426
PHOSPHOLIPASE A 2
[40]
[40] P h o s p h o l i p a s e A c t i v i t y o f Lecithin-Cholesterol Acyltransferase
By
CHRISTOPHER J. F I E L D I N G a n d XAVIER COLLET
Introduction Lecithin-cholesterol acyltransferase (LCAT; EC 2.3.1.43) in mammalian plasma reacts with high-density lipoprotein (HDL) to catalyze the transfer of an acyl group from lecithin to the 3-position of cholesterol with the production of lysolecithin and cholesteryl ester (Table I). Shortly after the first isolation of the enzyme protein, 1 it was determined that, in the absence of sterol, LCAT could also catalyze the hydrolysis of lecithin with the production of lysolecithin and unesterified fatty acid. 2'3 Subsequently it was shown that the LCAT protein was also responsible for the lecithin-lysolecithin acyltransferase (LLAT) exchange reaction which had been reported earlier to catalyze the exchange of acyl chains between lecithin and lysolecithin in plasma. 4'5 LCAT also shows esterase activity with short- and medium-chain p-nitrophenyl esters. 6 The LCAT gene has been cloned and sequenced and the predicted amino acid sequence of the enzyme described. 7'8 Identical information was subsequently obtained by direct peptide sequencing. 9 The amino acid sequence of LCAT shows some local similarities to other lipases, particularly lipoprotein lipase and the heparin-released hepatic triglyceride lipase. 10,11Like these, LCAT appears to be a classic serine hydrolase. The i j. j. Albers, V. G. Cabana, and Y. D. B. Stahl, Biochemistry 15, 1084 (1976). 2 U. Piran and T. Nishida, J. Biochem. (Tokyo) 80, 887 (1976). 3 L. Aron, S. Jones, and C. J. Fielding, J. Biol. Chem. 253, 7220 (1978) 4 p. V. Subbaiah, this series, Vol. 129 [47]. 5 p. V. Subbaiah, J. J. Albers, C. H. Chen, and J. D. Bagdade, J. Biol. Chem. 255, 9275 (1980). 6 F. S. Bonelli and A. Jonas, J. Biol. Chem. 264, 14723, (1989). 7 j. McLean, C. Fielding, D. Drayna, H. Dieplinger, B. Baer, W. Kohr, W. Henzei, and R. Lawn, Proc. Natl. Acad. Sci. U.S.A. 83, 2335 (1986). 8 j. McLean, K. Wion, D. Drayna, C. Fielding, and R. Lawn, Nucleic Acids Res. 14, 9397 (1986). 9 C. Y. Yang, D. Manoogian, Q. Pao, F. S. Lee, R. D. Knapp, A. M. Gotto, and H. J. Pownall, J. Biol. Chem. 262, 3086 (1987). 10 K. Wion, T. D. Kirchgessner, A. J. Lusis, M. C. Schotz, and R. M. Lawn, Science 235, 1638 (1987).
METHODS IN ENZYMOLOGY,VOL. 197
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any form reserved.
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LECITHIN-CHOLESTEROL ACYLTRANSFERASE
427
TABLE I REACTIONS CATALYZEDBY LECITHIN-CHOLESTEROLACYLTRANSFERASE Phospholipid substrate
Phospholipid product
Acylated product
Lysolecithin
Lecithin
Alcohols (sterois, long-chain alcohols) Water
Lecithin
Lysolecithin
Lysolecithin
Cholesteryl esters, long-chain esters Unesterified fatty acid Lecithin
Lecithin
-OH substrate
Lysolecithin
presence of active site serine and histidine residues has been demonstrated by chemical modification, 12'13and it is likely that the active site of LCAT involves the classic triad of serine, histidine, and aspartate residues. The phospholipase activity of LCAT has some unusual features. It is Ca 2+ independent, as shown by its undiminished activity in the presence of high concentrations of EGTA or EDTA. 3 LCAT phospholipase and transferase activities show predominant 2-position specificity3,14 but not the absolute specificity shown by many phospholipases (see other chapters in this volume). Finally the phospholipase activity of LCAT, like its acyltransferase activity, is markedly dependent on the presence oflipoprotein apolipoproteins, most effectively apolipoprotein A-I (apoA-I), the major protein of the physiological substrate, HDL. Pure LCAT shows very little catalytic activity with phospholipid vesicles in the absence of apoA-I. 3 As is the case with other phospholipases, the activity of LCAT is highly dependent on the physical structure of the lipid interface. Studies with various phospholipids dissolved in a nonhydrolyzable phosphatidyl ether matrix show that much of the substrate specificity observed when LCAT reacts with pure phospholipid dispersions reflects this type of effect, rather than true substrate specificity.15 Two recent reviews summarize current information about LCAT biochemistry. One summarizes the gene and protein structure and mechanism
11 G. A. Martin, S. Busch, G. D. Meredith, A. D. Cardin, D. T. Blankenship, S. T. J. Mao, A. E. Rechlin, C. W. Woods, M. M. Racke, M. P. Schafer, M. C. Fitzgerald, D. M. Burke, M. A. Flanagan, and R. L. Jackson, J. Biol. Chem. 263, 10907 (1988). i2 M. Jauhiainen and P. J. Dolphin, J. Biol. Chem. 261, 7032 (1986). 13 M. Jauhiainen, N. D. Ridgeway, and P. J. Dolphin, Biochim. Biophys. Acta 918, 175 (1987). J4 G. Assmann, G. Schmitz, N. Donath, and D. Lekim, Scand. J. Clin. Invest. 38 (Suppl. 150), 16 (1978). 15 H. J. Pownall, Q. Pao, and J. B. Massey, J. Biol. Chem. 260, 2146 (1985).
