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from 7 to 8 for platelet membranes 1 and from 7.5 to 8.5 for the heart microsomal fraction. 6 The enzyme activity was shown to be sensitive to detergents. Triton X-100 at concentrations above 0.1 mg/ml in platelet membranes ~ and above 0.2 mg/ml in macrophage microsomes 9 totally inhibited the transacylation activity. A similar but weaker inhibition was observed for cholate. Several sulfhydryl agents such as N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and p-chloromercuribenzenesulfonic acid (pCMBS) also inhibited the enzyme reaction, 1,6,9 suggesting that the SH group(s) in the enzyme protein is important for maintaining the catalytic activity. On the other hand, the reaction was not influenced by the presence of EDTA or EGTA, indicating that the presence of divalent cations such as Ca 2+ and Mg 2÷ is not required for enzyme activity. The addition of Ca 2+ to the incubation mixture did not markedly affect the transacylation from endogenous membrane phospholipids, whereas the transfer from exogenous phospholipids was somewhat enhanced by Ca2+.25 The reason for this discrepancy is as yet unknown. A possible explanation is that Ca 2+ may alter the affinity of exogenous substrates for the membrane-bound enzyme. Kinetic constants have also been reported by several investigators. Robinson et al. 8 compared the activities of three acylation systems toward 1-alkyl-GPC using relatively low concentrations of 1-alkyl-GPC and macrophage microsomes. They concluded that the CoA-independent transacylation system has the highest affinity for 1-alkyl-GPC in comparison with the CoA-dependent transacylation system and acyl-CoA: 1-alkylGPC acyltransferase. The apparent K m value for 1-alkyl-GPC was 1.1 /~M, and the apparent Vmax was 3.2 nmol/min/mg. In the case of platelet membranes, the apparent K m value for 1-alkyl-GPC was calculated to be 12/xM, and Vmax was 0.87 nmol/min/mg. 2 25T. Sugiura, unpublished results, 1990.
[8] L y s o p h o s p h a t i d y l c h o l i n e A c y l t r a n s f e r a s e B y PATRICK C. aHOY, PAUL G. TARDI, and J. J. MUKrtElUEE
Introduction Structural analysis of phosphatidylcholine (PC) indicates that saturated fatty acids are usually esterified at the C-1 position and unsaturated fatty METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
[8]
1-ACYL-GPC : ACYL-CoA ACYLTRANSFERASE
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acids at the C-2 position, l The observed fatty acid distribution does not seem to result from the limited selectivity of CDP-choline : 1,2-diacylglycerol phosphocholinetransferase (EC 2.7.8.2, cholinephosphotransferase) for diacylglycerol species during the de nooo biosynthesis of P C . 2 The remodeling of newly synthesized PC via deacylation-reacylationhas been regarded as an important mechanism for the selection of PC acyl groups in mammalian tissues. The pathway for the remodeling of PC was first identified by Lands) In this pathway, PC is first deacylated, and the resultant lyso-PC is acylated back to PC by the action of lyso-PC : acyl-CoA acyltransferase as follows: Lyso-PC + acyl-CoA--> PC + CoA-SH
Lyso-PC:acyl-CoA acyltransferase (EC 2.3.1.23, 1-acylglycerophosphocholine acyltransferase) activity has been detected in various mammalian tissues, 1 bacteria, protozoa, and plants. 4 The transfer of acyl groups from acyl-CoA to 1-acylglycerophosphocholine (1-acyl-GPC) is catalyzed by 1-acyl-GPC : acyl-CoA acyltransferase, whereas transfer of fatty acids to 2-acyl-GPC is catalyzed by 2-acyl-GPC:acyl-CoA acyltransferase. These two reactions appear to be carded out by separate enzymes.5,6 Most of the studies on acyltransferase have focused on the acylation of l-acyl-GPC, and only limited information is available on the acylation of 2-acyl-GPC. The procedures described here are for the assay of 1-acyl-GPC : acyl-CoA acyltransferase. Assay Methods Assay with Radioactive Substrate Principle. Enzyme activity is determined by the incorporation of a radiolabeled acyl group into 1-acyl-GPC to form 1,2-diacyl-GPC. The radiolabeled product is isolated from the substrate by phase separation followed by thin-layer chromatography.7 Preparation of Substrate. 1-AcyI-GPC is available from commercial 1 p. C. Choy and G. Arthur, in "Phosphatidylcholine Metabolism" (D. E. Vance, ed.), p. 87. CRC Press, Boca Raton, Florida, 1989. 2 G. Arthur and P. C. Choy, Biochim. Biophys. Acta 795, 221 (1984). 3 W. E. M. Lands, J. Biol. Chem. 235, 2233 (1960). 4 K. A. Devor and J. B. Mudd, J. Lipid Res. 12, 412 (1971). 5 W. E. M. Lands and P. Hart, J. Biol. Chem. 240, 1905 (1965). 6 G. Arthur, Biochem. J. 261, 575 (1989). 7 G. Arthur and P. C. Choy, Biochem. J. 236, 481 (1986).
