[39]
ERYTHROCYTEPAF ACETYLHYDROLASE
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isoforms is fully activated by physiologic concentrations of calcium ion, exhibiting 30 and 90% of maximal enzymatic activity in the presence of 200 and 800 nM calcium, respectively (Fig. 4). In contrast, the less acidic phospholipase A2 [cytosolic phospholipase A2 (a)] requires over 1 mM CaCI2to reach half-maximal activity, exhibiting nominal activity at calcium concentrations less than 100/zM. Thus, phospholipase A2 (fl) is the major phospholipase A 2 activity in platelet cytosol, is comprised of dimeric polypeptides which are exquisitely sensitive to physiologic alterations in calcium ion concentration (200-800 nM), and demonstrates a substantial preference for ether-linked phospholipid substrates. Since the majority of platelet arachidonic acid mass is contained in plasmalogen molecular species, these results suggest that activation of this phospholipase A 2 (13) during platelet stimulation by physiologic alterations in calcium ion concentration (500-800 nM) results in the selective release of arachidonic acid and contributes to the synthesis of platelet-activating factor. Acknowledgments This research was supported by Grants HL34839 and HL35864. RWG is the recipient of an Established Investigator Award from the American Heart Association.
[39] P l a t e l e t - A c t i v a t i n g F a c t o r A c e t y l h y d r o l a s e in Human Erythrocytes B y DIANA M. STAFFORINI, STEPHEN M. PRESCOTT, THOMAS M. MCINTYRE
and
Introduction Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) is a phospholipid that has been shown to possess potent biological activity. PAF induces hypotension, leukopenia, and thrombocytopenia 1 and increases vascular permeability. 2 PAF also activates platelets, neutrophils, and macrophages. A variety of cells, for example, neutrophils, macrophages, and endothelial cells, synthesize PAF on appropriate
1 M. Halonen, J. D. Palmer, I. C. Lohman, L. M. McManus, and R. N. Pinckard, Annu. Rev. Resp. Dis. 122, 915 (1980). 2 j. Bjork and G. Smedegard, J. Allergy Clin. Immunol. 71, 145 (1983).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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PHOSPHOLIPASEA 2
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stimulation. 3 Some inflammatory cells release most of the PAF that they synthesize (e.g., monocytes), 4 in contrast to vascular cells (e.g., endothelial cells) 5,6 which do not. PAF has been identified in human blood, saliva, urine, and amniotic fluid. 7-9 The rate of removal of PAF is likely an important means of regulating the bioactivity of this compound. Farr and co-workers 1°-12 were the first to demonstrate the occurrence, in mammalian plasma, of an enzyme that inactivates PAF by removal of the acetyl group esterifying the sn-2 position of glycerol, to produce lyso-PAF and acetate, which are biologically inactive. This reaction is catalyzed by a specific PAF acetylhydrolase (1-alkyl2-acetylglycerophosphocholine esterase, EC 3.1.1.47, 1-alkyl-2-acetyl-snglycero-3-phosphocholine acetohydrolase). The plasma PAF acetylhydrolase efficiently hydrolyzes PAF released by inflammatory cells such as monocytes. 13 In addition, the plasma enzyme is capable of inactivating PAF synthesized (but not released) by stimulated endothelial cells. 14Thus, the plasma PAF acetylhydrolase acts as a scavenger of circulating and cell-associated PAF. In addition to the plasma enzyme, intracellular PAF acetylhydrolases have been described in a variety of tissues and blood cells. 15,16 Blank et
3 S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre, Proc. Natl. Acad. Sci. U.S.A. 81, 3534 (1984). 4 M. R. Elstad, S. M. Prescott, T. M. McIntyre, and G. A. Zimmerman, J. lmmunol. 140, 1618 (1988). 5 S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre, Proc. Natl. Acad. Sci. U.S.A. 81, 3534 (1984). 6 F. Bussolino, F. Breviario, C. Tetta, M. Aglietta, A. Mantovani, and E. Dejana, J. Clin. Invest. 77, 2027 (1986). 7 K. E. Grandei, R. S. Farr, A. A. Wanderer, T. C. Eisenstadt, and S. I. Wasserman, N. Engl. J. Med. 313, 405 (1985). s C. P. Cox, M. L. Wardlow, R. Jorgensen, and R. S. Farr, J. lmmunol. 127, 46 (1981). 9 M. M. Billah and J. M. Johnston, Biochem. Biophys. Res. Commun. 113, 51 (1983). i0 R. S. Farr, C. P. Cox, M. L. Wardlow, and R. Jorgensen, Clin. Immunol. Immunopathol. 15, 318 (1980). 11 R. S. Farr, M. L. Wardlow, C. P. Cox, K. E. Meng, and D. E. Greene, Fed. Proc., Fed. Am. Soc. Exp. Biol. 42, 3120 (1983). 12M. L. Wardlow, C. P. Cox, K. E. Meng, D. Greene, and R. S. Farr, J. lmmunol. 136, 3441 (1986). 13D. M. Stafforini,M. R. Elstad, T. M. Mclntyre, G. A. Zimmerman, and S. M. Prescott, J. Biol. Chem. 265, 9682 (1990). 14G. A. Zilnmerman, T. M. McIntyre, M. Mehra, and S. M. Prescott,J. Ceil Biol. U0, 529 (1990). 15 R. Yanoshita, I. Kudo, K. Ikizawa, H. W. Chang, S. Kobayashi, M. Ohno, S. Nojima, and K. Inoue, J. Biochem. (Tokyo) 103, 815 (1988). 16T.-c. Lee, B. Malone, S. I. Wasserman, V. Fitzgerald,and F. Snyder, Biochem. Biophys. Res. C o m m u n . 105, 1303 (1982).