428
PHOSPHOLIPASEA2
[40]
of LCAT.16 The other provides a comprehensive account of biophysical aspects of substrate structure and mechanism.17 This chapter focuses on the phospholipase reactions of LCAT.
Isolation of Lecithin-Cholesterol Acyltransferase Protein Human plasma contains LCAT protein at a concentration of about 6 /zg/ml TMand has been used as source for almost all purification procedures described. The available methods are referenced in an earlier chapter in this series. 19 The following method is suitable for routine use. Step 1: Ultracentrifugal Flotation. Plasma (1000 ml) from blood collected in a final concentration of l0 mM sodium citrate (pH 7.0) is brought to 1 mM with disodium EDTA and to a solvent density of 1.21 g/ml with solid KBr (0.33 g/ml plasma) that has been maintained in a desiccator at 120°. Centrifugation is carried out for 48 hr at 40,000 rpm in a Beckman ultracentrifuge at 00-4 °. The floating colored lipoprotein layer is first removed by suction. The clear intermediate zone is then collected for further purification. Step 2: Phenyi-Agarose Affinity Chromatography. The material from Step 1 is applied to a column (2.5 × 20 cm) of phenyl-agarose (PharmaciaLKB, Uppsala, Sweden) equilibrated with 3 M NaC1, 1 mM EDTA. After washing with 500 ml of the same solution, the column is washed with 0.15 M NaC1, l mM EDTA, until the OD2s0 of the eluate is below 0.05. The remaining bound protein is then eluated with distilled water. Step 3: DEAE-Cellulose Chromatography. The eluate from Step 2 is applied to a column (1 × 10 cm) of DEAE-cellulose [Whatman (Clifton, NJ) DE-52] equilibrated with l0 mM Tris-HC1 (pH 7.4) and fractionated on a 250-rnl linear gradient of 0-0.35 M NaC1 in the same buffer. The enzyme activity is eluted in approximately 0.15 M NaCl. Step 4: Hydroxylapatite Chromatography. The DEAE-cellulose fractions containing LCAT activity are pooled and passed on to a column (1.5 × 4 cm) of hydroxylapatite (BioGel HT, Bio-Rad, Richmond, CA) equilibrated with l0 rnM Tris-HCl buffer, pH 7.0. The column is then eluted with a gradient (0-15 raM) of phosphate in the same buffer. Different batches of hydroxylapatite show considerably different binding properties 16 C. J. Fielding, in "Advances in Cholesterol Research" (M. Esfahani and J. Swaney, eds.), p. 271. Telford Press, New Jersey, 1990. 17 A. Jonas, in "Plasma Lipoproteins" (A. M. Gotto, ed.), p. 299. Elsevier, Amsterdam, 1989. 18 j. j. Albers, J. L. Adolphson, and C. H. Chen, J. Clin. Invest. 67, 141 (1981). 19j. j. Albers, C. H. Chen, and A. G. Lacko, this series, Vol. 129 [45].
[40]
LECITHIN-CHOLESTEROL ACYLTRANSFERASE
429
and must be standardized. LCAT activity is typically eluted as a sharp peak at phosphate concentrations of about 5 mM. The product, essentially pure by sensitive silver staining following sodium dodecyl sulfate (SDS)-gel electrophoresis, is recovered in a yield of about 10% purified about 20,000-fold from the original plasma.