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sources as lysophosphatidylcholine. Usually, the preparation consists of 95-99% 1-acyl-GPC with the remainder as 2-acyl-GPC. Synthetic 1-acylGPC with specific acyl groups are also commercially available. Both labeled and unlabeled oleoyl-CoA are available in sufficiently pure form (over 95% purity) from commercial sources. However, the purity of arachidonoyl-CoA must be carefully assessed prior to use. Procedure. The reaction mixture contains 80 mM Tris-HCl (pH 7.4), 100/zM [ l-14C]oleoyl-CoA [ 1000 disintegrations/min (dpm)/nmol], 150/zM 1-acyl-GPC, and enzyme source in a final volume of 0.7 ml. The mixture is preincubated at 25 ° for 5 min, and the reaction is initiated by the addition of enzyme. The reaction mixture is incubated in a shaking water bath at 25° for l0 min. The reaction is terminated by the addition of 3 ml of chloroform-methanol (2 : 1, v/v). Phase separation is caused by the addition of 0.8 ml of 0.9% KCI to the mixture. After brief centrifugation, the upper phase is removed, and aliquots of the lower phase are applied onto a 20 × 20 cm thin-layer chromatographic plate (Silica Gel G, Fisher Scientific, Ottawa, Ontario) etched with 2-cm lanes. Unlabeled PC (pig liver) is used as a carrier. The plate is developed in chloroform-methanol-water-acetic acid (70 : 30 : 4 : 2, v/v). After development, the PC fraction on the plate (Rf 0.43) is visualized by exposure of the plate to iodine vapor. The silica gel containing the PC fraction is removed from the plate and placed in a scintillation vial for radioactivity determination. The specific activity of the enzyme in rat liver 8 and heart 9 have been reported to be 58 and 13 nmol/min/mg protein, respectively.
Spectrophotometric Assay Principle. The spectrophotometric assay is dependent on the release of CoA-SH from the enzyme reaction, which reacts with 5,5'-dithiobis(2nitrobenzoic acid) (DTNB) to form thionitrobenzoic acid (TNB). The amount of TNB produced is monitored spectrophotometrically at 412 nm.5 Procedure. The complete assay mixture contains 40 mM Tris-HCl (pH 7.4), 50/zM oleoyl-CoA, 100 ~M 1-acyl-GPC, 0.1 mM DTNB, and 10-20 /xl of enzyme preparation in a final volume of 1 ml. The DTNB stock solution (1 mM) is prepared fresh prior to the assay. The reaction is carried out at 25 ° in a spectrophotometric cuvette (1-cm path length), and the change of absorbance is continuously monitored at 412 nm. A molar extinction coefficient of 13,600 is used to quantitate the amount of DTNB reduced. Since DTNB might also react slowly with the thiol groups of 8 y . Kawashima, T. Matsunaga, A. Hirose, T. Ogata, and H. Kozuka, Biochim. Biophys. Acta 11106, 214 (1989). 9 p. C. Choy, K. O, R. Y. K. Man, and A. C. Chan, Biochirn. Biophys. Acta 1005, 225 (1989).
[8]
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83
proteins and with CoA-SH formed by some specific or unspecific hydrolysis of acyl-CoA, a control that contains all the assay components except 1-acyl-GPC is usually employed. 5 The control mixture is placed into the reference cuvette (in a double-beam spectrophotometer), and the absorbance due to nonspecific reduction of DTNB is automatically subtracted from the reaction mixture. Owing to the nonspecific reduction of DTNB, the use of crude tissue homogenate for this assay is not recommended. Any enzyme preparation containing large particles may interfere with the spectrophotometric assay.
Other Assays Assays for acyltransferase activity with 1-[1J4C]palmitoyl-GPC or a 1-[ IJ4C]stearyl-GPC and unlabeled acyl-CoA have been reported.~°,~l The use of these labeled substrates is not recommended when there is a substantial amount of lyso-PC : lyso-PC acyltransferase activity in the enzyme preparation.
