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Additional improvement in purification of mitochondrial glycerophosphate acyltranferase was achieved using affinity chromatography on palmityl-CoA-agarose or glycerol 3-phosphate-agarose. When a glycerol 3-phosphate agarose column was used instead of octyl-Sepharose CL-4B column, the overall purification of glycerophosphate acyltransferase was over 40-fold. The molecular weight of the native glycerophosphate acyltransferase was 60-85 kDa as determined by gel filtration on Sephacryl S-300 HR in 0.2% CHAPS. Comparison of this result with electrophoretic data strongly suggests the mitochondrial glycerophosphate acyltransferase to be a monomeric enzyme. SDS-PAGE of the purified glycerophosphate acyltransferase followed by Coomassie blue staining exhibited a single band with a molecular weight of 80-85 kDa. Acknowledgment This work was supported by a grant from the National Science Foundation (DCB8801535).
[7] C o e n z y m e A - I n d e p e n d e n t A c y l t r a n s f e r a s e
By TAKAYUKI SUGIURA and KEIZO WAKU Introduction The coenzyme A (CoA)-independent transacylation system was first described by Kramer and Deykin 1'2 for human platelets. Similar enzyme activity was also found for several mammalian tissues and cells 36 including rabbit alveolar macrophages. 7-9 This system catalyzes the transfer of fatty I R. M. Kramer and D. Deykin, J. Biol. Chem. 258, 13806 (1983). 2 R. M. Kramer, G. M. Patton, C. R. Pritzker, and D. Deykin, J. Biol. Chem. 259, 13316 (1984). P. V. Reddy and H. H. O. Schmid, Biochem. Biophys. Res. Commun. 129, 381 (1985). 4 y . Masuzawa, S. Okano, Y. Nakagawa, A. Ojima, and K. Waku, Biochim. Biophys. Acta 876, 80 (1986). 5 A. Ojima, Y. Nakagawa, T. Sugiura, Y. Masuzawa, and K. Waku, J. Neurochem. 48, 1403 (1987). 6 0 . V. Reddy and H. H. O. Schmid, Biochim. Biophys. Acta 879, 369 (1986). 7 T. Sugiura and K. Waku, Biochem. Biophys. Res. Commun. 127, 384 (1985). 8 M. Robinson, M. L. Blank, and F. Snyder, J. Biol. Chem. 260, 7889 (1985). 9 T. Sugiura, Y. Masuzawa, Y. Nakagawa, and K. Waku, J. Biol. Chem. 262, 1199 (1987).
METHODS IN ENZYMOLOGY,VOL. 209
Copyright© 1992by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[7]
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acids from diradyl phospholipids to various lysophospholipids in the absence of any cofactors, differing from the CoA-dependent transacylation reaction which requires the presence of CoA. 10-14Free fatty acids cannot be introduced into phospholipids via this system. The types of fatty acids transferred by the CoA-independent system are restricted to C20 and C22 polyunsaturated fatty acids esterified at the 2-position of diradyl phospholipids, especially diacylglycerophosphocholine(diacyl-GPC). This is quite different from either the CoA-dependent transacylation system or the lysophospholipase-mediated transacylation reaction. Furthermore, the distribution among tissues and the subcellular localization of CoA-independent transacylation activity are also considerably different from those of CoA-dependent transacylation and lysophospholipase-mediated transacylation activities. Thus, CoA-independent transacylation is presumed to be a novel enzyme reaction which may play important roles in the remodeling of phospholipids in mammalian cells. It has been demonstrated 15-21 that C20 and C22 polyunsaturated fatty acids are gradually transferred from diacyl-GPC to ether-containing phospholipids in several inflammatory cells, endothelial cells, and testis under various conditions. It appears that such transfers can be responsible for the accumulation of C20 and C22 polyunsaturated fatty acids in ether phospholipids in several cell types. Although the precise mechanism for this transfer observed in living cells is not fully understood, it has been postulated that the CoA-independent transacylation system could be involved, at least in part, in the gradual transfer of polyunsaturated fatty acids between phospholipids. In fact, several lines of evidence suggest that the CoA-independent transacylation system is important in the reacylation of ether-containing lysophospholipids in mammalian tissues to provide polyunsaturated fatty acid-containing ether phospholipids as follows: (1) 10 R. F. Irvine and R. M. C. Dawson, Biochem. Biophys. Res. Commun. 91, 1399 (1979). ii R. M. Kramer, C. R. Pritzker, and D. Deykin, J. Biol. Chem. 259, 2403 (1984). n O. Colard, M. Breton, and G. Bereziat, Biochim. Biophys. Acta 793, 42 (1984). 13 j. Trotter, I. Flesch, B. Schmidt, and E. Ferber, J. Biol. Chem. 257, 1816 (1982). 14 T. Sugiura, Y. Masuzawa, and K. Waku, J. Biol. Chem. 263, 17490 (1988). Is M. L. Blank, R. L. Wykle, and F. Snyder, Biochim. Biophys. Acta 316, 28 (1973). t6 S. Rittenhouse-Simmons, F. A. Russell, and D. Deykin, Biochim. Biophys. Acta 488, 370 (1977). 17 T. Sugiura, O. Katayama, J. Fukui, Y. Nakagawa, and K. Waku, FEBS Lett. 165, 273 (1984). 18 T. Sugiura, Y. Masuzawa, and K. Waku, Biochem. Biophys. Res. Commun. 133, 574 (1985). 19 O. Colard, M. Breton, and G. Bereziat, Biochem. J. 222, 657 (1984). 2o F. H. Chilton and R. C. Murphy, J. Biol. Chem. 261, 7771 (1986). 21 F. H. Chilton, J. S. Hadley, and R. C. Murphy, Biochim. Biophys. Acta 917, 48 (1987).