Assay of Lecithin-Cholesterol Acyltransferase Phospholipase Activity Principle. LCAT phospholipase is assayed as the production of unesterified fatty acid from synthetic lecithins labeled in the acyl moiety with 3H or 14C. Lecithin + H20 ~ lysolecithin + fatty acid
Sources of Labeled Lecithins. A variety of synthetic labeled lecithins can now be purchased commercially. Double-labeled lecithins or unusual lecithin species unavailable commercially are readily synthesized. Labeled unesterified fatty acid is converted to its anhydride with cyclohexylcarbodiimide, and the labeled anhydride is then reacted with anhydrous glycerylphosphorylcholine-CdC12 complex or unlabeled 1-acyllysolecithin under vacuum at 70 ° for 72 hr. 3 The lecithin product is purified from fatty acid or unconverted lysolecithin by extraction into chloroform-methanol and thin-layer chromatography on silica gel layers developed in chloroform-methanol-water (65 : 35 : 5, v/v). Although pure dipalmitoyllecithin vesicles are a poor substrate for LCAT, the commercially available di[3H] palmitoyl)lecithin is readily hydrolyzed codispersed with a synthetic phosphatidylcholine ether or with egg lecithin and in this form is the most economical substrate for the routine phospholipase assay of LCAT. Sources of Activator Apolipoprotein A-I. ApoA-I is routinely prepared in the laboratory from human plasma HDL by delipidation with ethanol and ether followed by molecular sieve and DEAE-cellulose chromatography in buffer solutions containing 8 M urea. 2° It is now also available commercially as a lyophilized powder (Sigma, St. Louis, MO) which dissolves readily at a concentration of 1 mg/ml in 2 mM phosphate buffer, pH 7.4, and gives an activation of LCAT with synthetic phospholipid vesicles that is comparable with that obtained with locally prepared flashfrozen apoA-I in the same buffer solution. Preparation of Single-Walled Lecithin Vesicles. A convenient French Press method has been described in detail by Hamilton et al.,21 who also 2o C. Edelstein, C. T. Lim, and A. M. Scanu, J. Biol. Chem. 247, 5842 (1972). 21 R. L. Hamilton, J. Goerke, L. S. S. Guo, M. C. Williams, and R. J. Havel, J. Lipid Res. 21, 981 (1980).
430
PHOSPHOLIPASE A 2
[40]
described the size and structure of the lecithin dispersions obtained. The instrument (Aminco, Silver Spring, MD) consists of a titanium cell with a small orifice and a press used to force the lecithin dispersion through the orifice at pressures of up to 2000 pounds per square inch. Lecithin (1-5 mg/ml, 0.5-5.0 ml) is mixed to an opalescent dispersion by hand vortexing in distilled water, then transferred to the cell and dispersed by 3 cycles of pressure and release. The clear product consists largely of uniform singlewalled vesicles of diameter about 200/~.22 If required the product can be purified by subsequent molecular sieve chromatography on a column (0.9 × 20 cm) of Sepharose 4B (Pharmacia-LKB) in 0.1 M Tris-HC1 buffer (pH 7.4), although (as discussed below) activation of these vesicles with apoA-I involves conversion of the single-walled vesicles to a characteristic discoidal structure. 22 Single-walled vesicles can also be obtained with a sonifier (Heat Systems-Utrasonics, Plainview, NY) 23 The crude lecithin dispersion is sonicated for 3 min under nitrogen with cooling. It is usually necesary to remove titanium fragments from the probe by centrifugation (1000 g, 15 min) before use. Lecithin in solvents or detergent solutions vesiculate spontaneously when dialyzed, 24,25and such vesicles have been used as effective substrates for LCAT activity. Advantages of these methods include their utility, as specialized equipment is not needed. A potential disadvantage may be the retention of traces of dilute trapped solvent or detergent in the vesicles. This can be overcome by appropriate control procedures, for example by demonstrating complete removal of labeled detergent following dialysis. Assay Media for LCAT Phospholipase Activity. The kinetic characteristics and cofactor requirements of LCAT phospholipase activity are very similar to those of the corresponding acyltransferase activity. Assay media contain the following components (volumes given are per assay): 50 ~l 2palmitoyl[9,10-3H]phosphatidylcholine in egg lecithin [final concentration to /.~mol/ml; specific activity 0.5-1.0 x 106 disintegrations per minute (dpm)//xmol] dispersed in distilled water and 50/~l apolipoprotein A-I (0.25 mg/ml) reconstituted in 1 mM phosphate buffer (pH 7.4). These reagents are preincubated for 60 min at 37° to complete apoA-I-lecithin discoidal lipoprotein formation. 22 After incubation the following are added: 50/.d recrystallized human albumin solution (100 mg/ml) in 0.15 M NaCl, pH 22 L. S. S. Guo, R. L. Hamilton, J. Goerke, J. N. Weinstein, and R. J. Havel, J. Lipid Res. 21, 993 (1980). 23 C. H. Huang, Biochemistry 8, 344 (1969). C. H. Chen and J. J. Albers, J. Lipid Res. 23, 680 (1982). 25 S. Batzri and E. D. Korn, Biochim. Biophys. Acta 298, 1015 (1973).