Specificity of Assays The assay with radioactive acyl-CoA is very specific for the enzyme reaction. The spectrophotometric assay is more rapid, but the assay is based on the determination of a thiol group, and therefore is less specific. The data obtained from the spectrophotometric assay must be confirmed by a more specific assay. SubceUular Localization and Purification 1-Acyl-GPC : acyl-CoA acyltransferase has been found in all mammalian organs. Most of the enzyme activity is located in the microsomal fraction, lz The enzyme appears to have a transmembrane orientation in the microsomal membrane vesicle.~3 Acyltransferase activity has also been reported in the mitochondrial and plasma membrane fractions. 14'15 Low enzyme activity has been detected in the cytosolic fraction of the rabbit heart. ~6 10 B. J. Holub, J. A. MacNaughton, and J. Piekarski, Biochim. Biophys. Acta 572, 413 (1979). tl N. Deka, G. Y. Sun, and R. MacQuarrie, Arch. Biochem. Biophys. 246, 554 (1986). J2 H. Eibl, E. E. Hill, and W. E. M. Lands, Eur. J. Biochem. 9, 250 (1969). 13 W. Renooij and F. Snyder, Biochim. Biophys. Acta 666, 468 (1981). 14 G. Arthur, L. L. Page, C. L. Zaborniak, and P. C. Choy, Biochem. J. 242, 171 (1987). 15 O. Colard, D. Bard, G. Bereziat, and J. Polonovski, Biochim. Biophys. Acta 6111,88 (1980). 16 p. Needleman, A. Wyche, H. Sprecher, W. J. Elliott, and A. Evers, Biochim. Biophys. Acta 836, 267 (1985).
84
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Attempts to purify the 1-acyl-GPC:acyl-CoA acyltransferase were hampered by difficulties in its solubilization from the membrane domain. The use of detergent for this purpose has not been very effective since enzyme activity is inevitably inhibited by the presence of detergents. For example, when microsomes were treated with 1% cholate, 0.25% deoxycholate, or 0.05% Triton X-100, over 90% of enzyme activity was inhibited. The solubilization of the enzyme with 1-acyl-GPC and its analogs appears to be a more viable approach.17 The purification of the enzyme from bovine brain and bovine heart has been reported. 11'18
Acyl Specificity Since dipalmitoyl-PC is the major form of PC in the lung, the selectivity of the acyltransferase toward palmitoyl-CoA was expected to be higher than that for oleoyl-CoA. However, no difference in specificity of the enzyme toward palmitoyl-CoA and oleoyl-CoA has been detected. 1 Interestingly, 1-palmitoyl-GPC appears to be a better acyl acceptor than 1-stearyl-GPC.19 In rat liver, the enzyme has a definite specificity toward acyl-CoA with unsaturated acyl groups, 15,2°and 1-palmitoyl-GPC is more effective than l-stearyl-GPC as an acyl acceptor) ° In guinea pig heart, the microsomal enzyme is more active toward unsaturated acyl-CoAs, but displays little selectivity with respect to the degree of unsaturation. 7 Interestingly, the mitochondrial enzyme appears to have a very high specificity toward linoleoyl-CoA.14 The acyl specificity of the enzyme appears to be affected by the presence of detergent. 21 The acyl specificity is also affected during enzyme purification. For example, the purified acyltransferase from bovine brain displays a higher degree of selectivity toward arachidonate than the microsomal enzyme.11 Stimulation of smooth muscle cells with phorbol myristate results in the enhancement of arachidonate incorporation into 1-acylG P C . 22
17 H. U. Weltzien, G. Richter, and E. Ferber, J. Biol. Chem. 254, 3652 (1979). 18 M. Sanjanwala, G. Y. Sun, M. A. Cutrera, and R. A. MacQuarrie, Arch. Biochem. Biophys. 2,65, 476 (1988). 19 G. P. H. van Heusden, H. P. J. M. Noteborn, and H. van den Bosch, Biochim. Biophys. Acta 664, 49 (1981). 20 H. van den Bosch, L. M. G. van Golde, H. Eibl, and L. L. M. van Deenen, Biochim. Biophys. Acta 144, 613 (1967). 21 H. Okuyama, K. Yamada, and H. Ikezawa, J. Biol. Chem. 250, 1710 (1975). 22 T. Kanzaki, N. Morisaki, Y. Saito, and S. Yoshida, Lipids 24, 1024 (1989).