[39]
ERYTHROCYTEPAF ACETYLHYDROLASE
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al. ,7 described a PAF acetylhydrolase activity in the cytosolic fraction of mammalian tissues, Nijssen et al. 18 reported the presence of a similar
activity in rat lung cytosol, and we have studied the properties of PAF acetylhydrolases in human tissues and blood cells. 19 These activities belong to a family of intracellular PAF acetylhydrolases that, like the plasma enzyme, are constitutively active, require a short-chain residue at the sn-2 position, and are calcium independent. However, the intracellular activities can be differentiated from the plasma enzyme by a variety of biochemical criteria. 19'2° The function(s) of these activities is yet to be determined, but they may play an important role in decreasing the accumulation of intracellular PAF. For example, human macrophages regulate PAF accumulation by increasing the levels of intracellular PAF acetylhydrolase activity21; this may limit the activation of cells that respond to the lipid. In addition, the plasma 22 and intracellular23 PAF acetylhydrolases catalyze the degradation of oxidatively fragmented phospholipids. This may have major pathological significance since oxidatively fragmented phospholipids with toxic and biological activity have been shown to occur in vivo. 24'25 Thus, the PAF acetylhydrolases may play an important role as scavengers of PAF and toxic phospholipids. Human erythrocytes contain a PAF acetylhydrolase activity which is highly specific for hydrolysis of phospholipids with short-chain acyl groups, much like the human plasma and tissue PAF acetylhydrolases. 19.26 However, the erythrocyte activity is due to a distinct protein with biochemical properties that clearly differentiate it from the plasma and other tissue activities. 19 This chapter describes in detail the procedure to assay this activity and a partial purification that allows the investigator to perform preliminary characterization studies. In addition, we describe the bio17 M. L. Blank, T.-c. Lee, V. Fitzgerald, and F. Snyder, J. Biol. Chem. 256, 175 (1981). z8 j. G. Nijssen, C. F. P. Roosenbloom, and H. van den Bosch, Biochim. Biophys. Acta 876, 611 (1986). 19D. M. Sta~orini, S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre, Lipids, in press (1990). 20 M. L. Blank, M. N. Hall, E. A. Cress, and F. Snyder, Biochem. Biophys. Res. Commun. 113, 666 (1983). 21 M. R. Eistad, D. M. Stafforini, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman, J. Biol. Chem. 264, 8467 (1989). 22 K. E. Stremler, D. M. Stafforini, S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre, J. Biol. Chem. 264, 5331 (1989). 23 D. M. Stafforini, S. M. Prescott, and T. M. McIntyre, unpublished results. 24 A.Tokumura, K. Takauchi, T. Asai, K. Kamiyasu, T. Ogawa, and H. Tsukatani, J. Lipid Res. 30, 219 (1989). 2~ A. Tokumura, T. Asai, K. Takauchi, K. Kamiyasu, T. Ogawa, and H. Tsukatani, Biochem. Biophys. Res. Commun. 155, 863 (1988). 26 D. M. Stafforini, S. M. Prescott, and T. M. McIntyre, FASEB J. 2, A1375 (1988).
414
PHOSPHOLIPASEA
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chemical characteristics of the partially purified enzyme and compare this activity to the plasma PAF acetylhydrolase. Assay of Human Erythrocyte Platelet-Activating Factor Acetylhydrolase
Principle. To assay PAF acetylhydrolase activity, we use PAF labeled in the acetyl group. The amount of radioactivity released from the sn-2 position of the glycerol backbone is a direct measure of h y d r o l y s i s . 27'2s The released acetate can be easily separated from remaining substrate by reversed-phase column chromatography on disposable cartridges. The assay can be carried out easily and inexpensively. In addition, other shortchain glycerophospholipids labeled at the sn-2 position can be used as substrates. However, the only commercially available phospholipid with these features is PAF; thus, it is convenient to use this compound initially as a substrate for purification and characterization purposes. Reagents [acetyl-3H]PAF: We usually prepare 4 ml of a 0.1 mM solution by first mixing 400 nmol of PAF (supplied in chloroform, Avanti Polar Lipids, Birmingham, AL) with 4.5/zCi of hexadecyl-2-acetyl-snglyceryl-3-phosphorycholine, 1-O-[acetyl-3H(N)] (supplied in ethanol, New England Nuclear, Boston, MA). After evaporation of the solvents (by a stream of nitrogen) we add 4 ml of HEPES buffer, and the solution then is sonicated for 5 min at 4° and 100 W, using a 4-mm needle probe in a Braun Sonicator (Model 1510). The solution should be stored frozen to avoid nonenzymatic hydrolysis; it can be used for at least 1 week. We repeat the sonication step each time the substrate is thawed. Duplicate aliquots should be counted to determine the specific radioactivity of the substrate prepared each time; our working solutions are 100/.tM with 10,000 counts per minute (cpm)/nmol. HEPES buffer: 0.1 M, adjust the pH to 7.6 with 1 M potassium hydroxide. Prepare 100 ml. Acetic acid: prepare 100 ml of a l0 M solution. Add 57.1 ml of glacial acetic acid slowly to 42.9 ml of distilled water. Sodium acetate: 0.1 M. Prepare 500 ml. Octadecylsilica gel cartridges: purchased from Baker Chemical Co. (Phillipsburg, NJ). Each assay requires an individual column. They can be reused several times, provided that they are properly regen27 D. M. Stafforini, S. M. Prescott, and T. M. McIntyre, J. Biol. Chem. 262, 4223 (1987). 2s D. M. Stafforini, T. M. McIntyre, and S. M. Prescott, this series, Vol. 187, p. 344.
[39]
ERYTHROCYTEPAF ACETYLHYDROLASE
415
erated. Before each use, wash each column with 3 ml of chloroform/ methanol (1:2, v/v), followed by 3 ml of 95% ethanol, and, finally, 3 ml of water. Standard Assay Procedure. Human erythrocytes, obtained by drawing blood in EDTA or citrate, are lysed by dilution into NH4C1 (final concentration 0.84%, w/v) for 10 min at room temperature. The erythrocyte lysate then can be separated from white blood cells by centrifugation at 2000 g for 20 min. Aliquots (5/zl) of the samples to be assayed (diluted in HEPES buffer, if necessary) are mixed with 5/xl of 400 mM dithioerythritol or dithiothreitol and 40/zl of 0. I mM [acetyl-3H]PAF in polypropylene tubes and then incubated for 15 min at 37°. Glass tubes should be avoided since substrates will bind to the glass surface. After incubation, 50/zl of acetic acid is added to stop the reaction, followed by 1.5 ml of sodium acetate solution. Each reaction mixture is then passed through an octadecylsilica gel cartridge, and the filtrates are collected in 15-ml scintillation vials. Each assay tube then is washed with an additional 1.5 ml of sodium acetate solution, and the wash is also passed through the cartridge and combined with the original effluent. Ten milliliters of Opti-Fluor (Packard Instruments Co.) is added to the vials, and the amount of radioactivity is determined in a liquid scintillation counter. When there are many samples, it is convenient to use a multiplace vacuum manifold to allow several samples to be processed simultaneously. If only a few samples need to be assayed, one can get satisfactory results by manually pushing the product of the reaction through a syringe attached to an octadecylsilica gel cartridge. Expression of Results. The amount of enzymatic activity present is expressed after correction for quenching (see below), incubation time, dilution factors, and the amount of enzyme present in the assay. Using the procedure detailed above we found that the hydrolysis of PAF by a human erythrocyte lysate was linear with time (up to 15-30 min, Fig. 1A) and. with protein concentration (up to 0.4/xl of packed erythrocytes per assay, Fig. 1B). The apparent Km for PAF in lysed red blood cells was 32 /xM (Fig. I C); the K m of a partially purified preparation of the enzyme (see below) was 10.2/xM. The optimal amount of reducing agent to be added varies slightly from preparation to preparation. Thus, it is advisable to first establish the optimal concentration of reducing agent for a given fraction before performing an experiment. When assaying erythrocyte lysates or crude samples containing substantial amounts of hemoglobin, there can be artifacts due to quenching. Appropriate corrections should be made by comparison with a quenching curve where a constant amount of [3H]acetate is counted in the absence or presence of the colored sample after the latter is passed through an octadecylsilica cartridge, as above.
