it is genetically and biochemically distinct from acyl-CoA synthetase and glycerolphosphate and 1-acylglycerol-phosphate acyltransferases. The enzyme specifically catalyzes the transfer of fatty acids to the 1-position; l-acylGPE is not a substrate. The enzyme acylates 2-acylglycerophosphocholine as readily as 2-acyl-GPE but does not acylate 2-acylglycerophosphoglycerol. The enzyme prefers saturated fatty acids (palmitate and myristate) as substrates and is less active on shorter (decanoate) and unsaturated (oleate) fatty acids. Poor substrates have both a higher Km and lower Vmax . The acyltransferase binds ACP with high affinity (Kd --60 nM) and catalyzes the acyltransferase reaction without the dissociation of ACP or acyl-ACP from the enzyme. ACP is dissociated from the protein complex by exposure to high salt. This property accounts for the accumulation of acyl-ACP in assays performed in the presence of 0.4 M LiCI and explains the acyl-ACP synthetase activity expressed by the acyltransferase. There is little doubt that the protein functions only as an acyltranferase in v i v o and does not catalyze the formation ofacyl-ACP that can be made available to other proteins. Genetics and Molecular Biology A replica print procedure was used to isolate mutants that lacked both 2-acyl-GPE acyltransferase and acyl-ACP synthetase activities. This mutation maps to the 61-rain region of the E . c o l i chromosome, and the allele is designated a a s . ~4 The a a s mutants were then employed to isolate 14 L. Hsu, S. Jackowski, and C. O. Rock, J. Biol. Chem. 266, 13783 (1991).
[13]
2-ACYL-GPE ACYLTRANSFERASEFROME. coli
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plasmids that carry the a a s gene. One such plasmid, pLCH3, contains the full a a s coding sequence, and strains that harbor this plasmid possess 10fold higher specific activities of both 2-acyl-GPE acyltransferase and acylACP synthetase) 4 Thus, strains harboring this plasmid are an excellent source for purifying the acyltransferase/synthetase either for biochemical characterization or in routine preparation of the enzyme for the synthesis of acyl-ACPs. Synthesis of AcyI-ACP Although the acyltransferase/synthetase functions only as an acyltransferase in vivo, its ability to synthesize acyl-ACP in vitro has made it a useful tool for the preparation of defined acyl-ACPs to be used as substrates for other enzymes. 15 Our laboratory still uses the techniques outlined in a previous volume in this series 15 to synthesize acyl-ACP using the acyltransferase/synthetase, but there is a useful modification introduced by others 16: that warrants consideration. These investigators 16,17have taken advantage of the tight binding of the acyltransferase to Blue-Sepharose and the fact that the synthetase remains active when coupled to the resin. 6,16: The enzymatically active column is used directly to catalyze the formation of acyl-ACP by the continuous cycling of a reaction mixture ( - 6 0 ml) over the column for 24 hr with a peristaltic pump at 4°. 17 The reaction mixture contains 0.6 mg/ml ACP, 0.1 M TrisHCI, pH 8.0, 0.4 M LiCI, 10 mM ATP, 10 mM MgCI2,2 mM dithiothreitol, 0.07% Triton X-100, and 100/xM of the desired fatty acid) 7 Yields of acyl-ACP up to 90% or greater have been reported, and the acyl-ACP synthetase/Blue Sepharose column remains stable for at least 4 months at 4oC. 17
Acknowledgments This work was supported by National Institutes of Health Grant GM 28035, Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities. We thank Pare Jackson for excellent technical assistance.
t5 See this series, Vol. 72 [27]; see also C. O. Rock and J. L. Garwin, J. Biol. Chem. 254, 7123 (1979). 16 p. R. Green, A. H. Merrill, Jr., and R. M. Bell, J. Biol. Chem. 256, 11151 (1981). 17 M. S. Anderson, C. E. Bulawa, and C. R. H. Raetz, J. Biol. Chem. 260, 15536 (1985).