[8]
I-ACYL-GPC : ACYL-CoA ACYLTRANSFERASE
85
Characteristics and Kinetics The acyltransferase reaction is reversible and is dependent on the concentrations of the substrates. The pH optimum for the mammalian enzyme is between 7 and 8 and may be dependent on the transfer of a specific acyl group. Using the partially purified enzyme from rat liver, the transfer of oleoyl-CoA was found to be more active at pH 7, whereas arachidonoyl-CoA transfer was more rapid at a higher p H . 23 Recently, the molecular weight of the purified enzyme was determined to be 43,000 for bovine brain n and 64,000 for bovine heart. 18The partially purified enzyme from rabbit lung displayed an iso-Ping-Pong mechanism, and the K mvalues for palmitoyl-CoA and 1-acyl-GPC were found to be 8.5 and 61 /~M, respectively. 24In human platelets, the Km values for saturated and unsaturated acyl-CoA vary from 1.05 to 5.70/~M in the presence of 100/~M 1-acyl-GPC.25 In most mammalian tissues, the Km values for acyl-CoA and 1-acyl-GPC vary between 1-15/~M and 50-100/~M, respectively. Regulation of Enzyme Activity 1-AcyI-GPC acyltransferase activity in rat lung was inhibited by high concentrations of 1-acyl-GPC, but the inhibition by high concentrations of acyl-CoAs was not prominent. 26 Enzyme activity in the liver microsomes was relatively unaffected by sulfhydryl-binding reagents such as iodoacetate, N-ethylmaleimide, and p-chloromercuriphenylsulfonic acid, but the activity of the partially purified enzyme was inhibited by these reagents. 23 The compounds WY-14643 and clofibric acid were found to inhibit mitochondrial 1-acyl-GPC:acyl-CoA acyltransferase. 27 Methyllidocaine, a local anesthetic, caused the inhibition of enzyme activity in mammalian heart. 2s In addition, divalent cations such as Mg 2÷ and Ca 2÷ were inhibitory to enzyme activity. 7,29 Acyltransferase activity in vivo was affected by long-term administration of clofibric acid and chronic administration of isoproterenol, s'29 Enzyme activity in rat liver, rat heart, and rabbit gastric mucosa microsomes was inhibited by detergents and 23 H. Hasegawa-Sasaki and K. Ohno, Biochim. Biophys. Acta 617, 205 (1980). 24 R. Arche, P. Estrada, and C. Acebal, Arch. Biochem. Biophys. 257, 131 (1987). 25 M. L. McKean, J. B. Smith, and M. J. Silver, J. Biol. Chem. 257, 11278 (1982). 26 H. Hasegawa-Sasaki and K. Ohno, Biochim. Biophys. Acta 380, 486 (1975). 27 W. W. Riley and D. R. Pfeiffer, J. Biol. Chem. 761, 14018 (1986). :s p. G. Tardi, R. Y. K. Man, C. R. McMaster, and P. C. Choy, Biochem. Cell Biol. 68, 745 (1990). 29 K. Yashiro, Y. Kameyama, M. Mizuno, S. Hayashi, Y. Sakashita, and Y. Yokota, Biochin*. Biophys. Acta 1005, 56 (1989).
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inhibitors of cyclic nucleotide phosphodiesterase. 3° The regulation of enzyme activity by a phosphorylation-dephosphorylation cycle has been postulated. 31 Concluding Remarks Since the discovery of acyltransferases it has been postulated that there may be separate enzymes for each acyl group. Indirect evidence with Triton X-100-treated microsomes indicates that there may be a specific acyltransferase for oleoyl-CoA. 21 The identification of an acyltransferase with an absolute specificity for lineoleoyl-CoA lends support to this hypothesis. 14Purification of the enzyme from bovine brain causes an enrichment of specificity toward arachidonoyl-CoA. 11 Thus, acyl specificities previously determined in subcellular fractions could simply reflect the quantitative distribution of different acyltransferases in these fractions. Direct evidence for the existence of multiple forms of the acyltransferase is still lacking. 3o W. T. Shier, Biochem. Biophys. Res. Commun. 75, 186 (1977). 31 S. L. Reinhold, G. A. Zimmerman, S. M. Prescott, and T. M. McIntyre, J. Biol. Chem. 264, 21652 (1989).
[9] 1 - A l k y l - a n d
1-Alkenylglycerophosphocholine Acyltransferases
B y PATRICK C. CHOY a n d CHRISTOPHER R . M C M A S T E R
Introduction Three forms of choline-containing phospholipids are found in mammalian tissues. 1 The 1,2-diacyl form (phosphatidylcholine) is the most abundant form and is present as the major phospholipid in all mammalian tissues. The l-alkyl-2-acyl form (plasmanylcholine) is found in significant amounts in circulating cells such as neutrophils and macrophages but in low amounts in other tissues. The 1-alkenyl-2-acyl form (plasmenylcholine) comprises up to 40% of the choline-containing phospholipids in electrically active tissues such as the heart. The major catabolic pathway for 1-alkyl-2-acylglycerophosphocholine (1-alkyl-2-acyl-GPC) or 1-alkenyl-2-acylglycerophosphocholine 1 L. A. Horrocks and M. Sharma, in "Phospholipids" (J. N. Hawthorne and G. B. Ansell, eds.), p. 51. Elsevier Biomedical Press, Amsterdam, New York, and Oxford, 1982.