0.3
A
0.2
== e0.1
0.0
I
I
I
I
15
30
45
60
Time, min 0.8
B
0.6 0
0r.
qm
0 0 Q E ¢m
0.4
0.2
0.0
V
J
0.0
I
0.5
I
1.0
I
1.5
I
2.0
Erythrocyte Lysate, i~1 FIG. I. Dependence of PAF hydrolysis on (A) time, (B) protein, and (C) substrate concentration.
[39]
ERYTHROCYTEPAF ACETYLHYDROLASE
417
0.4
C
0.3
c
~"
0.1
0.0 0
I
I
I
I
20
40
60
80
PAF, ~M FIG. 1. (continued)
Comments. We have also used another octadecylsilica gel cartridge, the Waters (Milford, MA) Sep-Pak, but have found the Baker product to give less variable results. However, both types of cartridges can be reused at least I0 times without loss of binding capacity. The amount of protein permissible in the solution to be applied to the cartridges should be determined, since large amounts of protein will prevent binding of PAF to the resin. This results in what appears to be high activity since the product becomes contaminated with substrate. This should be examined by carrying out mock assays terminated at zero time. The Baker cartridge has a higher capacity than the Waters cartridge. Finally, following any change in procedure, one should verify that the apparent product is not contaminated with substrate. The effluent should be extracted29 and examined by thinlayer chromatography (TLC)3° or high-performance liquid chromatography (HPLC). 31
29 E. G. Bligh and W. F. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 3o H. W. Mueller, J. T. O'Flaherty, and R. L. Wykle, J. Biol. Chem. 2,58, 6213 (1983). 31 A. R. Brash, C. D. Ingram, and T. M. Harris, Biochemistry 26, 546 (1987).
418
PHOSPHOLIPASE A 2
[39]
Partial Purification of Human Erythrocyte Platelet-Activating Factor Acetylhydrolase The PAF acetylhydrolase activity present in human erythrocytes can initially be purified from a human erythrocyte lysate by ion-exchange chromatography on DEAE-Sepharose CL-6B, as described below. This procedure separates the enzyme from hemoglobin and yields a preparation that is 485-fold purified from erythrocytes.
Reagents Human blood cells: draw 200 ml of blood, using EDTA or sodium citrate as an anticoagulant. Keep the blood at 4° throughout the remaining steps. EDTA: prepare 100 ml of a 0.25 M solution. Adjust the pH to 7.0 with NaOH. Phosphate buffer: prepare 100 ml of a 1 M sodium phosphate stock by weighing equimolar amounts of NaH2PO 4 and Na2HPO 4. Make the necessary amount of 5 mM sodium phosphate buffer; if prepared as described above, the pH of this solution will be 6.85. Saline: prepare 1 liter of 0.9% NaCl (9 g of solid NaCl in 1 liter of water). DEAE-Sepharose CL-6B: 600 ml. Equilibrate the gel in 5 mM sodium phosphate buffer (pH 6.85) following the instructions provided by the manufacturer (Pharmacia, Piscataway, N J). Other reagents: KCI, NH4C1, aprotinin, and dithioerythritol. All of these reagents can be purchased from Sigma (St. Louis, MO). Procedure. Centrifuge 200 ml of freshly drawn blood for 20 min at 2000 g. Discard the supernatant, suspend the blood cells in 0.9% NaC1 (3 volumes of saline per volume of packed cells), and centrifuge as above. Discard the straw-colored supernatant and wash the cells twice more or until the washes are free of protein [determined by the Bio-Rad (Richmond, CA) protein assay] and free of plasma PAF acetylhydrolase activity. Discard the white buffy coat remaining on top of the red blood cells; to 100 ml of packed cells add 8.3 g of NH4C1, 38.5 mg of dithioerythritol, aprotinin (1.2 trypsin inhibitor units), 4 ml of 0.25 M EDTA, and water to a final volume of 1 liter. Stir the cells overnight at 4° and then dilute the preparation 5-fold by adding 4 liters of 5 mM sodium phosphate buffer (pH 6.85). Add 300 ml of DEAE-Sepharose CL-6B equilibrated in the same buffer and stir at 4° until an aliquot of the supernatant contains no PAF acetylhydrolase activity (3-4 hr). Decant the gel, discard the supernatant, and pack the slurry in a column of wide diameter (we use a 5 x 90 cm glass column). Wash the gel with 1 liter of the 5 mM sodium phosphate buffer; discard the reddish flow-through and the wash, which contain most of the hemoglobin
[39]
ERYTHROCYTEPAF ACETYLHYDROLASE
419
present in the original cell lysate. Wash the column with 500 ml of 0.3 M KCI in buffer and collect five 100 ml-fractions; most of the activity is present in the third and fourth KCI washes. Pool the active fractions (-200 ml), dilute them 5-fold with sodium phosphate buffer, add dithioerythritol to a final concentration of 250/zM, and load the preparation (usually 1 liter) on a 2.5 x 60 cm DEAE-Sepharose CL-6B column at a flow rate of 100 ml/hr. Wash the column with 600 ml of buffer containing 250 ~M dithioerythritol and elute the PAF acetylhydrolase with a 500-ml lifiear gradient of KC1 (0-0.3 M) in buffer, at a flow rate of 35-40 ml/hr. If necessary, the column can be chased with buffer containing 0.3 M KC1. The active fractions can be concentrated by ultrafiltration using a PMI0 Diaflo ultrafiltration membrane (Amicon, Danvers, MA). The preparation should be dialyzed to eliminate the KC1, and it is stable for at least several weeks if stored frozen in the presence of dithioerythritol (250/xM). Comments. The specific activity in a fresh human erythrocyte lysate is 3.15 nmol/hr/mg protein. The effluent from the first DEAE step is 143fold purified (specific activity 450 nmol/ml/hr). The second DEAE step yields a preparation that is 485-fold purified (specific activity 1530 nmol/ ml/hr). The recovery of activity is close to 100%. The buffer and pH used during chromatography on DEAE is critical. In Tris-HCl (pH 7.5) we have found that hemoglobin binds to the resin and copurifies with the erythrocyte PAF acetylhydrolase. Thus, sodium phosphate buffer (pH 6.85) is the optimal choice for this initial separation. Properties of Human Erythrocyte Platelet-Activating Factor Acetylhydrolase The human erythrocyte PAF acetylhydrolase has a broad range of pH in which it is active. Maximal hydrolysis is observed at pH 7.6 with HEPES buffer, but the enzyme is also active when assayed in Tris-HCl (pH 7.5) and phosphate buffers (pH 6.8). The enzyme is stable when stored at - 70 ° in phosphate and Tris-HC1 buffers. It exhibits a half-life of 144 min at 37°. Substrate Specificity. The human erythrocyte PAF acetylhydrolase has a high specificity for glycerophospholipids with short-chain acyl groups at the sn-2 position of glycerol. Phosphatidylcholines containing very longchain acyl groups (i.e., arachidonoyl) at the sn-2 position are not inhibitors of PAF hydrolysis by the purified enzyme. In addition, 1-palmitoyl-2hexanoylglycerophosphocholine is not a substrate of the purified enzyme. 1-Palmitoyl-2-glutaroylglycerophosphocholinewas hydrolyzed by the partially purified enzyme, albeit at 50% of the rate at which PAF was hydrolyzed. It remains to be established whether the suitability of this compound as a substrate is due to the oxidized nature of the substituent at the sn-2
420
[39]
PHOSPHOLIPASE A 2
TABLE I SULFHYDRYL GROUPS ESSENTIAL FOR ACTIVITY OF HUMAN ERYTHROCYTE P A F ACETYLHYDROLASE a
Relative acetylhydrolase activity (%) Additions b
Preincubation c (min)
Erythrocyte
Plasma
Dithiothreitol (1 mM) Dithioerythritol (1 raM) DTNB (2 mM) Iodoacetic acid (2 raM) Iodoacetic acid (20 raM)
10 10 30 10 10
152 136 4 48.4 5.2
94 N.D. d 83 90.4 67.7
Partially purified preparations of the acetylhydrolases were used for these studies. b Final concentrations in the assay mixtures. c At 37°. a N.D., Not determined.