[40]
LECITHIN---CHOLESTEROL ACYLTRANSFERASE
431
7.4 (to bind product unesterified fatty acid and lysolecithin), 50/zl of 50 mM Tris-HC1 buffer (pH 7.4), and 200/zl of 0.15 M NaCI. The reaction is started by addition of 50/zl of purified LCAT solution or 50/zl of the same buffer without enzyme. Incubation is normally linear for at least 60 min, if less than 5% of the initial substrate is hydrolyzed. At the end of the incubation, the assays are chilled in ice water, and an equal volume of methanol containing 0.5 M H2SO4 is added, then the same volume of chloroform. After vortexing to mix the phases, 250/xl of the lower chloroform phase is taken for thin-layer chromatography on silica gel G layers on plastic sheets (Merck, Darmstadt, FRG) developed in hexane-diethyl ether-acetic acid (83 : 16 : I, v/v/v). The simultaneous and reproducible application of multiple samples to the silica gel layers is conveniently automated with an TLC Multispotter (AIS, Libertyville, IL) or similar instrument. After development and drying (5-10 min) in a fume hood, the unesterified fatty acid region of the silica gel (Rf ~0.4) is identified with minimal exposure in an iodine vapor tank. After the removal of visible iodine in the fume hood, the fatty acid region is cut out and counted with a liquid scintillation counter. In this system unhydrolyzed lecithin and lysolecithin both remain near the origin. The recovery of unesterified fatty acid is over 90%. Enzymatic Properties of Lecithin-Cholesterol AcyltransferaseDependent Phospholipase Activity S u b s t r a t e S p e c i f i c i t y . As assayed in a nonhydrolyzable matrix of phosphatidyl ether, LCAT hydrolyzes lecithin and phosphatidylethanolamine at roughly equivalent rates. Other phospholipids are hydrolyzed more slowly. There is no detectable activity against the Noacyl residue of sphingomyelin. 26 In reaction with pure substrates of long-chain lecithins LCAT shows the greatest reactivity with mono- and diunsaturated lecithins and less activity with the corresponding saturated species. ~5,~8,24 However, this probably relates mainly to the physical structure of the substrate since dipalmitoyllecithin, a very poor substrate as a pure lipid, is relatively reactive in a dimyristoylphosphatidyl ether matrix.15 Although LCAT shows a predominant 2-positional specificity, this is not absolute 3:5 (Table II). The generation of minor amounts of 1-position acylation products may be the result of acyl migration catalyzed by the LLAT reaction during the course of hydrolysis:
1-Palmitoyl-2-oleyllecithin--,1-palmitoyllysolecithin+ oleic acid 26C. J. Fielding, Scand. J. Clin, Lab. Invest. 33 (Suppl. 137), 15 (1974).
(1)
432
[40]
PHOSPHOLIPASE A 2 TABLE II POSITIONAL SPECIFICITY OF LCAT PHOSPHOLIPASEAND ACYLTRANSFERASE ACTIVITIESa
Percentage of fatty acids released Unesterified fatty acids
Cholesteryl ester fatty acids
Lecithin substrate
16:0
18:1
16:0
18:1
l-Palmitoyl-2-oleyl
13.4 90.8
86.6 9.2
23.3 90.4
76.7 9.6
l-Oleyl-2-palmitoyl a Data from Ref. 3.