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
ACYLTRANSFERASES
[8]
from 7 to 8 for platelet membranes 1 and from 7.5 to 8.5 for the heart microsomal fraction. 6 The enzyme activity was shown to be sensitive to detergents. Triton X-100 at concentrations above 0.1 mg/ml in platelet membranes ~ and above 0.2 mg/ml in macrophage microsomes 9 totally inhibited the transacylation activity. A similar but weaker inhibition was observed for cholate. Several sulfhydryl agents such as N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and p-chloromercuribenzenesulfonic acid (pCMBS) also inhibited the enzyme reaction, 1,6,9 suggesting that the SH group(s) in the enzyme protein is important for maintaining the catalytic activity. On the other hand, the reaction was not influenced by the presence of EDTA or EGTA, indicating that the presence of divalent cations such as Ca 2+ and Mg 2÷ is not required for enzyme activity. The addition of Ca 2+ to the incubation mixture did not markedly affect the transacylation from endogenous membrane phospholipids, whereas the transfer from exogenous phospholipids was somewhat enhanced by Ca2+.25 The reason for this discrepancy is as yet unknown. A possible explanation is that Ca 2+ may alter the affinity of exogenous substrates for the membrane-bound enzyme. Kinetic constants have also been reported by several investigators. Robinson et al. 8 compared the activities of three acylation systems toward 1-alkyl-GPC using relatively low concentrations of 1-alkyl-GPC and macrophage microsomes. They concluded that the CoA-independent transacylation system has the highest affinity for 1-alkyl-GPC in comparison with the CoA-dependent transacylation system and acyl-CoA: 1-alkylGPC acyltransferase. The apparent K m value for 1-alkyl-GPC was 1.1 /~M, and the apparent Vmax was 3.2 nmol/min/mg. In the case of platelet membranes, the apparent K m value for 1-alkyl-GPC was calculated to be 12/xM, and Vmax was 0.87 nmol/min/mg. 2 25T. Sugiura, unpublished results, 1990.
[8] L y s o p h o s p h a t i d y l c h o l i n e A c y l t r a n s f e r a s e B y PATRICK C. aHOY, PAUL G. TARDI, and J. J. MUKrtElUEE
Introduction Structural analysis of phosphatidylcholine (PC) indicates that saturated fatty acids are usually esterified at the C-1 position and unsaturated fatty METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
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1-ACYL-GPC : ACYL-CoA ACYLTRANSFERASE
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acids at the C-2 position, l The observed fatty acid distribution does not seem to result from the limited selectivity of CDP-choline : 1,2-diacylglycerol phosphocholinetransferase (EC 2.7.8.2, cholinephosphotransferase) for diacylglycerol species during the de nooo biosynthesis of P C . 2 The remodeling of newly synthesized PC via deacylation-reacylationhas been regarded as an important mechanism for the selection of PC acyl groups in mammalian tissues. The pathway for the remodeling of PC was first identified by Lands) In this pathway, PC is first deacylated, and the resultant lyso-PC is acylated back to PC by the action of lyso-PC : acyl-CoA acyltransferase as follows: Lyso-PC + acyl-CoA--> PC + CoA-SH
Lyso-PC:acyl-CoA acyltransferase (EC 2.3.1.23, 1-acylglycerophosphocholine acyltransferase) activity has been detected in various mammalian tissues, 1 bacteria, protozoa, and plants. 4 The transfer of acyl groups from acyl-CoA to 1-acylglycerophosphocholine (1-acyl-GPC) is catalyzed by 1-acyl-GPC : acyl-CoA acyltransferase, whereas transfer of fatty acids to 2-acyl-GPC is catalyzed by 2-acyl-GPC:acyl-CoA acyltransferase. These two reactions appear to be carded out by separate enzymes.5,6 Most of the studies on acyltransferase have focused on the acylation of l-acyl-GPC, and only limited information is available on the acylation of 2-acyl-GPC. The procedures described here are for the assay of 1-acyl-GPC : acyl-CoA acyltransferase. Assay Methods Assay with Radioactive Substrate Principle. Enzyme activity is determined by the incorporation of a radiolabeled acyl group into 1-acyl-GPC to form 1,2-diacyl-GPC. The radiolabeled product is isolated from the substrate by phase separation followed by thin-layer chromatography.7 Preparation of Substrate. 1-AcyI-GPC is available from commercial 1 p. C. Choy and G. Arthur, in "Phosphatidylcholine Metabolism" (D. E. Vance, ed.), p. 87. CRC Press, Boca Raton, Florida, 1989. 2 G. Arthur and P. C. Choy, Biochim. Biophys. Acta 795, 221 (1984). 3 W. E. M. Lands, J. Biol. Chem. 235, 2233 (1960). 4 K. A. Devor and J. B. Mudd, J. Lipid Res. 12, 412 (1971). 5 W. E. M. Lands and P. Hart, J. Biol. Chem. 240, 1905 (1965). 6 G. Arthur, Biochem. J. 261, 575 (1989). 7 G. Arthur and P. C. Choy, Biochem. J. 236, 481 (1986).