74
ACYLTRANSFERASES
[7]
the enzyme activities of acyl-CoA : 1-alkyl-GPC and acyl-CoA : 1-alkenylGPC acyltransferases are usually low, and (2) fatty acid specificities of the acyl-CoA-mediated acylation of ether-containing lysophospholipids in vitro are different from those observed for the acylation profiles of ethercontaining lysophospholipids in living cells. In contrast, (3) the acylation profiles of the CoA-independent transacylation reaction observed in vitro closely resemble the acylation pattern of ether-containing lysophospholipids in living cells (see below), and (4) CoA-independent transacylation activity was shown to be present in microsomal fractions of various mammalian tissues and cells except for liver, in which ether-containing phospholipids are known to be almost absent. ~4 We suppose that the CoAindependent transacylation system will be effective in promptly disposing of ether-containing lysophospholipids generated within the cell to form nontoxic diradyl phospholipids. This might be different from the case of acyl-containing lysophospholipids which can be rapidly metabolized by lysophospholipase or by acyl-CoA : lysophospholipid acyltransferase. The properties of the CoA-independent transacylation reaction have been studied by several investigators. 1-9 However, a detailed mechanism of the reaction remains unclear. Purification and characterization of enzyme protein have not yet been successful. Thus, further efforts are required to investigate the properties of the enzyme involved in CoA-independent transacylation system and understand the precise mechanism of the reaction. In particular, detailed comparative studies of CoA-independent transacylation activity and phospholipase A 2 activity seem to be necessary in order to clarify the mechanism of the transacylation reaction, since the transacylation system involves the degradation of diradyl phospholipids in its initial step. In a previous study, we have shown that CoA-independent transacylation activity is almost negligible in macrophage cytosolic fractions in which a remarkably high activity of phospholipase A2 was noted, 9 indicating that cytosolic phospholipase A 2 is incapable of catalyzing the transacylation reaction. Furthermore, we found that the microsomal fraction obtained from the slug Incilaria bilineata does not contain CoA-independent transacylation activity, despite the presence of phospholipase A2 activity in this fraction and high amounts of ether phospholipids in this animal. 22 The absence of CoA-independent transacylation activity was also observed for the microsomal fraction from the earthworm Eisenia foetida, 22a suggesting that the acylation system for ether-containing lysophospholipids may be somewhat different between lower animals and mammals. In any event, it is obvious that phospholipase A2 lacks the ability to catalyze the 22 T. Sugiura, T. Ojima, T. Fukuda, K. Satouchi, K. Saito, and K. Waku, J. Lipid Res., in press. ~2a T. Sugiura, manuscript in preparation.
[7]
COENZYME A-INDEPENDENT ACYLTRANSFERASE
75
transacylation reaction in these cases. Recent progress in the structural analysis of phospholipase A2 in mammalian tissues provided the evidence for the occurrence of several types of phospholipase A2. There remains the possibility, therefore, that some forms can catalyze the transfer of polyunsaturated fatty acids between phospholipids. Alternatively, intrinsic enzyme protein other than phospholipase A2 may catalyze the reaction. Further detailed studies on this issue may answer the question and contribute to a better understanding of the regulation of polyunsaturated fatty acid metabolism in mammalian tissues. Procedures Dual-assay systems are possible, one containing radiolabeled exogenous or endogenous phospholipids as acyl donors and nonradiolabeled lysophospholipids as acyl acceptors, the other containing endogenous membrane phospholipids as acyl donors and radiolabeled lysophospholipids as acyl acceptors. Both systems have advantages and disadvantages.