position, or whether the specificity of the enzyme decreases abruptly as the length of the sn-2 acyl groups increases from five to six carbons. Sensitivity to Sulfhydryl Reagents. Three lines of evidence suggest the presence of sulfhydryl groups essential for activity of the human erythrocyte PAF acetylhydrolase. First, the addition of reducing agents is necessary for maximal hydrolysis (Table I). Second, the activity can be stabilized on storage by supplementing reducing agents (data not shown). In addition, PAF hydrolysis is inhibited by pretreatment with agents such as dithiobis(2-nitrobenzoic acid) (DTNB) and iodoacetic acid, which inactivate enzymes by reacting with free sulfhydryl groups (Table I). In contrast, the human plasma PAF acetylhydrolase does not require the addition of reducing agents for maximal activity, nor is the enzyme inhibited by DTNB or iodoacetic acid. Metals and Chelators. A variety of heavy metals are inhibitors of the erythrocyte PAF acetylhydrolase activity (Fig. 2). The presence of sulfhydryl groups essential for activity likely accounts for the inhibition exerted by heavy metals such as cadmium, lead t copper. PAF did not protect the enzyme from copper inhibition, sugg~oting that heavy metals and phospholipid substrates bind to different sites. The addition of calcium, an obligatory cofactor for activity of most phospholipases A2, is not necessary for PAF hydrolysis by the human erythrocyte PAF acetylhydrolase (Table II). Further, the divalent metal chelator EDTA does not significantly inhibit PAF hydrolysis. Histidine Inhibitors. The presence of essential histidine residues at the
[39]
ERYTHROCYTE PAF ACETYLHYDROLASE
>
1 2 0 - ~
421
Lead Chloride
P 8O
~E Q 0
40,
Cadmium Chloride
LI. D.
Cupric Sulfate O
I
0
I
I
50 100 150 Metal Chloride, I~M
I
200
FIG. 2. Effect of metals on human erythrocyte PAF acetylhydrolase activity.
TABLE 1I E F F E C T OF CALCIUM IONS AND
EDTA ON
HYDROLYSIS OF P A F CATALYZED BY
HUMAN ERYTHROCYTESa
Additions b
Relative activity (%)
CaC12 (0.5 m/~) CaC12 (1.0 raM) EDTA (12.5 mM) EDTA (25.0 mM)
64 42 88 75
a A washed erythrocyte lysate (5/zl of a 25-fold dilution) was preincubated for 30 min at 37° in the absence or presence of CaCI2 or EDTA, in a total volume of 10/.d. Then 40/zl of 0.1 mM [acetyl-3H]PAF was added, and incubations were continued for 30 min at 37°. b Final concentrations in the assay mixtures.
422
PHOSPHOLIPASE A 2
[39]
active site of the human erythrocyte PAF acetylhydrolase was examined by testing the effects of diethyl pyrocarbonate (DEPC) and p-bromophenacyl bromide (pBPB), a nucleophilic reagent that derivatizes a histidine at the active site of the phospholipase A 2 purified from snake venom. 32 The PAF acetylhydrolase activity from human erythrocytes was inhibited by both compounds; in contrast, the activity present in human plasma was resistant (Fig. 3). This suggests the presence of an essential histidine residue(s) at the active site of the erythrocyte, but not the plasma, activity. Serine Esterase Inhibitors. The presence of serine residues at the active site of the human erythrocyte PAF acetylhydrolase was examined by testing the effect of the serine esterase inhibitor diisopropyl fluorophosphate (DFP) on PAF hydrolysis. Preincubation with DFP (up to 1 mM) for 30 rain at 37° almost completely inhibited PAF hydrolysis by the human erythrocyte PAF acetylhydrolase. The plasma activity was inhibited to a lesser extent (Fig. 4). Susceptibility to Proteolysis. An additional line of evidence suggesting that the erythrocyte and plasma PAF acetylhydrolases are distinct enzymes is their different sensitivities to proteolysis. We incubated the erythrocyte and plasma PAF acetylhydrolases with various proteases and then examined the amount of activity remaining after protease treatment (Table III). The erythrocyte activity was sensitive to all the proteases tested, in contrast to the plasma activity which was resistant. Effect of Detergents. Since the phospholipids that are substrates of the erythrocyte PAF acetylhydrolase are hydrophobic, it may be necessary and often advisable to add detergents to the incubation assay, especially when testing phospholipids with sn-2 acyl chains longer than acetate. The detergent of choice must provide adequate substrate solubilization and not inhibit enzymatic activity. We examined the effect of detergents on PAF hydrolysis by the human erythrocyte PAF acetylhydrolase (Table IV). Tween 20 and decyl-fl-o-glucopyranoside are suitable choices since they do not significantly inhibit PAF hydrolysis. Comparison between Erythrocyte and Other Intracellular PAF Acetylhydrolases. The human erythrocyte PAF acetylhydrolase can clearly be distinguished from other intracellular PAF acetylhydrolases by several criteria. 19First, the erythrocyte activity is sensitive to sulfhydryl reagents, and it requires the addition of reducing agents for maximal activity, in contrast to other cellular PAF acetylhydrolase activities. Second, sodium fluoride inhibits only the erythrocyte activity. In addition, the erythrocyte activity is extremely sensitive to proteolysis, in contrast to other intracellular PAF acetylhydrolases which are only partially sensitive to treatment 32 M. F. Roberts, R. A. Deems, T. C. Mincey, and E. A. Dennis, J. Biol. Chem. 252, 2405 (1977).
