[34]
MITOCHONDRIAL PHOSPHOLIPASE A 2
365
a fluoroketone,21 or phosphonate 22 instead of an ester bond at the sn-2 position are also reversible inhibitors. Cobra venom phospholipase A 2 is completely inactivated by p-bromophenacyl bromide, which covalently modifies the active site histidine. 2a The sponge metabolite manoalide 24and its synthetic analog manoalogue 25 cause a partial irreversible inactivation of the enzyme by modification of lysine residues. Acknowledgments Support for this work was providedby the NationalInstitutes of Health (GM-20,501)and the National Science Foundation (DMB 89-17392). 21W. Yuan, R. J. Berman, and M. H. Gelb, J. Am. Chem. Soc. 1119,8071 (1987). W. Yuan and M. H. Gelb, J. Am. Chem. Soc. 110, 2665 (1988). 23M. F. Roberts, R. A. Deems, T. C. Mincey, and E. A. Dennis, J. Biol. Chem. 252, 2405 (1977). z4D. Lombardoand E. A. Dennis, J. Biol. Chem. 260, 7234 (1985). 2~L. J. Reynolds, B. P. Morgan, G. A. Hite, E. D. Mihelich, and E. A. Dennis, J. Am. Chem. Soc. 110, 5172 (1988).
[34] P h o s p h o l i p a s e A 2 f r o m R a t L i v e r M i t o c h o n d r i a B y H. VAN DEN BOSCH, J. G. N. DE JONG, and A. J. AARSMAN
Introduction Phospholipases A2 (EC 3.1.1.4) are abundantly present in pancreatic juice and snake venoms, and detailed insight into the structure and mechanism of these extracellular enzymes is available. 1 Intracellular forms of phospholipase A 2 have been reported for a great variety of cell types, with the enzymes occurring in both soluble form and in association with the membranes of different subcellular organelles. 2 A long standing question concerns the structural relationships between the enzymes that are present in different compartments of a given cell, between the intracellular phospholipases A2 from different cells and tissues, and between these cellular phospholipases A2 and the extracellular ones. One approach to these problems is to purify cellular phospholipases A2 for structural and enzymological characterization. This chapter deals with various aspects of the I H. M. Verheij, A. J. Slotboom, and G. H. de Haas, Rev. Physiol. Biochem. Pharmacol. 91, 91 (1981). 2 H. van den Bosch , Biochim. Biophys. Acta 604, 191 (1980).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991by AcademicPress, Inc, All rights of reproduction in any form reserved.
366
PHOSPHOLIPASEA 2
[34]
purification and properties of rat liver mitochondrial phospholipase A2 as an example of a low abundancy cellular phospholipase A 2. Rat liver mitochondria are one of the first subcellular organelles reported to contain phospholipase A2, 3 and partial purifications of the enzyme have been described. 4,5 Assay Method Principle. E n z y m e activity can be assayed by measuring the release of a radioactive fatty acid from the sn-2 position of radiolabeled phospholipids. The released fatty acid can be conveniently extracted by a modified Dole extraction procedure, 6 thus avoiding cumbersome thin-layer chromatographic (TLC) techniques. H o w e v e r , depending on the nature and unsaturation of the phospholipid substrate, the heptane layer of the Dole extraction procedure may contain up to 25% of the substrate. This can easily be removed by chromatographing an aliquot of the heptane layer over a minicolumn of 300 mg silica gel in a cotton wool-plugged Pasteur pipette. N u m e r o u s assay methods for cellular phospholipases AE have been described and reviewed. 6,7 Many of these, including the method described here, cannot be applied indiscriminately for the measurement of phospholipase A2 activity in crude (sub)cellular fractions because the consecutive action of phospholipase AI and lysophospholipase may also release the fatty acid from the sn-2 position of phospholipids. H o w e v e r , rat liver mitochondria do not contain phospholipase A1 or lysophospholipases to any appreciable extent. 8'9 Although different radiolabeled phospholipids are in use for measurement of cellular phospholipases A2, we prefer phosphatidylethanolamine. This substrate, especially in the presence of Ca a÷ , easily associates with biomembranes and can reach the membrane-associated phospholipases, presumably through lateral diffusion.l° These properties make this phospholipid the substrate of choice to compare the activity
3 p. BjCrnstad, J. Lipid Res. 7, 612 (1960). 4 M. Waite and P. Sisson, Biochemistry 10, 2377 (1971). 5 y. Natori, K. Karasawa, H. Arai, Y. Tamori-Natori, and S. Nojima, J. Biochem. (Tokyo) 93, 631 (1983). 6 H. van den Bosch and A. J. Aarsman, Agents Actions 9, 382 (1979). 7 L. J. Reynolds, W. N. Washburn, R. A. Deems, and E. A. Dennis, this volume [1]. s G. L. Scherphof, M. Waite, and L. L. M. van Deenen, Biochim. Biophys. Acta 125, 406 (1966). 9 j. M. de Winter, G. M. Vianen, and H. van den Bosch, Biochim. Biophys. Acta 712, 332 (1982). lo H. B. M. Lenting, F. W. Neys, and H. van den Bosch, Biochim. Biophys. Acta 917~ 178 (1987).