Transacylation from Exogenous Phospholipids CoA-independent transacylation activity is mainly found in the microsomal fraction (105,000 g pellet fraction) in various mammalian tissues. The mitochondrial fraction (7000 g pellet fraction) has also been shown to contain some activity, whereas the cytosolic fraction (105,000 g supernatant fraction) has not. We used, therefore, the microsomal fraction or membrane fraction as the enzyme s o u r c e . 4'5'7'9'18'23 The assay system for the transacylation of 1-alkyl-GPC by the macrophage microsomal fraction consists of a microsomal fraction (0.2 mg of protein), 5 nmol of 1-alkyl-GPC [lyso-platelet-activating factor (lyso-PAF)] (final 20/xM), 14C-labeled fatty acid-containing phospholipids [20,000 disintegrations/min (dpm)], and 250/~1 of 0. I M Tris-HC1 buffer (pH 7.4) containing 5 mM EGTA. 914C-Labeled fatty acid-containing phospholipids are sonicated in advance in distilled water with a Branson Sonifier (Danbury, CT) (40 W, 4 periods of 10 sec each). Alternatively, labeled phospholipids can be previously dissolved in ethanol and then directly added to the incubation mixture. Similar results are obtained in either case. If the ethanolamine-containing 1-alkyl(alkenyl)-GPE or 1-acyl-GPE is used as acceptor instead of choline-containing lysophospholipids, it is also necessary to sonicate these lysophospholipids in distilled water. The incubation is carried out at 37° for various periods of time. The 53 T. Sugiura, T. Fukuda, Y. Masuzawa, and K. Waku, Biochim. Biophys. Acta 1047, 223 (1990).
76
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reaction is linear at least up to 30-60 min, which may be dependent on the concentration of the microsomal protein. If higher concentrations of l-alkyl-GPC are employed, the reaction is rather reduced, probably owing to the detergent effect of 1-alkyl-GPC. In the case of ethanolamine-containing lysophospholipids such as 1-alkenyl(alkyl)-GPE, we could not find strong inhibition. The incubation is stopped by adding chloroform-methanol, and total lipids are extracted by the method of Bligh and Dyer. Individual phospholipids are separated by two-dimensional thin-layer chromatography (TLC), first with chloroform-methanol-28% NH4OH (65 : 35 : 5, v/v) and second with chloroform-acetone-methanol-acetic acid-water (5 : 2 : 1 : 1.5 : 0.5, v/v). To measure the distribution of radioactivities among subclasses of individual phospholipids (especially for CGP and EGP), they are hydrolyzed with phospholipase C and subsequently acetylated with acetic anhydride and pyridine. The resultant 1,2-diradyl-3-acetylglycerols are separated by TLC developed first with petroleum ether-diethyl ether-acetic acid (90 : l0 : 1, v/v) and then with toluene. This technique enables the resolution of individual phospholipids into alkenylacyl, alkylacyl, and diacyl subclasses. Such separation is essential for the analysis of the transacylation reaction where donor phospholipids and acceptor lysophospholipids have the same polar head groups. To estimate the transfer of ~4C-labeled fatty acids between the same phospholipid class (e.g., between diacyl-GPC and 1-acyl-GPC), different fatty acyl moieties at the 1-position are selected for donor and acceptor phospholipids. The reaction product (transacylated phospholipids) can be separated from the original material (donor phospholipids) by high-performance liquid chromatography (HPLC) on the basis of differences of the fatty acids at the 1-position after conversion to 1,2-diradyl-3-acetylglycerol derivatives as described below. The assay method with exogenous donor phospholipids has the advantage of using chemically defined phospholipids with various types of fatty acids at the I- and 2-positions. However, the estimation by this method might be influenced to some extent by the amount and composition of endogenous phospholipids in the microsomal (membrane) fraction used as the enzyme sources. Furthermore, physicochemical properties of donor phospholipids in the incubation mixture must be taken into account. In order to exclude such possibilities, we mix the donor phospholipids containing different fatty acids and then used this mixture in some experiments. Similar fatty acid specificities are observed even in the case of the mixed donor phospholipids. Moreover, the fatty acid specificity determined using the exogenous phospholipids coincides with that obtained with endogenous membrane phospholipids. 5'9
[7]
COENZYME A-INDEPENDENT ACYLTRANSFERASE
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Transacylation from Endogenous Phospholipids The assay system contains a microsomal fraction (0.2 mg of protein) and 5 nmol of various radiolabeled lysophospholipids such as 1-[3H]alkyl GPC (40,000 dpm, final 20/xM) in 250 ~1 of 0. I M Tris-HCl buffer (pH 7.4) containing 5 mM EGTA. 9 The incubation is carried out at 37° for various periods of time. The incubation is stopped by adding chloroform-methanol, and total lipids are then extracted by the method of Bligh and Dyer. The individual phospholipids are separated by two-dimensional TLC, and the subclasses are separated from each other as described above. The enzyme activity is calculated from the radioactivity found in the product (the acylated lysophospholipid) and that in the original lysophospholipid. To examine the fatty acid specificity of the reaction, the purified 1,2diradyl-3-acetylglycerols are further fractionated into molecular species by reversed-phase HPLC according to the method of Nakagawa and Horrocks.24 Briefly, the sample, dissolved in 20/~1 of methanol, is injected into a HPLC system (Shimadzu, Kyoto, Japan, LC-6A) equipped with a 4.6 mm x 25 cm Zorbax ODS column (Du Pont Co., Wilmington, DE) and an ultraviolet detector which is operated at 205 nm. Alkenylacyl and alkylacyl compounds are eluted with acetonitrile-2-propanol-methyl tertbutyl ether-water (63 : 28 : 7 : 2, v/v), and diacyl compounds are separated with a solvent system of acetonitrile-2-propanol-methyl tert-butyl ether-water (72 : 18 : 8 : 2, v/v). Figure 1 shows typical acylation profiles of 1-[3H]alkyl(18 : 0)-GPC by intact cells (Fig. la) and by the microsomal fraction in the absence of cofactors (Fig. lb). The values are the radioactivities from individual fractions (tube/40 sec) collected between 12 and 52 min after injection of the samples. In this method, the enzyme utilizes endogenous phospholipids as acyl donors, making it unnecessary to prepare various types of phospholipids having different radiolabeled fatty acids. Furthermore, the physicochemical effects on the enzyme activity of dispersing donor phospholipids into water can be neglected. On the other hand, the disadvantages of this method may be that the fatty acid composition of the membrane phospholipids affect the fatty acid specificity of the reaction. In addition, it seems important to take special care in the interpretation of the results, especially in experiments where acyl-containing lysophospholipids are used as acyl acceptors, since acyl-containing lysophospholipids can be acylated either via the CoA-independent transacylation reaction or via the lysophospholipase-mediated transacylation reaction. In addition to the above types of 24 y . Nakagawa and L. A. Horrocks, J. Lipid Res. 24, 1268 (1983).