1-Palmitoyl-2-oleyllecithin + l-palmitoyllecithin ~ 1,2-dipalmitoyllecithin + l-oleyllecithin (2) 1,2-Dipalmitoyllecithin ~ 1-palmitoyllecithin + palmitic acid (3)
The relative proportions of 1- and 2-position fatty acids would then depend on the relative rates of the LCAT and LLAT reactions of the enzyme with a given substrate. As the rate of the LLAT reaction is L~creased about 10fold in the presence of low-density lipoprotein (LDL),5 this type of reaction may be more significant quantitatively in native plasma than in experiments with pure reconstituted reagents as shown in Table II. Dependence of LCAT Phospholipase Activity on ApoA-I. Both acyltransferase and phospholipase activities of LCAT with long-chain lecithins TABLE III ApoA-I DEPENDENCE OF LCAT PHOSPHOLIPASE ACTIVITYa ApoA-I (/zg/ml assay)
Oleic acid released (nmol/ml/hr)
0 2.5 5.0 10.0 15.0 20.0 25.0 50.0
PHOSPHOLIPASE A 2
[40]
[40] P h o s p h o l i p a s e A c t i v i t y o f Lecithin-Cholesterol Acyltransferase
By
CHRISTOPHER J. F I E L D I N G a n d XAVIER COLLET
Introduction Lecithin-cholesterol acyltransferase (LCAT; EC 2.3.1.43) in mammalian plasma reacts with high-density lipoprotein (HDL) to catalyze the transfer of an acyl group from lecithin to the 3-position of cholesterol with the production of lysolecithin and cholesteryl ester (Table I). Shortly after the first isolation of the enzyme protein, 1 it was determined that, in the absence of sterol, LCAT could also catalyze the hydrolysis of lecithin with the production of lysolecithin and unesterified fatty acid. 2'3 Subsequently it was shown that the LCAT protein was also responsible for the lecithin-lysolecithin acyltransferase (LLAT) exchange reaction which had been reported earlier to catalyze the exchange of acyl chains between lecithin and lysolecithin in plasma. 4'5 LCAT also shows esterase activity with short- and medium-chain p-nitrophenyl esters. 6 The LCAT gene has been cloned and sequenced and the predicted amino acid sequence of the enzyme described. 7'8 Identical information was subsequently obtained by direct peptide sequencing. 9 The amino acid sequence of LCAT shows some local similarities to other lipases, particularly lipoprotein lipase and the heparin-released hepatic triglyceride lipase. 10,11Like these, LCAT appears to be a classic serine hydrolase. The i j. j. Albers, V. G. Cabana, and Y. D. B. Stahl, Biochemistry 15, 1084 (1976). 2 U. Piran and T. Nishida, J. Biochem. (Tokyo) 80, 887 (1976). 3 L. Aron, S. Jones, and C. J. Fielding, J. Biol. Chem. 253, 7220 (1978) 4 p. V. Subbaiah, this series, Vol. 129 [47]. 5 p. V. Subbaiah, J. J. Albers, C. H. Chen, and J. D. Bagdade, J. Biol. Chem. 255, 9275 (1980). 6 F. S. Bonelli and A. Jonas, J. Biol. Chem. 264, 14723, (1989). 7 j. McLean, C. Fielding, D. Drayna, H. Dieplinger, B. Baer, W. Kohr, W. Henzei, and R. Lawn, Proc. Natl. Acad. Sci. U.S.A. 83, 2335 (1986). 8 j. McLean, K. Wion, D. Drayna, C. Fielding, and R. Lawn, Nucleic Acids Res. 14, 9397 (1986). 9 C. Y. Yang, D. Manoogian, Q. Pao, F. S. Lee, R. D. Knapp, A. M. Gotto, and H. J. Pownall, J. Biol. Chem. 262, 3086 (1987). 10 K. Wion, T. D. Kirchgessner, A. J. Lusis, M. C. Schotz, and R. M. Lawn, Science 235, 1638 (1987).
METHODS IN ENZYMOLOGY,VOL. 197
Copyright© 1991by AcademicPress, Inc. All rightsof reproductionin any form reserved.
[40]
LECITHIN-CHOLESTEROL ACYLTRANSFERASE
427
TABLE I REACTIONS CATALYZEDBY LECITHIN-CHOLESTEROLACYLTRANSFERASE Phospholipid substrate
Phospholipid product
Acylated product
Lysolecithin
Lecithin
Alcohols (sterois, long-chain alcohols) Water
Lecithin
Lysolecithin
Lysolecithin
Cholesteryl esters, long-chain esters Unesterified fatty acid Lecithin
Lecithin
-OH substrate
Lysolecithin
presence of active site serine and histidine residues has been demonstrated by chemical modification, 12'13and it is likely that the active site of LCAT involves the classic triad of serine, histidine, and aspartate residues. The phospholipase activity of LCAT has some unusual features. It is Ca 2+ independent, as shown by its undiminished activity in the presence of high concentrations of EGTA or EDTA. 3 LCAT phospholipase and transferase activities show predominant 2-position specificity3,14 but not the absolute specificity shown by many phospholipases (see other chapters in this volume). Finally the phospholipase activity of LCAT, like its acyltransferase activity, is markedly dependent on the presence oflipoprotein apolipoproteins, most effectively apolipoprotein A-I (apoA-I), the major protein of the physiological substrate, HDL. Pure LCAT shows very little catalytic activity with phospholipid vesicles in the absence of apoA-I. 3 As is the case with other phospholipases, the activity of LCAT is highly dependent on the physical structure of the lipid interface. Studies with various phospholipids dissolved in a nonhydrolyzable phosphatidyl ether matrix show that much of the substrate specificity observed when LCAT reacts with pure phospholipid dispersions reflects this type of effect, rather than true substrate specificity.15 Two recent reviews summarize current information about LCAT biochemistry. One summarizes the gene and protein structure and mechanism
11 G. A. Martin, S. Busch, G. D. Meredith, A. D. Cardin, D. T. Blankenship, S. T. J. Mao, A. E. Rechlin, C. W. Woods, M. M. Racke, M. P. Schafer, M. C. Fitzgerald, D. M. Burke, M. A. Flanagan, and R. L. Jackson, J. Biol. Chem. 263, 10907 (1988). i2 M. Jauhiainen and P. J. Dolphin, J. Biol. Chem. 261, 7032 (1986). 13 M. Jauhiainen, N. D. Ridgeway, and P. J. Dolphin, Biochim. Biophys. Acta 918, 175 (1987). J4 G. Assmann, G. Schmitz, N. Donath, and D. Lekim, Scand. J. Clin. Invest. 38 (Suppl. 150), 16 (1978). 15 H. J. Pownall, Q. Pao, and J. B. Massey, J. Biol. Chem. 260, 2146 (1985).