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sources as lysophosphatidylcholine. Usually, the preparation consists of 95-99% 1-acyl-GPC with the remainder as 2-acyl-GPC. Synthetic 1-acylGPC with specific acyl groups are also commercially available. Both labeled and unlabeled oleoyl-CoA are available in sufficiently pure form (over 95% purity) from commercial sources. However, the purity of arachidonoyl-CoA must be carefully assessed prior to use. Procedure. The reaction mixture contains 80 mM Tris-HCl (pH 7.4), 100/zM [ l-14C]oleoyl-CoA [ 1000 disintegrations/min (dpm)/nmol], 150/zM 1-acyl-GPC, and enzyme source in a final volume of 0.7 ml. The mixture is preincubated at 25 ° for 5 min, and the reaction is initiated by the addition of enzyme. The reaction mixture is incubated in a shaking water bath at 25° for l0 min. The reaction is terminated by the addition of 3 ml of chloroform-methanol (2 : 1, v/v). Phase separation is caused by the addition of 0.8 ml of 0.9% KCI to the mixture. After brief centrifugation, the upper phase is removed, and aliquots of the lower phase are applied onto a 20 × 20 cm thin-layer chromatographic plate (Silica Gel G, Fisher Scientific, Ottawa, Ontario) etched with 2-cm lanes. Unlabeled PC (pig liver) is used as a carrier. The plate is developed in chloroform-methanol-water-acetic acid (70 : 30 : 4 : 2, v/v). After development, the PC fraction on the plate (Rf 0.43) is visualized by exposure of the plate to iodine vapor. The silica gel containing the PC fraction is removed from the plate and placed in a scintillation vial for radioactivity determination. The specific activity of the enzyme in rat liver 8 and heart 9 have been reported to be 58 and 13 nmol/min/mg protein, respectively.
Spectrophotometric Assay Principle. The spectrophotometric assay is dependent on the release of CoA-SH from the enzyme reaction, which reacts with 5,5'-dithiobis(2nitrobenzoic acid) (DTNB) to form thionitrobenzoic acid (TNB). The amount of TNB produced is monitored spectrophotometrically at 412 nm.5 Procedure. The complete assay mixture contains 40 mM Tris-HCl (pH 7.4), 50/zM oleoyl-CoA, 100 ~M 1-acyl-GPC, 0.1 mM DTNB, and 10-20 /xl of enzyme preparation in a final volume of 1 ml. The DTNB stock solution (1 mM) is prepared fresh prior to the assay. The reaction is carried out at 25 ° in a spectrophotometric cuvette (1-cm path length), and the change of absorbance is continuously monitored at 412 nm. A molar extinction coefficient of 13,600 is used to quantitate the amount of DTNB reduced. Since DTNB might also react slowly with the thiol groups of 8 y . Kawashima, T. Matsunaga, A. Hirose, T. Ogata, and H. Kozuka, Biochim. Biophys. Acta 11106, 214 (1989). 9 p. C. Choy, K. O, R. Y. K. Man, and A. C. Chan, Biochirn. Biophys. Acta 1005, 225 (1989).
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proteins and with CoA-SH formed by some specific or unspecific hydrolysis of acyl-CoA, a control that contains all the assay components except 1-acyl-GPC is usually employed. 5 The control mixture is placed into the reference cuvette (in a double-beam spectrophotometer), and the absorbance due to nonspecific reduction of DTNB is automatically subtracted from the reaction mixture. Owing to the nonspecific reduction of DTNB, the use of crude tissue homogenate for this assay is not recommended. Any enzyme preparation containing large particles may interfere with the spectrophotometric assay.
Other Assays Assays for acyltransferase activity with 1-[1J4C]palmitoyl-GPC or a 1-[ IJ4C]stearyl-GPC and unlabeled acyl-CoA have been reported.~°,~l The use of these labeled substrates is not recommended when there is a substantial amount of lyso-PC : lyso-PC acyltransferase activity in the enzyme preparation.