[39]
ERYTHROCYTE P A F ACETYLHYDROLASE
if,
423
A
12°t o
.m
>
lasma
o
ERYTHROCYTEPAF ACETYLHYDROLASE
411
isoforms is fully activated by physiologic concentrations of calcium ion, exhibiting 30 and 90% of maximal enzymatic activity in the presence of 200 and 800 nM calcium, respectively (Fig. 4). In contrast, the less acidic phospholipase A2 [cytosolic phospholipase A2 (a)] requires over 1 mM CaCI2to reach half-maximal activity, exhibiting nominal activity at calcium concentrations less than 100/zM. Thus, phospholipase A2 (fl) is the major phospholipase A 2 activity in platelet cytosol, is comprised of dimeric polypeptides which are exquisitely sensitive to physiologic alterations in calcium ion concentration (200-800 nM), and demonstrates a substantial preference for ether-linked phospholipid substrates. Since the majority of platelet arachidonic acid mass is contained in plasmalogen molecular species, these results suggest that activation of this phospholipase A 2 (13) during platelet stimulation by physiologic alterations in calcium ion concentration (500-800 nM) results in the selective release of arachidonic acid and contributes to the synthesis of platelet-activating factor. Acknowledgments This research was supported by Grants HL34839 and HL35864. RWG is the recipient of an Established Investigator Award from the American Heart Association.
[39] P l a t e l e t - A c t i v a t i n g F a c t o r A c e t y l h y d r o l a s e in Human Erythrocytes B y DIANA M. STAFFORINI, STEPHEN M. PRESCOTT, THOMAS M. MCINTYRE
and
Introduction Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) is a phospholipid that has been shown to possess potent biological activity. PAF induces hypotension, leukopenia, and thrombocytopenia 1 and increases vascular permeability. 2 PAF also activates platelets, neutrophils, and macrophages. A variety of cells, for example, neutrophils, macrophages, and endothelial cells, synthesize PAF on appropriate
1 M. Halonen, J. D. Palmer, I. C. Lohman, L. M. McManus, and R. N. Pinckard, Annu. Rev. Resp. Dis. 122, 915 (1980). 2 j. Bjork and G. Smedegard, J. Allergy Clin. Immunol. 71, 145 (1983).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
412
PHOSPHOLIPASEA 2
[39]
stimulation. 3 Some inflammatory cells release most of the PAF that they synthesize (e.g., monocytes), 4 in contrast to vascular cells (e.g., endothelial cells) 5,6 which do not. PAF has been identified in human blood, saliva, urine, and amniotic fluid. 7-9 The rate of removal of PAF is likely an important means of regulating the bioactivity of this compound. Farr and co-workers 1°-12 were the first to demonstrate the occurrence, in mammalian plasma, of an enzyme that inactivates PAF by removal of the acetyl group esterifying the sn-2 position of glycerol, to produce lyso-PAF and acetate, which are biologically inactive. This reaction is catalyzed by a specific PAF acetylhydrolase (1-alkyl2-acetylglycerophosphocholine esterase, EC 3.1.1.47, 1-alkyl-2-acetyl-snglycero-3-phosphocholine acetohydrolase). The plasma PAF acetylhydrolase efficiently hydrolyzes PAF released by inflammatory cells such as monocytes. 13 In addition, the plasma enzyme is capable of inactivating PAF synthesized (but not released) by stimulated endothelial cells. 14Thus, the plasma PAF acetylhydrolase acts as a scavenger of circulating and cell-associated PAF. In addition to the plasma enzyme, intracellular PAF acetylhydrolases have been described in a variety of tissues and blood cells. 15,16 Blank et
3 S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre, Proc. Natl. Acad. Sci. U.S.A. 81, 3534 (1984). 4 M. R. Elstad, S. M. Prescott, T. M. McIntyre, and G. A. Zimmerman, J. lmmunol. 140, 1618 (1988). 5 S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre, Proc. Natl. Acad. Sci. U.S.A. 81, 3534 (1984). 6 F. Bussolino, F. Breviario, C. Tetta, M. Aglietta, A. Mantovani, and E. Dejana, J. Clin. Invest. 77, 2027 (1986). 7 K. E. Grandei, R. S. Farr, A. A. Wanderer, T. C. Eisenstadt, and S. I. Wasserman, N. Engl. J. Med. 313, 405 (1985). s C. P. Cox, M. L. Wardlow, R. Jorgensen, and R. S. Farr, J. lmmunol. 127, 46 (1981). 9 M. M. Billah and J. M. Johnston, Biochem. Biophys. Res. Commun. 113, 51 (1983). i0 R. S. Farr, C. P. Cox, M. L. Wardlow, and R. Jorgensen, Clin. Immunol. Immunopathol. 15, 318 (1980). 11 R. S. Farr, M. L. Wardlow, C. P. Cox, K. E. Meng, and D. E. Greene, Fed. Proc., Fed. Am. Soc. Exp. Biol. 42, 3120 (1983). 12M. L. Wardlow, C. P. Cox, K. E. Meng, D. Greene, and R. S. Farr, J. lmmunol. 136, 3441 (1986). 13D. M. Stafforini,M. R. Elstad, T. M. Mclntyre, G. A. Zimmerman, and S. M. Prescott, J. Biol. Chem. 265, 9682 (1990). 14G. A. Zilnmerman, T. M. McIntyre, M. Mehra, and S. M. Prescott,J. Ceil Biol. U0, 529 (1990). 15 R. Yanoshita, I. Kudo, K. Ikizawa, H. W. Chang, S. Kobayashi, M. Ohno, S. Nojima, and K. Inoue, J. Biochem. (Tokyo) 103, 815 (1988). 16T.-c. Lee, B. Malone, S. I. Wasserman, V. Fitzgerald,and F. Snyder, Biochem. Biophys. Res. C o m m u n . 105, 1303 (1982).