428
PHOSPHOLIPASEA2
[40]
of LCAT.16 The other provides a comprehensive account of biophysical aspects of substrate structure and mechanism.17 This chapter focuses on the phospholipase reactions of LCAT.
Isolation of Lecithin-Cholesterol Acyltransferase Protein Human plasma contains LCAT protein at a concentration of about 6 /zg/ml TMand has been used as source for almost all purification procedures described. The available methods are referenced in an earlier chapter in this series. 19 The following method is suitable for routine use. Step 1: Ultracentrifugal Flotation. Plasma (1000 ml) from blood collected in a final concentration of l0 mM sodium citrate (pH 7.0) is brought to 1 mM with disodium EDTA and to a solvent density of 1.21 g/ml with solid KBr (0.33 g/ml plasma) that has been maintained in a desiccator at 120°. Centrifugation is carried out for 48 hr at 40,000 rpm in a Beckman ultracentrifuge at 00-4 °. The floating colored lipoprotein layer is first removed by suction. The clear intermediate zone is then collected for further purification. Step 2: Phenyi-Agarose Affinity Chromatography. The material from Step 1 is applied to a column (2.5 × 20 cm) of phenyl-agarose (PharmaciaLKB, Uppsala, Sweden) equilibrated with 3 M NaC1, 1 mM EDTA. After washing with 500 ml of the same solution, the column is washed with 0.15 M NaC1, l mM EDTA, until the OD2s0 of the eluate is below 0.05. The remaining bound protein is then eluated with distilled water. Step 3: DEAE-Cellulose Chromatography. The eluate from Step 2 is applied to a column (1 × 10 cm) of DEAE-cellulose [Whatman (Clifton, NJ) DE-52] equilibrated with l0 mM Tris-HC1 (pH 7.4) and fractionated on a 250-rnl linear gradient of 0-0.35 M NaC1 in the same buffer. The enzyme activity is eluted in approximately 0.15 M NaCl. Step 4: Hydroxylapatite Chromatography. The DEAE-cellulose fractions containing LCAT activity are pooled and passed on to a column (1.5 × 4 cm) of hydroxylapatite (BioGel HT, Bio-Rad, Richmond, CA) equilibrated with l0 rnM Tris-HCl buffer, pH 7.0. The column is then eluted with a gradient (0-15 raM) of phosphate in the same buffer. Different batches of hydroxylapatite show considerably different binding properties 16 C. J. Fielding, in "Advances in Cholesterol Research" (M. Esfahani and J. Swaney, eds.), p. 271. Telford Press, New Jersey, 1990. 17 A. Jonas, in "Plasma Lipoproteins" (A. M. Gotto, ed.), p. 299. Elsevier, Amsterdam, 1989. 18 j. j. Albers, J. L. Adolphson, and C. H. Chen, J. Clin. Invest. 67, 141 (1981). 19j. j. Albers, C. H. Chen, and A. G. Lacko, this series, Vol. 129 [45].
[40]
LECITHIN-CHOLESTEROL ACYLTRANSFERASE
429
and must be standardized. LCAT activity is typically eluted as a sharp peak at phosphate concentrations of about 5 mM. The product, essentially pure by sensitive silver staining following sodium dodecyl sulfate (SDS)-gel electrophoresis, is recovered in a yield of about 10% purified about 20,000-fold from the original plasma.