Specificity of Assays The assay with radioactive acyl-CoA is very specific for the enzyme reaction. The spectrophotometric assay is more rapid, but the assay is based on the determination of a thiol group, and therefore is less specific. The data obtained from the spectrophotometric assay must be confirmed by a more specific assay. SubceUular Localization and Purification 1-Acyl-GPC : acyl-CoA acyltransferase has been found in all mammalian organs. Most of the enzyme activity is located in the microsomal fraction, lz The enzyme appears to have a transmembrane orientation in the microsomal membrane vesicle.~3 Acyltransferase activity has also been reported in the mitochondrial and plasma membrane fractions. 14'15 Low enzyme activity has been detected in the cytosolic fraction of the rabbit heart. ~6 10 B. J. Holub, J. A. MacNaughton, and J. Piekarski, Biochim. Biophys. Acta 572, 413 (1979). tl N. Deka, G. Y. Sun, and R. MacQuarrie, Arch. Biochem. Biophys. 246, 554 (1986). J2 H. Eibl, E. E. Hill, and W. E. M. Lands, Eur. J. Biochem. 9, 250 (1969). 13 W. Renooij and F. Snyder, Biochim. Biophys. Acta 666, 468 (1981). 14 G. Arthur, L. L. Page, C. L. Zaborniak, and P. C. Choy, Biochem. J. 242, 171 (1987). 15 O. Colard, D. Bard, G. Bereziat, and J. Polonovski, Biochim. Biophys. Acta 6111,88 (1980). 16 p. Needleman, A. Wyche, H. Sprecher, W. J. Elliott, and A. Evers, Biochim. Biophys. Acta 836, 267 (1985).
84
ACYLTRANSFERASES
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Attempts to purify the 1-acyl-GPC:acyl-CoA acyltransferase were hampered by difficulties in its solubilization from the membrane domain. The use of detergent for this purpose has not been very effective since enzyme activity is inevitably inhibited by the presence of detergents. For example, when microsomes were treated with 1% cholate, 0.25% deoxycholate, or 0.05% Triton X-100, over 90% of enzyme activity was inhibited. The solubilization of the enzyme with 1-acyl-GPC and its analogs appears to be a more viable approach.17 The purification of the enzyme from bovine brain and bovine heart has been reported. 11'18
Acyl Specificity Since dipalmitoyl-PC is the major form of PC in the lung, the selectivity of the acyltransferase toward palmitoyl-CoA was expected to be higher than that for oleoyl-CoA. However, no difference in specificity of the enzyme toward palmitoyl-CoA and oleoyl-CoA has been detected. 1 Interestingly, 1-palmitoyl-GPC appears to be a better acyl acceptor than 1-stearyl-GPC.19 In rat liver, the enzyme has a definite specificity toward acyl-CoA with unsaturated acyl groups, 15,2°and 1-palmitoyl-GPC is more effective than l-stearyl-GPC as an acyl acceptor) ° In guinea pig heart, the microsomal enzyme is more active toward unsaturated acyl-CoAs, but displays little selectivity with respect to the degree of unsaturation. 7 Interestingly, the mitochondrial enzyme appears to have a very high specificity toward linoleoyl-CoA.14 The acyl specificity of the enzyme appears to be affected by the presence of detergent. 21 The acyl specificity is also affected during enzyme purification. For example, the purified acyltransferase from bovine brain displays a higher degree of selectivity toward arachidonate than the microsomal enzyme.11 Stimulation of smooth muscle cells with phorbol myristate results in the enhancement of arachidonate incorporation into 1-acylG P C . 22
17 H. U. Weltzien, G. Richter, and E. Ferber, J. Biol. Chem. 254, 3652 (1979). 18 M. Sanjanwala, G. Y. Sun, M. A. Cutrera, and R. A. MacQuarrie, Arch. Biochem. Biophys. 2,65, 476 (1988). 19 G. P. H. van Heusden, H. P. J. M. Noteborn, and H. van den Bosch, Biochim. Biophys. Acta 664, 49 (1981). 20 H. van den Bosch, L. M. G. van Golde, H. Eibl, and L. L. M. van Deenen, Biochim. Biophys. Acta 144, 613 (1967). 21 H. Okuyama, K. Yamada, and H. Ikezawa, J. Biol. Chem. 250, 1710 (1975). 22 T. Kanzaki, N. Morisaki, Y. Saito, and S. Yoshida, Lipids 24, 1024 (1989).