[34]
MITOCHONDRIAL PHOSPHOLIPASE A 2
367
of membrane-associated phospholipases A2 before and after solubilization, while avoiding the use of detergents.
Reagents 1-Acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphoethanolamine, 1 mM, sonicate in water H Tris-HC1 buffer, 0.5 M, pH 8.5 CaCI z, 0.1 M Dole's extraction medium 12 (2-propanol, n-heptane, 1 N H2SO4, 40 : 10 : l, v/v) n-Heptane Silica gel (Silic AR cc-4, Mallinckrodt, St. Louis, MO) 13 Procedure. The standard incubation mixture contains 0.2 mM substrate, 10 m M CaC1 z, and varying amounts of enzyme preparations in 0.5 ml of 0.1 M Tris-HC1 (pH 8.5). After a 30-min incubation at 37°, the reaction is stopped by addition of 2.5 ml of Dole's extraction medium, followed by addition of 1.5 ml each of n-heptane and distilled water with vortexing for 15 sec after each addition. An aliquot of 1.0 ml of the upper heptane phase (total volume 2.0 ml) is chromatographed over a 300-mg silica gel minicolumn in a Pasteur pipette. The heptane eluate and a 1.0ml diethyl ether wash are collected directly in scintillation vials containing 3.7 ml emulsifier (Packard) scintillation fluid for radioactivity measurements. Blanks containing no enzyme are included in each series to correct for nonenzymatic hydrolysis.
Enzyme Purification
Isolation of Mitochondria. Crude mitochondrial fractions are prepared from 10 or 20% (w/v) rat liver homogenates in 0.25 M sucrose, 1 mM EDTA, 1 m M phenylmethanesulfonyl fluoride, and 20 mM Tris-HCl (pH 7.4). The homogenate is first spun for 10 min at 1000 g and then for 10 min at 8000 g. The final pellet is resuspended in 50 mM Tris-HC1 (pH 8.0) at II This substrate can be prepared biosynthetically from [1-14C]linoleic acid and l-acyllysophosphatidylethanolamine using rat liver microsomes [H. van den Bosch, A. J. Aarsman, and L. L. M. van Deenen, Biochim. Biophys. Acta 348, 197 (1974)]. Alternatively, commercially available preparations can be used. Routinely, the labeled substrates are diluted with either rat liver or egg yolk phosphatidylethanolamine to give a final specific activity of 300 dpm/nmol. Stock sonicates can be stored frozen and can be reused after thawing and brief resonication. t2 V. P. Dole, J. Clin. Invest. 35, 150 (1956). 13 This type of silica gel gives optimal results with respect to fatty acid recovery and removal of excess substrate from the heptane layer.