78
ACYLTRANSFERASES
[7]
"7,
a I
~5
X
A
..':r o4
6
I
Lf~ O4 . . . . . .
t.o ..
(N i
o~
~(N i
o
.O.
i
o o
ooo~
20
40
.~.$
60 f r a c t i o n number
b
x
~o
.5 =
1
20
40
60 f r a c t i o n number
FIG. I. Chromatographic analysis of the acylation profiles of 1-[3-H]alkyl(18 : 0)-GPC by intact macrophages (a) and by macrophage microsomes (b).9 I-[3H]-Alkyl-GPC was incubated with intact macrophages (a) and macrophage microsomes alone (b) at 37° for 60 min. The acylated product was purified and then analyzed following suitable modifications.
[7]
COENZYME A-INDEPENDENT ACYLTRANSFERASE
79
experiments, it is also possible to use prelabeled endogenous phospholipids as acyl donors. Kramer and Deykin I studied the CoA-independent transacylation reaction using 3H-prelabeled platelet membranes. This method is also very useful, although the donor phospholipid may not be accurately specified in this case compared with the experiments where chemically defined donor phospholipids are employed. Properties The CoA-independent transacylation system was shown to catalyze the transfer of various C2o and C22 polyunsaturated fatty acids either from exogenously added phospholipids or from endogenous membrane phospholipids.1-9'23 Both n-6 and n-3 acids can be transferred. On the other hand, the transfer rates for C~6 and CIS acids such as 16 : 0, 18 : 0, 18 : 1, and 18:2 were low, if any. Thus, the CoA-independent transacylation system appears to have high specificity with respect to the transferable fatty acyl residues. Concerning the types of donor phospholipids, CGP, especially diacyl-GPC, was shown to be the most preferred substrate. ~,9 Although diacyl-GPE also serves as the donor phospholipid to some extent, diacylglycerophosphoinositol (diacyl-GPI) does not act at least in the case where l-alkyl-GPC is used as an acceptor. As for the acceptor lysophospholipids, three subclasses of choline-containing lysophospholipids (1-alkenyl-GPC, 1-alkyl-GPC, and 1-acyl-GPC) were shown to be effective acceptors, 1-alkyl-GPC being most rapidly acylated among them. 9 1-Alkenyl-GPE, 1-alkyl-GPE, and 1-acyl-GPE were also acylated with 20:4 transferred from diacyl-GPC. On the other hand, 1-acyl-GPI and 1-acylglycerophosphoserine (1-acyl-GPS), as well as 1-acylglycerophosphate, do not serve as effective acceptors. ~,9 Free fatty acids once liberated from donor phospholipids could not be involved in the transacylation reaction, since exogenously added free 3Hlabeled 20:4 acids failed to be incorporated into phospholipids in the absence of cofactors. 7 Furthermore, Kramer and Deykin I demonstrated that the addition of unlabeled 20 : 4 acids did not affect the arachidonoyl transacylation reaction at least at lower concentrations, though a slight inhibition was observed at higher concentrations. We concluded that such inhibition could be attributed to the nonspecific physicochemical effects of free fatty acids, since the addition of 18 : 2 acids also gave a similar inhibition curve. 9 It has already been suggested that the possible formation of the arachidonoyl enzyme intermediate may take place before the reesterification of 20:4 acids into lysophospholipids, ~ although the precise mechanism is still unknown. The CoA-independent transacylation has a broad pH optimum, ranging
80
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.