Assay of Lecithin-Cholesterol Acyltransferase Phospholipase Activity Principle. LCAT phospholipase is assayed as the production of unesterified fatty acid from synthetic lecithins labeled in the acyl moiety with 3H or 14C. Lecithin + H20 ~ lysolecithin + fatty acid
Sources of Labeled Lecithins. A variety of synthetic labeled lecithins can now be purchased commercially. Double-labeled lecithins or unusual lecithin species unavailable commercially are readily synthesized. Labeled unesterified fatty acid is converted to its anhydride with cyclohexylcarbodiimide, and the labeled anhydride is then reacted with anhydrous glycerylphosphorylcholine-CdC12 complex or unlabeled 1-acyllysolecithin under vacuum at 70 ° for 72 hr. 3 The lecithin product is purified from fatty acid or unconverted lysolecithin by extraction into chloroform-methanol and thin-layer chromatography on silica gel layers developed in chloroform-methanol-water (65 : 35 : 5, v/v). Although pure dipalmitoyllecithin vesicles are a poor substrate for LCAT, the commercially available di[3H] palmitoyl)lecithin is readily hydrolyzed codispersed with a synthetic phosphatidylcholine ether or with egg lecithin and in this form is the most economical substrate for the routine phospholipase assay of LCAT. Sources of Activator Apolipoprotein A-I. ApoA-I is routinely prepared in the laboratory from human plasma HDL by delipidation with ethanol and ether followed by molecular sieve and DEAE-cellulose chromatography in buffer solutions containing 8 M urea. 2° It is now also available commercially as a lyophilized powder (Sigma, St. Louis, MO) which dissolves readily at a concentration of 1 mg/ml in 2 mM phosphate buffer, pH 7.4, and gives an activation of LCAT with synthetic phospholipid vesicles that is comparable with that obtained with locally prepared flashfrozen apoA-I in the same buffer solution. Preparation of Single-Walled Lecithin Vesicles. A convenient French Press method has been described in detail by Hamilton et al.,21 who also 2o C. Edelstein, C. T. Lim, and A. M. Scanu, J. Biol. Chem. 247, 5842 (1972). 21 R. L. Hamilton, J. Goerke, L. S. S. Guo, M. C. Williams, and R. J. Havel, J. Lipid Res. 21, 981 (1980).
430
PHOSPHOLIPASE A 2
[40]
described the size and structure of the lecithin dispersions obtained. The instrument (Aminco, Silver Spring, MD) consists of a titanium cell with a small orifice and a press used to force the lecithin dispersion through the orifice at pressures of up to 2000 pounds per square inch. Lecithin (1-5 mg/ml, 0.5-5.0 ml) is mixed to an opalescent dispersion by hand vortexing in distilled water, then transferred to the cell and dispersed by 3 cycles of pressure and release. The clear product consists largely of uniform singlewalled vesicles of diameter about 200/~.22 If required the product can be purified by subsequent molecular sieve chromatography on a column (0.9 × 20 cm) of Sepharose 4B (Pharmacia-LKB) in 0.1 M Tris-HC1 buffer (pH 7.4), although (as discussed below) activation of these vesicles with apoA-I involves conversion of the single-walled vesicles to a characteristic discoidal structure. 22 Single-walled vesicles can also be obtained with a sonifier (Heat Systems-Utrasonics, Plainview, NY) 23 The crude lecithin dispersion is sonicated for 3 min under nitrogen with cooling. It is usually necesary to remove titanium fragments from the probe by centrifugation (1000 g, 15 min) before use. Lecithin in solvents or detergent solutions vesiculate spontaneously when dialyzed, 24,25and such vesicles have been used as effective substrates for LCAT activity. Advantages of these methods include their utility, as specialized equipment is not needed. A potential disadvantage may be the retention of traces of dilute trapped solvent or detergent in the vesicles. This can be overcome by appropriate control procedures, for example by demonstrating complete removal of labeled detergent following dialysis. Assay Media for LCAT Phospholipase Activity. The kinetic characteristics and cofactor requirements of LCAT phospholipase activity are very similar to those of the corresponding acyltransferase activity. Assay media contain the following components (volumes given are per assay): 50 ~l 2palmitoyl[9,10-3H]phosphatidylcholine in egg lecithin [final concentration to /.~mol/ml; specific activity 0.5-1.0 x 106 disintegrations per minute (dpm)//xmol] dispersed in distilled water and 50/~l apolipoprotein A-I (0.25 mg/ml) reconstituted in 1 mM phosphate buffer (pH 7.4). These reagents are preincubated for 60 min at 37° to complete apoA-I-lecithin discoidal lipoprotein formation. 22 After incubation the following are added: 50/.d recrystallized human albumin solution (100 mg/ml) in 0.15 M NaCl, pH 22 L. S. S. Guo, R. L. Hamilton, J. Goerke, J. N. Weinstein, and R. J. Havel, J. Lipid Res. 21, 993 (1980). 23 C. H. Huang, Biochemistry 8, 344 (1969). C. H. Chen and J. J. Albers, J. Lipid Res. 23, 680 (1982). 25 S. Batzri and E. D. Korn, Biochim. Biophys. Acta 298, 1015 (1973).