368
PHOSPHOLIPASE A 2
[34]
a protein concentration of approximately 20 mg/ml. All these and further operations are carried out at 0-4 ° . Solubilization. The resuspended mitochondria are poured into 10 times their volume of ice-cold acetone containing 0.125 ml of concentrated ammonia per liter acetone. 4 After stirring for 5 min, the mixture is centrifuged for 5 min at 16,000 g. The supernatant is decanted, and the pellet is briefly dried under a stream of nitrogen and resuspended in extraction buffer consisting of 20 mM Tris-HC1 buffer (pH 7.4) containing 2 mM EDTA, 1 M KC1, and 10% (v/v) glycerol, 14 using a volume equivalent to one-half that of the original mitochondrial suspension. The suspension is stirred for 1 hr and then centrifuged for 20 min at 25,000 g to yield a clear extract. This can be stored frozen without any appreciable loss of activity. Removal of the salt by dialysis leads to the formation of protein precipitates that contain all phospholipase A2 activity. This excludes ion-exchange chromatography at this stage, and gel filtration in buffers containing 1 M KC1 is used as the next purification step. 15The drawback of the low capacity of gel filtration as a first purification step is compensated by an unusually high purification factor due to the low molecular weight of the enzyme. Gel Filtration. The extract (24 ml, 215 mg protein, 1600 mU) is filtered over an AcA 54 column (3.5 x 145 cm), equilibrated and eluted with extraction buffer, at a flow rate of 40 ml/hr. Fractions of 20 ml are collected (Fig. 1). Enzyme after AcA 54 chromatography is stable when stored at 4° (less than 25% activity loss in 6 months) and forms a convenient starting material for further purification. Hydroxyapatite Chromatography. Pooled fractions from the gel-filtration step (240 ml) are concentrated 2-fold in a dialysis bag put in 300 g Aquacide F (Calbiochem, La Jolla, CA) and dialyzed overnight against 10 volumes of 3 mM potassium phosphate (pH 7.4), 0.2 M KCI, and 10% (v/v) glycerol. The dialyzate is pumped onto a hydroxyapatite column (1.8 × 4.5 cm) at a flow rate of 15 ml/hr. The column is rinsed with 1 bed volume of buffer and then eluted with a gradient consisting of 4 bed volumes each of 3 and 300 mM potassium phosphate (pH 7.4) in 0.2 M KCI and 10% (v/v) glycerol. Fractions of 4 ml are collected. Phospholipase A2 activity elutes at 100 mM phosphate, somewhat retarded with respect to the main protein peak. Matrex Gel Blue A Chromatography. Pooled fractions from the previous step (48 ml) are dialyzed against 50 volumes of 20 mM Tris-HCl (pH 7.4), 0.2 M KC1, and 10% (v/v) glycerol. The dialyzate is pumped onto a 14 Initially 10 m M 2-mercaptoethanol was included in this buffer, but this can be omitted. 15 For unknown reasons AcA 54 gives considerably higher recoveries than Sephadex G-75. When the AcA column is eluted with a buffer containing 0.15 rather than 1 M KCI, phospholipase A2 activity elutes in the void volume protein peak without much increase in specific activity.
[34]
MITOCHONDRIAL PHOSPHOLIPASE A 2 I
I
I
I
I
I
I
I
[
I
I
369
q-! /
q12~
0
~:2o 40 8O IO0
2 "~ I
15 20 25 30 35 40 45 50 55 6 0 6 5 FRACTION NUMBER
FIG. 1. AcA 54 chromatography of phospholipase A 2 solubilized from rat liver mitochondria. For details, see text. The eluent containing phospholipase A 2represents a stable enzyme preparation that can be used for further purification by either hydroxyapatite and Matrex gel Blue A chromatography, ligand affinity chromatography, or immunoaffinity chromatography. [Reproduced with permission from J. G. N. de Jong, H. Amesz, A. J. Aarsman, H. B. M. Lenting, and H. van den Bosch, Eur. J. Biochem. 164, 129 (1987).]
Matrex gel Blue A column (0.8 × 7 cm) at a flow rate of 10 ml/hr. The column is previously treated with 0.5 N NaOH/8 M urea according to the instructions of the supplier (Amicon, Danvers, MA) and equilibrated with the above dialysis buffer. After sample application the column is rinsed with 2 bed volumes of the same buffer and then eluted with a linear gradient consisting of 7 bed volumes each of 0.2 and 1.0 M KC1 in 20 mM TrisHC1 (pH 7.4) and 10% (v/v) glycerol. Fractions of 2.3 ml are collected. Phospholipase A2 elutes at 0.75 M KC1. Table I summarizes the purification procedure.
TABLE I PURIFICATION
OF
PHOSPHOLIPASE
A 2 FROM
RAT
LIVER
MITOCHONDRIA
Step
Total protein a (rag)
Total activity b (mU)
Specific activity (mU/mg)
Recovery (%)
Purification (-fold)
Mitochondria Extract AcA 54 Hydroxyapatite Matrex gel Blue A
1343 216 4.5 0.47 0.095
2283 1598 1440 1233 822
1.7 7.4 320 2630 8650
100 71 63 54 36
4 188 1550 5090
a Protein is measured according to O. H. Lowry, N. J. Rosebrough, A. L. Fan', and R. J. Randall, J. Biol. Chem. 193, 265 (1951). Dilute samples, namely, those from A c A 54 fraction on, were first precipitated with trichloroacetic acid (7%, w/v, final) in the presence of 0.02% (w/v) sodium deoxycholate. b One milliunit (mU) represents the release of 1 nmol fatty acid/rain.