ACYLTRANSFERASES
[7]
Additional improvement in purification of mitochondrial glycerophosphate acyltranferase was achieved using affinity chromatography on palmityl-CoA-agarose or glycerol 3-phosphate-agarose. When a glycerol 3-phosphate agarose column was used instead of octyl-Sepharose CL-4B column, the overall purification of glycerophosphate acyltransferase was over 40-fold. The molecular weight of the native glycerophosphate acyltransferase was 60-85 kDa as determined by gel filtration on Sephacryl S-300 HR in 0.2% CHAPS. Comparison of this result with electrophoretic data strongly suggests the mitochondrial glycerophosphate acyltransferase to be a monomeric enzyme. SDS-PAGE of the purified glycerophosphate acyltransferase followed by Coomassie blue staining exhibited a single band with a molecular weight of 80-85 kDa. Acknowledgment This work was supported by a grant from the National Science Foundation (DCB8801535).
[7] C o e n z y m e A - I n d e p e n d e n t A c y l t r a n s f e r a s e
By TAKAYUKI SUGIURA and KEIZO WAKU Introduction The coenzyme A (CoA)-independent transacylation system was first described by Kramer and Deykin 1'2 for human platelets. Similar enzyme activity was also found for several mammalian tissues and cells 36 including rabbit alveolar macrophages. 7-9 This system catalyzes the transfer of fatty I R. M. Kramer and D. Deykin, J. Biol. Chem. 258, 13806 (1983). 2 R. M. Kramer, G. M. Patton, C. R. Pritzker, and D. Deykin, J. Biol. Chem. 259, 13316 (1984). P. V. Reddy and H. H. O. Schmid, Biochem. Biophys. Res. Commun. 129, 381 (1985). 4 y . Masuzawa, S. Okano, Y. Nakagawa, A. Ojima, and K. Waku, Biochim. Biophys. Acta 876, 80 (1986). 5 A. Ojima, Y. Nakagawa, T. Sugiura, Y. Masuzawa, and K. Waku, J. Neurochem. 48, 1403 (1987). 6 0 . V. Reddy and H. H. O. Schmid, Biochim. Biophys. Acta 879, 369 (1986). 7 T. Sugiura and K. Waku, Biochem. Biophys. Res. Commun. 127, 384 (1985). 8 M. Robinson, M. L. Blank, and F. Snyder, J. Biol. Chem. 260, 7889 (1985). 9 T. Sugiura, Y. Masuzawa, Y. Nakagawa, and K. Waku, J. Biol. Chem. 262, 1199 (1987).
METHODS IN ENZYMOLOGY,VOL. 209
Copyright© 1992by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[7]
COENZYME A-INDEPENDENT ACYLTRANSFERASE
73
acids from diradyl phospholipids to various lysophospholipids in the absence of any cofactors, differing from the CoA-dependent transacylation reaction which requires the presence of CoA. 10-14Free fatty acids cannot be introduced into phospholipids via this system. The types of fatty acids transferred by the CoA-independent system are restricted to C20 and C22 polyunsaturated fatty acids esterified at the 2-position of diradyl phospholipids, especially diacylglycerophosphocholine(diacyl-GPC). This is quite different from either the CoA-dependent transacylation system or the lysophospholipase-mediated transacylation reaction. Furthermore, the distribution among tissues and the subcellular localization of CoA-independent transacylation activity are also considerably different from those of CoA-dependent transacylation and lysophospholipase-mediated transacylation activities. Thus, CoA-independent transacylation is presumed to be a novel enzyme reaction which may play important roles in the remodeling of phospholipids in mammalian cells. It has been demonstrated 15-21 that C20 and C22 polyunsaturated fatty acids are gradually transferred from diacyl-GPC to ether-containing phospholipids in several inflammatory cells, endothelial cells, and testis under various conditions. It appears that such transfers can be responsible for the accumulation of C20 and C22 polyunsaturated fatty acids in ether phospholipids in several cell types. Although the precise mechanism for this transfer observed in living cells is not fully understood, it has been postulated that the CoA-independent transacylation system could be involved, at least in part, in the gradual transfer of polyunsaturated fatty acids between phospholipids. In fact, several lines of evidence suggest that the CoA-independent transacylation system is important in the reacylation of ether-containing lysophospholipids in mammalian tissues to provide polyunsaturated fatty acid-containing ether phospholipids as follows: (1) 10 R. F. Irvine and R. M. C. Dawson, Biochem. Biophys. Res. Commun. 91, 1399 (1979). ii R. M. Kramer, C. R. Pritzker, and D. Deykin, J. Biol. Chem. 259, 2403 (1984). n O. Colard, M. Breton, and G. Bereziat, Biochim. Biophys. Acta 793, 42 (1984). 13 j. Trotter, I. Flesch, B. Schmidt, and E. Ferber, J. Biol. Chem. 257, 1816 (1982). 14 T. Sugiura, Y. Masuzawa, and K. Waku, J. Biol. Chem. 263, 17490 (1988). Is M. L. Blank, R. L. Wykle, and F. Snyder, Biochim. Biophys. Acta 316, 28 (1973). t6 S. Rittenhouse-Simmons, F. A. Russell, and D. Deykin, Biochim. Biophys. Acta 488, 370 (1977). 17 T. Sugiura, O. Katayama, J. Fukui, Y. Nakagawa, and K. Waku, FEBS Lett. 165, 273 (1984). 18 T. Sugiura, Y. Masuzawa, and K. Waku, Biochem. Biophys. Res. Commun. 133, 574 (1985). 19 O. Colard, M. Breton, and G. Bereziat, Biochem. J. 222, 657 (1984). 2o F. H. Chilton and R. C. Murphy, J. Biol. Chem. 261, 7771 (1986). 21 F. H. Chilton, J. S. Hadley, and R. C. Murphy, Biochim. Biophys. Acta 917, 48 (1987).