[8]
I-ACYL-GPC : ACYL-CoA ACYLTRANSFERASE
85
Characteristics and Kinetics The acyltransferase reaction is reversible and is dependent on the concentrations of the substrates. The pH optimum for the mammalian enzyme is between 7 and 8 and may be dependent on the transfer of a specific acyl group. Using the partially purified enzyme from rat liver, the transfer of oleoyl-CoA was found to be more active at pH 7, whereas arachidonoyl-CoA transfer was more rapid at a higher p H . 23 Recently, the molecular weight of the purified enzyme was determined to be 43,000 for bovine brain n and 64,000 for bovine heart. 18The partially purified enzyme from rabbit lung displayed an iso-Ping-Pong mechanism, and the K mvalues for palmitoyl-CoA and 1-acyl-GPC were found to be 8.5 and 61 /~M, respectively. 24In human platelets, the Km values for saturated and unsaturated acyl-CoA vary from 1.05 to 5.70/~M in the presence of 100/~M 1-acyl-GPC.25 In most mammalian tissues, the Km values for acyl-CoA and 1-acyl-GPC vary between 1-15/~M and 50-100/~M, respectively. Regulation of Enzyme Activity 1-AcyI-GPC acyltransferase activity in rat lung was inhibited by high concentrations of 1-acyl-GPC, but the inhibition by high concentrations of acyl-CoAs was not prominent. 26 Enzyme activity in the liver microsomes was relatively unaffected by sulfhydryl-binding reagents such as iodoacetate, N-ethylmaleimide, and p-chloromercuriphenylsulfonic acid, but the activity of the partially purified enzyme was inhibited by these reagents. 23 The compounds WY-14643 and clofibric acid were found to inhibit mitochondrial 1-acyl-GPC:acyl-CoA acyltransferase. 27 Methyllidocaine, a local anesthetic, caused the inhibition of enzyme activity in mammalian heart. 2s In addition, divalent cations such as Mg 2÷ and Ca 2÷ were inhibitory to enzyme activity. 7,29 Acyltransferase activity in vivo was affected by long-term administration of clofibric acid and chronic administration of isoproterenol, s'29 Enzyme activity in rat liver, rat heart, and rabbit gastric mucosa microsomes was inhibited by detergents and 23 H. Hasegawa-Sasaki and K. Ohno, Biochim. Biophys. Acta 617, 205 (1980). 24 R. Arche, P. Estrada, and C. Acebal, Arch. Biochem. Biophys. 257, 131 (1987). 25 M. L. McKean, J. B. Smith, and M. J. Silver, J. Biol. Chem. 257, 11278 (1982). 26 H. Hasegawa-Sasaki and K. Ohno, Biochim. Biophys. Acta 380, 486 (1975). 27 W. W. Riley and D. R. Pfeiffer, J. Biol. Chem. 761, 14018 (1986). :s p. G. Tardi, R. Y. K. Man, C. R. McMaster, and P. C. Choy, Biochem. Cell Biol. 68, 745 (1990). 29 K. Yashiro, Y. Kameyama, M. Mizuno, S. Hayashi, Y. Sakashita, and Y. Yokota, Biochin*. Biophys. Acta 1005, 56 (1989).
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inhibitors of cyclic nucleotide phosphodiesterase. 3° The regulation of enzyme activity by a phosphorylation-dephosphorylation cycle has been postulated. 31 Concluding Remarks Since the discovery of acyltransferases it has been postulated that there may be separate enzymes for each acyl group. Indirect evidence with Triton X-100-treated microsomes indicates that there may be a specific acyltransferase for oleoyl-CoA. 21 The identification of an acyltransferase with an absolute specificity for lineoleoyl-CoA lends support to this hypothesis. 14Purification of the enzyme from bovine brain causes an enrichment of specificity toward arachidonoyl-CoA. 11 Thus, acyl specificities previously determined in subcellular fractions could simply reflect the quantitative distribution of different acyltransferases in these fractions. Direct evidence for the existence of multiple forms of the acyltransferase is still lacking. 3o W. T. Shier, Biochem. Biophys. Res. Commun. 75, 186 (1977). 31 S. L. Reinhold, G. A. Zimmerman, S. M. Prescott, and T. M. McIntyre, J. Biol. Chem. 264, 21652 (1989).
[9] 1 - A l k y l - a n d
1-Alkenylglycerophosphocholine Acyltransferases
B y PATRICK C. CHOY a n d CHRISTOPHER R . M C M A S T E R
Introduction Three forms of choline-containing phospholipids are found in mammalian tissues. 1 The 1,2-diacyl form (phosphatidylcholine) is the most abundant form and is present as the major phospholipid in all mammalian tissues. The l-alkyl-2-acyl form (plasmanylcholine) is found in significant amounts in circulating cells such as neutrophils and macrophages but in low amounts in other tissues. The 1-alkenyl-2-acyl form (plasmenylcholine) comprises up to 40% of the choline-containing phospholipids in electrically active tissues such as the heart. The major catabolic pathway for 1-alkyl-2-acylglycerophosphocholine (1-alkyl-2-acyl-GPC) or 1-alkenyl-2-acylglycerophosphocholine 1 L. A. Horrocks and M. Sharma, in "Phospholipids" (J. N. Hawthorne and G. B. Ansell, eds.), p. 51. Elsevier Biomedical Press, Amsterdam, New York, and Oxford, 1982.
METHODS IN ENZYMOLOGY, VOL. 209
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