[39]
ERYTHROCYTEPAF ACETYLHYDROLASE
413
al. ,7 described a PAF acetylhydrolase activity in the cytosolic fraction of mammalian tissues, Nijssen et al. 18 reported the presence of a similar
activity in rat lung cytosol, and we have studied the properties of PAF acetylhydrolases in human tissues and blood cells. 19 These activities belong to a family of intracellular PAF acetylhydrolases that, like the plasma enzyme, are constitutively active, require a short-chain residue at the sn-2 position, and are calcium independent. However, the intracellular activities can be differentiated from the plasma enzyme by a variety of biochemical criteria. 19'2° The function(s) of these activities is yet to be determined, but they may play an important role in decreasing the accumulation of intracellular PAF. For example, human macrophages regulate PAF accumulation by increasing the levels of intracellular PAF acetylhydrolase activity21; this may limit the activation of cells that respond to the lipid. In addition, the plasma 22 and intracellular23 PAF acetylhydrolases catalyze the degradation of oxidatively fragmented phospholipids. This may have major pathological significance since oxidatively fragmented phospholipids with toxic and biological activity have been shown to occur in vivo. 24'25 Thus, the PAF acetylhydrolases may play an important role as scavengers of PAF and toxic phospholipids. Human erythrocytes contain a PAF acetylhydrolase activity which is highly specific for hydrolysis of phospholipids with short-chain acyl groups, much like the human plasma and tissue PAF acetylhydrolases. 19.26 However, the erythrocyte activity is due to a distinct protein with biochemical properties that clearly differentiate it from the plasma and other tissue activities. 19 This chapter describes in detail the procedure to assay this activity and a partial purification that allows the investigator to perform preliminary characterization studies. In addition, we describe the bio17 M. L. Blank, T.-c. Lee, V. Fitzgerald, and F. Snyder, J. Biol. Chem. 256, 175 (1981). z8 j. G. Nijssen, C. F. P. Roosenbloom, and H. van den Bosch, Biochim. Biophys. Acta 876, 611 (1986). 19D. M. Sta~orini, S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre, Lipids, in press (1990). 20 M. L. Blank, M. N. Hall, E. A. Cress, and F. Snyder, Biochem. Biophys. Res. Commun. 113, 666 (1983). 21 M. R. Eistad, D. M. Stafforini, T. M. McIntyre, S. M. Prescott, and G. A. Zimmerman, J. Biol. Chem. 264, 8467 (1989). 22 K. E. Stremler, D. M. Stafforini, S. M. Prescott, G. A. Zimmerman, and T. M. McIntyre, J. Biol. Chem. 264, 5331 (1989). 23 D. M. Stafforini, S. M. Prescott, and T. M. McIntyre, unpublished results. 24 A.Tokumura, K. Takauchi, T. Asai, K. Kamiyasu, T. Ogawa, and H. Tsukatani, J. Lipid Res. 30, 219 (1989). 2~ A. Tokumura, T. Asai, K. Takauchi, K. Kamiyasu, T. Ogawa, and H. Tsukatani, Biochem. Biophys. Res. Commun. 155, 863 (1988). 26 D. M. Stafforini, S. M. Prescott, and T. M. McIntyre, FASEB J. 2, A1375 (1988).
414
PHOSPHOLIPASEA
[39]
chemical characteristics of the partially purified enzyme and compare this activity to the plasma PAF acetylhydrolase. Assay of Human Erythrocyte Platelet-Activating Factor Acetylhydrolase
Principle. To assay PAF acetylhydrolase activity, we use PAF labeled in the acetyl group. The amount of radioactivity released from the sn-2 position of the glycerol backbone is a direct measure of h y d r o l y s i s . 27'2s The released acetate can be easily separated from remaining substrate by reversed-phase column chromatography on disposable cartridges. The assay can be carried out easily and inexpensively. In addition, other shortchain glycerophospholipids labeled at the sn-2 position can be used as substrates. However, the only commercially available phospholipid with these features is PAF; thus, it is convenient to use this compound initially as a substrate for purification and characterization purposes. Reagents [acetyl-3H]PAF: We usually prepare 4 ml of a 0.1 mM solution by first mixing 400 nmol of PAF (supplied in chloroform, Avanti Polar Lipids, Birmingham, AL) with 4.5/zCi of hexadecyl-2-acetyl-snglyceryl-3-phosphorycholine, 1-O-[acetyl-3H(N)] (supplied in ethanol, New England Nuclear, Boston, MA). After evaporation of the solvents (by a stream of nitrogen) we add 4 ml of HEPES buffer, and the solution then is sonicated for 5 min at 4° and 100 W, using a 4-mm needle probe in a Braun Sonicator (Model 1510). The solution should be stored frozen to avoid nonenzymatic hydrolysis; it can be used for at least 1 week. We repeat the sonication step each time the substrate is thawed. Duplicate aliquots should be counted to determine the specific radioactivity of the substrate prepared each time; our working solutions are 100/.tM with 10,000 counts per minute (cpm)/nmol. HEPES buffer: 0.1 M, adjust the pH to 7.6 with 1 M potassium hydroxide. Prepare 100 ml. Acetic acid: prepare 100 ml of a l0 M solution. Add 57.1 ml of glacial acetic acid slowly to 42.9 ml of distilled water. Sodium acetate: 0.1 M. Prepare 500 ml. Octadecylsilica gel cartridges: purchased from Baker Chemical Co. (Phillipsburg, NJ). Each assay requires an individual column. They can be reused several times, provided that they are properly regen27 D. M. Stafforini, S. M. Prescott, and T. M. McIntyre, J. Biol. Chem. 262, 4223 (1987). 2s D. M. Stafforini, T. M. McIntyre, and S. M. Prescott, this series, Vol. 187, p. 344.
[39]
ERYTHROCYTEPAF ACETYLHYDROLASE
415
erated. Before each use, wash each column with 3 ml of chloroform/ methanol (1:2, v/v), followed by 3 ml of 95% ethanol, and, finally, 3 ml of water. Standard Assay Procedure. Human erythrocytes, obtained by drawing blood in EDTA or citrate, are lysed by dilution into NH4C1 (final concentration 0.84%, w/v) for 10 min at room temperature. The erythrocyte lysate then can be separated from white blood cells by centrifugation at 2000 g for 20 min. Aliquots (5/zl) of the samples to be assayed (diluted in HEPES buffer, if necessary) are mixed with 5/xl of 400 mM dithioerythritol or dithiothreitol and 40/zl of 0. I mM [acetyl-3H]PAF in polypropylene tubes and then incubated for 15 min at 37°. Glass tubes should be avoided since substrates will bind to the glass surface. After incubation, 50/zl of acetic acid is added to stop the reaction, followed by 1.5 ml of sodium acetate solution. Each reaction mixture is then passed through an octadecylsilica gel cartridge, and the filtrates are collected in 15-ml scintillation vials. Each assay tube then is washed with an additional 1.5 ml of sodium acetate solution, and the wash is also passed through the cartridge and combined with the original effluent. Ten milliliters of Opti-Fluor (Packard Instruments Co.) is added to the vials, and the amount of radioactivity is determined in a liquid scintillation counter. When there are many samples, it is convenient to use a multiplace vacuum manifold to allow several samples to be processed simultaneously. If only a few samples need to be assayed, one can get satisfactory results by manually pushing the product of the reaction through a syringe attached to an octadecylsilica gel cartridge. Expression of Results. The amount of enzymatic activity present is expressed after correction for quenching (see below), incubation time, dilution factors, and the amount of enzyme present in the assay. Using the procedure detailed above we found that the hydrolysis of PAF by a human erythrocyte lysate was linear with time (up to 15-30 min, Fig. 1A) and. with protein concentration (up to 0.4/xl of packed erythrocytes per assay, Fig. 1B). The apparent Km for PAF in lysed red blood cells was 32 /xM (Fig. I C); the K m of a partially purified preparation of the enzyme (see below) was 10.2/xM. The optimal amount of reducing agent to be added varies slightly from preparation to preparation. Thus, it is advisable to first establish the optimal concentration of reducing agent for a given fraction before performing an experiment. When assaying erythrocyte lysates or crude samples containing substantial amounts of hemoglobin, there can be artifacts due to quenching. Appropriate corrections should be made by comparison with a quenching curve where a constant amount of [3H]acetate is counted in the absence or presence of the colored sample after the latter is passed through an octadecylsilica cartridge, as above.