74
ACYLTRANSFERASES
[7]
the enzyme activities of acyl-CoA : 1-alkyl-GPC and acyl-CoA : 1-alkenylGPC acyltransferases are usually low, and (2) fatty acid specificities of the acyl-CoA-mediated acylation of ether-containing lysophospholipids in vitro are different from those observed for the acylation profiles of ethercontaining lysophospholipids in living cells. In contrast, (3) the acylation profiles of the CoA-independent transacylation reaction observed in vitro closely resemble the acylation pattern of ether-containing lysophospholipids in living cells (see below), and (4) CoA-independent transacylation activity was shown to be present in microsomal fractions of various mammalian tissues and cells except for liver, in which ether-containing phospholipids are known to be almost absent. ~4 We suppose that the CoAindependent transacylation system will be effective in promptly disposing of ether-containing lysophospholipids generated within the cell to form nontoxic diradyl phospholipids. This might be different from the case of acyl-containing lysophospholipids which can be rapidly metabolized by lysophospholipase or by acyl-CoA : lysophospholipid acyltransferase. The properties of the CoA-independent transacylation reaction have been studied by several investigators. 1-9 However, a detailed mechanism of the reaction remains unclear. Purification and characterization of enzyme protein have not yet been successful. Thus, further efforts are required to investigate the properties of the enzyme involved in CoA-independent transacylation system and understand the precise mechanism of the reaction. In particular, detailed comparative studies of CoA-independent transacylation activity and phospholipase A 2 activity seem to be necessary in order to clarify the mechanism of the transacylation reaction, since the transacylation system involves the degradation of diradyl phospholipids in its initial step. In a previous study, we have shown that CoA-independent transacylation activity is almost negligible in macrophage cytosolic fractions in which a remarkably high activity of phospholipase A2 was noted, 9 indicating that cytosolic phospholipase A 2 is incapable of catalyzing the transacylation reaction. Furthermore, we found that the microsomal fraction obtained from the slug Incilaria bilineata does not contain CoA-independent transacylation activity, despite the presence of phospholipase A2 activity in this fraction and high amounts of ether phospholipids in this animal. 22 The absence of CoA-independent transacylation activity was also observed for the microsomal fraction from the earthworm Eisenia foetida, 22a suggesting that the acylation system for ether-containing lysophospholipids may be somewhat different between lower animals and mammals. In any event, it is obvious that phospholipase A2 lacks the ability to catalyze the 22 T. Sugiura, T. Ojima, T. Fukuda, K. Satouchi, K. Saito, and K. Waku, J. Lipid Res., in press. ~2a T. Sugiura, manuscript in preparation.
[7]
COENZYME A-INDEPENDENT ACYLTRANSFERASE
75
transacylation reaction in these cases. Recent progress in the structural analysis of phospholipase A2 in mammalian tissues provided the evidence for the occurrence of several types of phospholipase A2. There remains the possibility, therefore, that some forms can catalyze the transfer of polyunsaturated fatty acids between phospholipids. Alternatively, intrinsic enzyme protein other than phospholipase A2 may catalyze the reaction. Further detailed studies on this issue may answer the question and contribute to a better understanding of the regulation of polyunsaturated fatty acid metabolism in mammalian tissues. Procedures Dual-assay systems are possible, one containing radiolabeled exogenous or endogenous phospholipids as acyl donors and nonradiolabeled lysophospholipids as acyl acceptors, the other containing endogenous membrane phospholipids as acyl donors and radiolabeled lysophospholipids as acyl acceptors. Both systems have advantages and disadvantages.