370
PHOSPHOLIPASEAz
[34]
OH
I TosCL Tos-O- (CH2)10- OH [ 1. POCL3 2. ChoLine TosyLate
O II + Tos- O- (CH2)10- O- ?- O-CH 2 -CH 2 -N (CH3)3 OE3
l
÷
CH2-O- P - O - C H 2- CH2-N (CH3)3 FIG. 2. Schematic representation of the synthesis of a ligand affinity adsorbent for phospholipase A2. For details, see text. [Reproduced with permission from A. J. Aarsman, F. Neys, and H. van den Bosch, Biochim. Biophys. Acta 792, 363 (1984).]
Affinity Purification of Phospholipase A 2 Alternative, single-step, purification procedures for rat liver mitochondrial phospholipase A 2 starting from the stable preparation obtained after AcA 54 gel filtration rely on affinity chromatography and can be distinguished in ligand affinity chromatography and immunoaffinity chromatography. The leading thought behind the development of the ligand affinity adsorbent represented in Fig. 2 has been that phospholipase A 2 is able to hydrolyze glycolphosphatidylcholines 16 and to bind n-alkylphosphocholines in a Ca2÷-dependent manner. 17 Thus, the e n z y m e binds to immobilized decane-l-O-phosphocholine in the presence of Ca 2+ and can be eluted in the presence of EDTA. TM Synthesis o f Ligand Affinity Adsorbent. The synthesis (Fig. 2) starts with the preparation of the monotosylated derivative of 1,10-decanediol. 16G. n. de Haas, N. M. Postema, W. Nieuwenhuizen, and L. L. M. van Deenen, Biochim. Biophys. Acta 159, 103 (1968). 17M. C. E. van Dam-Mieras, A. J. Slotboom, W. A. Pieterson, and G. H. de Haas, Biochemistry 14, 5387 (1975). 18A. J. Aarsman, F. Neys, and H. van den Bosch, Biochim. Biophys. Acta 792, 363 (1984).
[34]
MITOCHONDRIAL PHOSPHOLIPASE A 2
371
This is synthesized by adding p-toluenesulfonyl chloride (19.1 g, 100 mmol) in 20 ml pyridine dropwise to a solution of 17.4 g (100 mmol) of 1,10-decanediol in 40 ml pyridine at 0% After 1 hr at 0° and 3 hr at room temperature, the reaction is stopped by addition of ice-cold 1 N HC1, and the products are extracted with 400 ml of diethyl ether. The ether layer is washed successively with 300 ml each of a 5% (w/v) NaHCO3 solution and water. After drying on Na2SO 4 the ether layer is concentrated in vacuo, and the product is purified on a column (4 × 50 cm) of kieselgel 60 (Merck, Darmstadt, FRG) using petroleum ether/diethyl ether (3:7, v/v) as eluent. The monotosyl derivative of 1,10-decanediol is obtained in a yield of 70%. gf values on TLC [Schleicher and Schiill (Dassel, FRG) F 1500 (LS 254) in CHC13] are 0.07, 0.27, and 0.65 for 1,10-decanediol, monotosyl, and ditosyl derivatives, respectively. The tosylated derivatives can be detected under 256-nm UV light. The monotosyl derivative (1.71 g, 5.2 mmol) together with 12.5 mmol pyridine in 30 ml dry dichloromethane is added dropwise to a stirred solution of 843 mg (5.5 mmol) of POCI3 in 20 ml of dry dichloromethane at 0% Stirring is continued for 1 hr at room temperature after which TLC analysis shows the complete disappearance of the monotosyl derivative of decanediol. Pyridine (1 ml, 12.5 mmol) and choline toluene sulfonate (1.9 g, 6.9 mmol) are added, and stirring is continued for 16 hr. 19 After addition of 1 ml each of pyridine and water and stirring for 2 hr, water (50 ml) and methanol (100 ml) are added, and the pH is adjusted to 3 with 1 N HC1. The product is extracted twice with 50 ml CHCI 3, evaporated to dryness, and percolated over a mixed ion-exchange column (Amberlite IRA-45 and IRC-50) in chloroform/methanol/water (5:4:1, v/v). The product is further purified on kieselgel 60 by elution with chloroform/ methanol mixtures. The purified compound, namely, 10-O-p-toluenesulfonyldecane-l-O-phosphocholine, shows an Rf value of 0.33 on TLC (see above) with chloroform/methanol/water (60:40: 10, v/v) as developing solvent and is UV-, phosphor-, and choline-positive. The ligand is coupled to AH Sepharose 4B according to the procedure of Nilsson and Mosbach. 2° Ligand (245 mg, 0.5 mmol) is added to 2 g freeze-dried AH Sepharose 4B, swollen and washed as recommended by the supplier (Pharmacia, Uppsala, Sweden), in l0 ml of 0.2 M NaHCO 3 (pH 10.7) and allowed to react for 20 hr at 37° with gentle agitation. The gel is washed successively with 150-ml portions of water, 0.1 M sodium acetate buffer (pH 4.0), water, 0.5 M NaCl, water, 0.2 M sodium bicarbon-
19 H. Brockerhoff and N. K. W. Ayengar, Lipids 14, 88 (1979). 2o K. Nilsson and K. Mosbach, Biochem. Biophys. Res. Commun. 102, 449 (1981).