0.3
A
0.2
== e0.1
0.0
I
I
I
I
15
30
45
60
Time, min 0.8
B
0.6 0
0r.
qm
0 0 Q E ¢m
0.4
0.2
0.0
V
J
0.0
I
0.5
I
1.0
I
1.5
I
2.0
Erythrocyte Lysate, i~1 FIG. I. Dependence of PAF hydrolysis on (A) time, (B) protein, and (C) substrate concentration.
[39]
ERYTHROCYTEPAF ACETYLHYDROLASE
417
0.4
C
0.3
c
~"
0.1
0.0 0
I
I
I
I
20
40
60
80
PAF, ~M FIG. 1. (continued)
Comments. We have also used another octadecylsilica gel cartridge, the Waters (Milford, MA) Sep-Pak, but have found the Baker product to give less variable results. However, both types of cartridges can be reused at least I0 times without loss of binding capacity. The amount of protein permissible in the solution to be applied to the cartridges should be determined, since large amounts of protein will prevent binding of PAF to the resin. This results in what appears to be high activity since the product becomes contaminated with substrate. This should be examined by carrying out mock assays terminated at zero time. The Baker cartridge has a higher capacity than the Waters cartridge. Finally, following any change in procedure, one should verify that the apparent product is not contaminated with substrate. The effluent should be extracted29 and examined by thinlayer chromatography (TLC)3° or high-performance liquid chromatography (HPLC). 31
29 E. G. Bligh and W. F. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 3o H. W. Mueller, J. T. O'Flaherty, and R. L. Wykle, J. Biol. Chem. 2,58, 6213 (1983). 31 A. R. Brash, C. D. Ingram, and T. M. Harris, Biochemistry 26, 546 (1987).
418
PHOSPHOLIPASE A 2
[39]
Partial Purification of Human Erythrocyte Platelet-Activating Factor Acetylhydrolase The PAF acetylhydrolase activity present in human erythrocytes can initially be purified from a human erythrocyte lysate by ion-exchange chromatography on DEAE-Sepharose CL-6B, as described below. This procedure separates the enzyme from hemoglobin and yields a preparation that is 485-fold purified from erythrocytes.
Reagents Human blood cells: draw 200 ml of blood, using EDTA or sodium citrate as an anticoagulant. Keep the blood at 4° throughout the remaining steps. EDTA: prepare 100 ml of a 0.25 M solution. Adjust the pH to 7.0 with NaOH. Phosphate buffer: prepare 100 ml of a 1 M sodium phosphate stock by weighing equimolar amounts of NaH2PO 4 and Na2HPO 4. Make the necessary amount of 5 mM sodium phosphate buffer; if prepared as described above, the pH of this solution will be 6.85. Saline: prepare 1 liter of 0.9% NaCl (9 g of solid NaCl in 1 liter of water). DEAE-Sepharose CL-6B: 600 ml. Equilibrate the gel in 5 mM sodium phosphate buffer (pH 6.85) following the instructions provided by the manufacturer (Pharmacia, Piscataway, N J). Other reagents: KCI, NH4C1, aprotinin, and dithioerythritol. All of these reagents can be purchased from Sigma (St. Louis, MO). Procedure. Centrifuge 200 ml of freshly drawn blood for 20 min at 2000 g. Discard the supernatant, suspend the blood cells in 0.9% NaC1 (3 volumes of saline per volume of packed cells), and centrifuge as above. Discard the straw-colored supernatant and wash the cells twice more or until the washes are free of protein [determined by the Bio-Rad (Richmond, CA) protein assay] and free of plasma PAF acetylhydrolase activity. Discard the white buffy coat remaining on top of the red blood cells; to 100 ml of packed cells add 8.3 g of NH4C1, 38.5 mg of dithioerythritol, aprotinin (1.2 trypsin inhibitor units), 4 ml of 0.25 M EDTA, and water to a final volume of 1 liter. Stir the cells overnight at 4° and then dilute the preparation 5-fold by adding 4 liters of 5 mM sodium phosphate buffer (pH 6.85). Add 300 ml of DEAE-Sepharose CL-6B equilibrated in the same buffer and stir at 4° until an aliquot of the supernatant contains no PAF acetylhydrolase activity (3-4 hr). Decant the gel, discard the supernatant, and pack the slurry in a column of wide diameter (we use a 5 x 90 cm glass column). Wash the gel with 1 liter of the 5 mM sodium phosphate buffer; discard the reddish flow-through and the wash, which contain most of the hemoglobin
[39]
ERYTHROCYTEPAF ACETYLHYDROLASE
419
present in the original cell lysate. Wash the column with 500 ml of 0.3 M KCI in buffer and collect five 100 ml-fractions; most of the activity is present in the third and fourth KCI washes. Pool the active fractions (-200 ml), dilute them 5-fold with sodium phosphate buffer, add dithioerythritol to a final concentration of 250/zM, and load the preparation (usually 1 liter) on a 2.5 x 60 cm DEAE-Sepharose CL-6B column at a flow rate of 100 ml/hr. Wash the column with 600 ml of buffer containing 250 ~M dithioerythritol and elute the PAF acetylhydrolase with a 500-ml lifiear gradient of KC1 (0-0.3 M) in buffer, at a flow rate of 35-40 ml/hr. If necessary, the column can be chased with buffer containing 0.3 M KC1. The active fractions can be concentrated by ultrafiltration using a PMI0 Diaflo ultrafiltration membrane (Amicon, Danvers, MA). The preparation should be dialyzed to eliminate the KC1, and it is stable for at least several weeks if stored frozen in the presence of dithioerythritol (250/xM). Comments. The specific activity in a fresh human erythrocyte lysate is 3.15 nmol/hr/mg protein. The effluent from the first DEAE step is 143fold purified (specific activity 450 nmol/ml/hr). The second DEAE step yields a preparation that is 485-fold purified (specific activity 1530 nmol/ ml/hr). The recovery of activity is close to 100%. The buffer and pH used during chromatography on DEAE is critical. In Tris-HCl (pH 7.5) we have found that hemoglobin binds to the resin and copurifies with the erythrocyte PAF acetylhydrolase. Thus, sodium phosphate buffer (pH 6.85) is the optimal choice for this initial separation. Properties of Human Erythrocyte Platelet-Activating Factor Acetylhydrolase The human erythrocyte PAF acetylhydrolase has a broad range of pH in which it is active. Maximal hydrolysis is observed at pH 7.6 with HEPES buffer, but the enzyme is also active when assayed in Tris-HCl (pH 7.5) and phosphate buffers (pH 6.8). The enzyme is stable when stored at - 70 ° in phosphate and Tris-HC1 buffers. It exhibits a half-life of 144 min at 37°. Substrate Specificity. The human erythrocyte PAF acetylhydrolase has a high specificity for glycerophospholipids with short-chain acyl groups at the sn-2 position of glycerol. Phosphatidylcholines containing very longchain acyl groups (i.e., arachidonoyl) at the sn-2 position are not inhibitors of PAF hydrolysis by the purified enzyme. In addition, 1-palmitoyl-2hexanoylglycerophosphocholine is not a substrate of the purified enzyme. 1-Palmitoyl-2-glutaroylglycerophosphocholinewas hydrolyzed by the partially purified enzyme, albeit at 50% of the rate at which PAF was hydrolyzed. It remains to be established whether the suitability of this compound as a substrate is due to the oxidized nature of the substituent at the sn-2
420
[39]
PHOSPHOLIPASE A 2
TABLE I SULFHYDRYL GROUPS ESSENTIAL FOR ACTIVITY OF HUMAN ERYTHROCYTE P A F ACETYLHYDROLASE a
Relative acetylhydrolase activity (%) Additions b
Preincubation c (min)
Erythrocyte
Plasma
Dithiothreitol (1 mM) Dithioerythritol (1 raM) DTNB (2 mM) Iodoacetic acid (2 raM) Iodoacetic acid (20 raM)
10 10 30 10 10
152 136 4 48.4 5.2
94 N.D. d 83 90.4 67.7
Partially purified preparations of the acetylhydrolases were used for these studies. b Final concentrations in the assay mixtures. c At 37°. a N.D., Not determined.