Transacylation from Exogenous Phospholipids CoA-independent transacylation activity is mainly found in the microsomal fraction (105,000 g pellet fraction) in various mammalian tissues. The mitochondrial fraction (7000 g pellet fraction) has also been shown to contain some activity, whereas the cytosolic fraction (105,000 g supernatant fraction) has not. We used, therefore, the microsomal fraction or membrane fraction as the enzyme s o u r c e . 4'5'7'9'18'23 The assay system for the transacylation of 1-alkyl-GPC by the macrophage microsomal fraction consists of a microsomal fraction (0.2 mg of protein), 5 nmol of 1-alkyl-GPC [lyso-platelet-activating factor (lyso-PAF)] (final 20/xM), 14C-labeled fatty acid-containing phospholipids [20,000 disintegrations/min (dpm)], and 250/~1 of 0. I M Tris-HC1 buffer (pH 7.4) containing 5 mM EGTA. 914C-Labeled fatty acid-containing phospholipids are sonicated in advance in distilled water with a Branson Sonifier (Danbury, CT) (40 W, 4 periods of 10 sec each). Alternatively, labeled phospholipids can be previously dissolved in ethanol and then directly added to the incubation mixture. Similar results are obtained in either case. If the ethanolamine-containing 1-alkyl(alkenyl)-GPE or 1-acyl-GPE is used as acceptor instead of choline-containing lysophospholipids, it is also necessary to sonicate these lysophospholipids in distilled water. The incubation is carried out at 37° for various periods of time. The 53 T. Sugiura, T. Fukuda, Y. Masuzawa, and K. Waku, Biochim. Biophys. Acta 1047, 223 (1990).
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reaction is linear at least up to 30-60 min, which may be dependent on the concentration of the microsomal protein. If higher concentrations of l-alkyl-GPC are employed, the reaction is rather reduced, probably owing to the detergent effect of 1-alkyl-GPC. In the case of ethanolamine-containing lysophospholipids such as 1-alkenyl(alkyl)-GPE, we could not find strong inhibition. The incubation is stopped by adding chloroform-methanol, and total lipids are extracted by the method of Bligh and Dyer. Individual phospholipids are separated by two-dimensional thin-layer chromatography (TLC), first with chloroform-methanol-28% NH4OH (65 : 35 : 5, v/v) and second with chloroform-acetone-methanol-acetic acid-water (5 : 2 : 1 : 1.5 : 0.5, v/v). To measure the distribution of radioactivities among subclasses of individual phospholipids (especially for CGP and EGP), they are hydrolyzed with phospholipase C and subsequently acetylated with acetic anhydride and pyridine. The resultant 1,2-diradyl-3-acetylglycerols are separated by TLC developed first with petroleum ether-diethyl ether-acetic acid (90 : l0 : 1, v/v) and then with toluene. This technique enables the resolution of individual phospholipids into alkenylacyl, alkylacyl, and diacyl subclasses. Such separation is essential for the analysis of the transacylation reaction where donor phospholipids and acceptor lysophospholipids have the same polar head groups. To estimate the transfer of ~4C-labeled fatty acids between the same phospholipid class (e.g., between diacyl-GPC and 1-acyl-GPC), different fatty acyl moieties at the 1-position are selected for donor and acceptor phospholipids. The reaction product (transacylated phospholipids) can be separated from the original material (donor phospholipids) by high-performance liquid chromatography (HPLC) on the basis of differences of the fatty acids at the 1-position after conversion to 1,2-diradyl-3-acetylglycerol derivatives as described below. The assay method with exogenous donor phospholipids has the advantage of using chemically defined phospholipids with various types of fatty acids at the I- and 2-positions. However, the estimation by this method might be influenced to some extent by the amount and composition of endogenous phospholipids in the microsomal (membrane) fraction used as the enzyme sources. Furthermore, physicochemical properties of donor phospholipids in the incubation mixture must be taken into account. In order to exclude such possibilities, we mix the donor phospholipids containing different fatty acids and then used this mixture in some experiments. Similar fatty acid specificities are observed even in the case of the mixed donor phospholipids. Moreover, the fatty acid specificity determined using the exogenous phospholipids coincides with that obtained with endogenous membrane phospholipids. 5'9
[7]
COENZYME A-INDEPENDENT ACYLTRANSFERASE
77
Transacylation from Endogenous Phospholipids The assay system contains a microsomal fraction (0.2 mg of protein) and 5 nmol of various radiolabeled lysophospholipids such as 1-[3H]alkyl GPC (40,000 dpm, final 20/xM) in 250 ~1 of 0. I M Tris-HCl buffer (pH 7.4) containing 5 mM EGTA. 9 The incubation is carried out at 37° for various periods of time. The incubation is stopped by adding chloroform-methanol, and total lipids are then extracted by the method of Bligh and Dyer. The individual phospholipids are separated by two-dimensional TLC, and the subclasses are separated from each other as described above. The enzyme activity is calculated from the radioactivity found in the product (the acylated lysophospholipid) and that in the original lysophospholipid. To examine the fatty acid specificity of the reaction, the