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LECITHIN---CHOLESTEROL ACYLTRANSFERASE
431
7.4 (to bind product unesterified fatty acid and lysolecithin), 50/zl of 50 mM Tris-HC1 buffer (pH 7.4), and 200/zl of 0.15 M NaCI. The reaction is started by addition of 50/zl of purified LCAT solution or 50/zl of the same buffer without enzyme. Incubation is normally linear for at least 60 min, if less than 5% of the initial substrate is hydrolyzed. At the end of the incubation, the assays are chilled in ice water, and an equal volume of methanol containing 0.5 M H2SO4 is added, then the same volume of chloroform. After vortexing to mix the phases, 250/xl of the lower chloroform phase is taken for thin-layer chromatography on silica gel G layers on plastic sheets (Merck, Darmstadt, FRG) developed in hexane-diethyl ether-acetic acid (83 : 16 : I, v/v/v). The simultaneous and reproducible application of multiple samples to the silica gel layers is conveniently automated with an TLC Multispotter (AIS, Libertyville, IL) or similar instrument. After development and drying (5-10 min) in a fume hood, the unesterified fatty acid region of the silica gel (Rf ~0.4) is identified with minimal exposure in an iodine vapor tank. After the removal of visible iodine in the fume hood, the fatty acid region is cut out and counted with a liquid scintillation counter. In this system unhydrolyzed lecithin and lysolecithin both remain near the origin. The recovery of unesterified fatty acid is over 90%. Enzymatic Properties of Lecithin-Cholesterol AcyltransferaseDependent Phospholipase Activity S u b s t r a t e S p e c i f i c i t y . As assayed in a nonhydrolyzable matrix of phosphatidyl ether, LCAT hydrolyzes lecithin and phosphatidylethanolamine at roughly equivalent rates. Other phospholipids are hydrolyzed more slowly. There is no detectable activity against the Noacyl residue of sphingomyelin. 26 In reaction with pure substrates of long-chain lecithins LCAT shows the greatest reactivity with mono- and diunsaturated lecithins and less activity with the corresponding saturated species. ~5,~8,24 However, this probably relates mainly to the physical structure of the substrate since dipalmitoyllecithin, a very poor substrate as a pure lipid, is relatively reactive in a dimyristoylphosphatidyl ether matrix.15 Although LCAT shows a predominant 2-positional specificity, this is not absolute 3:5 (Table II). The generation of minor amounts of 1-position acylation products may be the result of acyl migration catalyzed by the LLAT reaction during the course of hydrolysis:
1-Palmitoyl-2-oleyllecithin--,1-palmitoyllysolecithin+ oleic acid 26C. J. Fielding, Scand. J. Clin, Lab. Invest. 33 (Suppl. 137), 15 (1974).
(1)
432
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PHOSPHOLIPASE A 2 TABLE II POSITIONAL SPECIFICITY OF LCAT PHOSPHOLIPASEAND ACYLTRANSFERASE ACTIVITIESa
Percentage of fatty acids released Unesterified fatty acids
Cholesteryl ester fatty acids
Lecithin substrate
16:0
18:1
16:0
18:1
l-Palmitoyl-2-oleyl
13.4 90.8
86.6 9.2
23.3 90.4
76.7 9.6
l-Oleyl-2-palmitoyl a Data from Ref. 3.
1-Palmitoyl-2-oleyllecithin + l-palmitoyllecithin ~ 1,2-dipalmitoyllecithin + l-oleyllecithin (2) 1,2-Dipalmitoyllecithin ~ 1-palmitoyllecithin + palmitic acid (3)
The relative proportions of 1- and 2-position fatty acids would then depend on the relative rates of the LCAT and LLAT reactions of the enzyme with a given substrate. As the rate of the LLAT reaction is L~creased about 10fold in the presence of low-density lipoprotein (LDL),5 this type of reaction may be more significant quantitatively in native plasma than in experiments with pure reconstituted reagents as shown in Table II. Dependence of LCAT Phospholipase Activity on ApoA-I. Both acyltransferase and phospholipase activities of LCAT with long-chain lecithins TABLE III ApoA-I DEPENDENCE OF LCAT PHOSPHOLIPASE ACTIVITYa ApoA-I (/zg/ml assay)
Oleic acid released (nmol/ml/hr)
0 2.5 5.0 10.0 15.0 20.0 25.0 50.0