position, or whether the specificity of the enzyme decreases abruptly as the length of the sn-2 acyl groups increases from five to six carbons. Sensitivity to Sulfhydryl Reagents. Three lines of evidence suggest the presence of sulfhydryl groups essential for activity of the human erythrocyte PAF acetylhydrolase. First, the addition of reducing agents is necessary for maximal hydrolysis (Table I). Second, the activity can be stabilized on storage by supplementing reducing agents (data not shown). In addition, PAF hydrolysis is inhibited by pretreatment with agents such as dithiobis(2-nitrobenzoic acid) (DTNB) and iodoacetic acid, which inactivate enzymes by reacting with free sulfhydryl groups (Table I). In contrast, the human plasma PAF acetylhydrolase does not require the addition of reducing agents for maximal activity, nor is the enzyme inhibited by DTNB or iodoacetic acid. Metals and Chelators. A variety of heavy metals are inhibitors of the erythrocyte PAF acetylhydrolase activity (Fig. 2). The presence of sulfhydryl groups essential for activity likely accounts for the inhibition exerted by heavy metals such as cadmium, lead t copper. PAF did not protect the enzyme from copper inhibition, sugg~oting that heavy metals and phospholipid substrates bind to different sites. The addition of calcium, an obligatory cofactor for activity of most phospholipases A2, is not necessary for PAF hydrolysis by the human erythrocyte PAF acetylhydrolase (Table II). Further, the divalent metal chelator EDTA does not significantly inhibit PAF hydrolysis. Histidine Inhibitors. The presence of essential histidine residues at the
[39]
ERYTHROCYTE PAF ACETYLHYDROLASE
>
1 2 0 - ~
421
Lead Chloride
P 8O
~E Q 0
40,
Cadmium Chloride
LI. D.
Cupric Sulfate O
I
0
I
I
50 100 150 Metal Chloride, I~M
I
200
FIG. 2. Effect of metals on human erythrocyte PAF acetylhydrolase activity.
TABLE 1I E F F E C T OF CALCIUM IONS AND
EDTA ON
HYDROLYSIS OF P A F CATALYZED BY
HUMAN ERYTHROCYTESa
Additions b
Relative activity (%)
CaC12 (0.5 m/~) CaC12 (1.0 raM) EDTA (12.5 mM) EDTA (25.0 mM)
64 42 88 75
a A washed erythrocyte lysate (5/zl of a 25-fold dilution) was preincubated for 30 min at 37° in the absence or presence of CaCI2 or EDTA, in a total volume of 10/.d. Then 40/zl of 0.1 mM [acetyl-3H]PAF was added, and incubations were continued for 30 min at 37°. b Final concentrations in the assay mixtures.
422
PHOSPHOLIPASE A 2
[39]
active site of the human erythrocyte PAF acetylhydrolase was examined by testing the effects of diethyl pyrocarbonate (DEPC) and p-bromophenacyl bromide (pBPB), a nucleophilic reagent that derivatizes a histidine at the active site of the phospholipase A 2 purified from snake venom. 32 The PAF acetylhydrolase activity from human erythrocytes was inhibited by both compounds; in contrast, the activity present in human plasma was resistant (Fig. 3). This suggests the presence of an essential histidine residue(s) at the active site of the erythrocyte, but not the plasma, activity. Serine Esterase Inhibitors. The presence of serine residues at the active site of the human erythrocyte PAF acetylhydrolase was examined by testing the effect of the serine esterase inhibitor diisopropyl fluorophosphate (DFP) on PAF hydrolysis. Preincubation with DFP (up to 1 mM) for 30 rain at 37° almost completely inhibited PAF hydrolysis by the human erythrocyte PAF acetylhydrolase. The plasma activity was inhibited to a lesser extent (Fig. 4). Susceptibility to Proteolysis. An additional line of evidence suggesting that the erythrocyte and plasma PAF acetylhydrolases are distinct enzymes is their different sensitivities to proteolysis. We incubated the erythrocyte and plasma PAF acetylhydrolases with various proteases and then examined the amount of activity remaining after protease treatment (Table III). The erythrocyte activity was sensitive to all the proteases tested, in contrast to the plasma activity which was resistant. Effect of Detergents. Since the phospholipids that are substrates of the erythrocyte PAF acetylhydrolase are hydrophobic, it may be necessary and often advisable to add detergents to the incubation assay, especially when testing phospholipids with sn-2 acyl chains longer than acetate. The detergent of choice must provide adequate substrate solubilization and not inhibit enzymatic activity. We examined the effect of detergents on PAF hydrolysis by the human erythrocyte PAF acetylhydrolase (Table IV). Tween 20 and decyl-fl-o-glucopyranoside are suitable choices since they do not significantly inhibit PAF hydrolysis. Comparison between Erythrocyte and Other Intracellular PAF Acetylhydrolases. The human erythrocyte PAF acetylhydrolase can clearly be distinguished from other intracellular PAF acetylhydrolases by several criteria. 19First, the erythrocyte activity is sensitive to sulfhydryl reagents, and it requires the addition of reducing agents for maximal activity, in contrast to other cellular PAF acetylhydrolase activities. Second, sodium fluoride inhibits only the erythrocyte activity. In addition, the erythrocyte activity is extremely sensitive to proteolysis, in contrast to other intracellular PAF acetylhydrolases which are only partially sensitive to treatment 32 M. F. Roberts, R. A. Deems, T. C. Mincey, and E. A. Dennis, J. Biol. Chem. 252, 2405 (1977).
[39]
ERYTHROCYTE P A F ACETYLHYDROLASE
if,
423
A
12°t o
.m
>
lasma
o