purified 1,2diradyl-3-acetylglycerols are further fractionated into molecular species by reversed-phase HPLC according to the method of Nakagawa and Horrocks.24 Briefly, the sample, dissolved in 20/~1 of methanol, is injected into a HPLC system (Shimadzu, Kyoto, Japan, LC-6A) equipped with a 4.6 mm x 25 cm Zorbax ODS column (Du Pont Co., Wilmington, DE) and an ultraviolet detector which is operated at 205 nm. Alkenylacyl and alkylacyl compounds are eluted with acetonitrile-2-propanol-methyl tertbutyl ether-water (63 : 28 : 7 : 2, v/v), and diacyl compounds are separated with a solvent system of acetonitrile-2-propanol-methyl tert-butyl ether-water (72 : 18 : 8 : 2, v/v). Figure 1 shows typical acylation profiles of 1-[3H]alkyl(18 : 0)-GPC by intact cells (Fig. la) and by the microsomal fraction in the absence of cofactors (Fig. lb). The values are the radioactivities from individual fractions (tube/40 sec) collected between 12 and 52 min after injection of the samples. In this method, the enzyme utilizes endogenous phospholipids as acyl donors, making it unnecessary to prepare various types of phospholipids having different radiolabeled fatty acids. Furthermore, the physicochemical effects on the enzyme activity of dispersing donor phospholipids into water can be neglected. On the other hand, the disadvantages of this method may be that the fatty acid composition of the membrane phospholipids affect the fatty acid specificity of the reaction. In addition, it seems important to take special care in the interpretation of the results, especially in experiments where acyl-containing lysophospholipids are used as acyl acceptors, since acyl-containing lysophospholipids can be acylated either via the CoA-independent transacylation reaction or via the lysophospholipase-mediated transacylation reaction. In addition to the above types of 24 y . Nakagawa and L. A. Horrocks, J. Lipid Res. 24, 1268 (1983).
78
ACYLTRANSFERASES
[7]
"7,
a I
~5
X
A
..':r o4
6
I
Lf~ O4 . . . . . .
t.o ..
(N i
o~
~(N i
o
.O.
i
o o
ooo~
20
40
.~.$
60 f r a c t i o n number
b
x
~o
.5 =
1
20
40
60 f r a c t i o n number
FIG. I. Chromatographic analysis of the acylation profiles of 1-[3-H]alkyl(18 : 0)-GPC by intact macrophages (a) and by macrophage microsomes (b).9 I-[3H]-Alkyl-GPC was incubated with intact macrophages (a) and macrophage microsomes alone (b) at 37° for 60 min. The acylated product was purified and then analyzed following suitable modifications.
[7]
COENZYME A-INDEPENDENT ACYLTRANSFERASE
79
experiments, it is also possible to use prelabeled endogenous phospholipids as acyl donors. Kramer and Deykin I studied the CoA-independent transacylation reaction using 3H-prelabeled platelet membranes. This method is also very useful, although the donor phospholipid may not be accurately specified in this case compared with the experiments where chemically defined donor phospholipids are employed. Properties The CoA-independent transacylation system was shown to catalyze the transfer of various C2o and C22 polyunsaturated fatty acids either from exogenously added phospholipids or from endogenous membrane phospholipids.1-9'23 Both n-6 and n-3 acids can be transferred. On the other hand, the transfer rates for C~6 and CIS acids such as 16 : 0, 18 : 0, 18 : 1, and 18:2 were low, if any. Thus, the CoA-independent transacylation system appears to have high specificity with respect to the transferable fatty acyl residues. Concerning the types of donor phospholipids, CGP, especially diacyl-GPC, was shown to be the most preferred substrate. ~,9 Although diacyl-GPE also serves as the donor phospholipid to some extent, diacylglycerophosphoinositol (diacyl-GPI) does not act at least in the case where l-alkyl-GPC is used as an acceptor. As for the acceptor lysophospholipids, three subclasses of choline-containing lysophospholipids (1-alkenyl-GPC, 1-alkyl-GPC, and 1-acyl-GPC) were shown to be effective acceptors, 1-alkyl-GPC being most rapidly acylated among them. 9 1-Alkenyl-GPE, 1-alkyl-GPE, and 1-acyl-GPE were also acylated with 20:4 transferred from diacyl-GPC. On the other hand, 1-acyl-GPI and 1-acylglycerophosphoserine (1-acyl-GPS), as well as 1-acylglycerophosphate, do not serve as effective acceptors. ~,9 Free fatty acids once liberated from donor phospholipids could not be involved in the transacylation reaction, since exogenously added free 3Hlabeled 20:4 acids failed to be incorporated into phospholipids in the absence of cofactors. 7 Furthermore, Kramer and Deykin I demonstrated that the addition of unlabeled 20 : 4 acids did not affect the arachidonoyl transacylation reaction at least at lower concentrations, though a slight inhibition was observed at higher concentrations. We concluded that such inhibition could be attributed to the nonspecific physicochemical effects of free fatty acids, since the addition of 18 : 2 acids also gave a similar inhibition curve. 9 It has already been suggested that the possible formation of the arachidonoyl enzyme intermediate may take place before the reesterification of 20:4 acids into lysophospholipids, ~ although the precise mechanism is still unknown. The CoA-independent transacylation has a broad pH optimum, ranging
80
ACYLTRANSFERASES
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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.