372
PHOSPHOLIPASEA2
[34]
ate, and water. Phosphor determination21 on destructed gels shows the coupling of 1.0-3.2/zmol ligand/ml gel for various preparations. Ligand Affinity Chromatography. The ligand affinity adsorbent is packed into a column (bed 0.5 x 3.5 cm) and equilibrated with 50 mM Tris-HCl (pH 7.4) containing 0.2 M KC1, 10% (v/v) glycerol, 10 mM CaCI2, and 1 mM EDTA. Pooled AcA 54 fractions 22 (see Fig. 1) are dialyzed against this equilibration buffer, and the dialyzate, containing up to 1 mg protein, is loaded on the column at a flow rate of 8 ml/hr. The column is rinsed with equilibration buffer until the eluate is free of material absorbing at 280 nm (approximately 10 bed volumes) and then eluted with the above buffer except that 0.2 M KC1 and 10 mM CaCI2 are replaced by 0.5 M KC1 and 50 mM EDTA, respectively. Fractions of 0.67 ml are collected at a flow rate of 8 ml/hr throughout. Phospholipase A 2 starts to elute after 2 to 5 bed volumes and is recovered in 80 to 100% yield in 10 to 15 bed volumes. Immunoaffinity Chromatography. Starting from pooled AcA 54 fractions the enzyme can be obtained in homogeneous and more concentrated form by immunoaffinity chromatography. Since this method depends on the availability of purified monoclonal antibodies to the enzyme23 it is not described here in detail. A full account of this procedure has been published recently. 24 Properties
Stability. In the presence of 10% (v/v) glycerol the enzyme is stable until AcA 54 purification. Thereafter, the enzyme preparations steadily lose activity on storage, presumably due to adsorption of the enzyme to glass and other surfaces at these low protein concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of stored or dialyzed enzyme solutions deafly shows a decrease of enzyme protein over time in solution. Rinsing of the tube or dialysis bag with SDS solutions shows the presence of adsorbed protein. The enzyme itself is fairly stable and can be treated for 24 hr with 5 M urea without loss of activity provided the urea concentration in the assay mixture remains less than 0.2 M. Purity. Pooled fractions from Matrex gel Blue A, 9 ligand affinity chro2t G. Rouser, S. Fleischer, and A. Yamamoto,Lipids 5, 494 (1970). 22Direct application of the mitochondrialextract to the affinitycolumnresults in partial elution of the phospholipase A2 activityin the break-throughpeak. 23j. G. N. de Jong, H. Amesz, A. J. Aarsman, H. B. M. Lenting,and H. van den Bosch, Eur. J. Biochem. 164, 129 (1987). 24A. J. Aarsman,J. G. N. de Jong, E. Arnoldussen,F. W. Neys, P. D. van Wassenaar,and H. van den Bosch, J. Biol. Chem. 264, 10008 (1989).
[35]
PLA 2 F R O M
HUMAN RHEUMATOID SYNOVIAL FLUID
373
matography, 18and immunoaffinity chromatography 24all give a single band on SDS-PAGE corresponding to a molecular weight of 14,000. A single amino acid sequence is obtained 24 for the N-terminal 24 residues, which indicates that the enzyme belongs to group II phospholipases A 2, lacking Cys-11. Enzymatic Properties. The enzyme is absolutely specific for the acyl ester bond at the sn-2 position of phospholipids, as could be deduced from the release of [~4C]linoleate and the production of [3H]palmitoyllysophosphatidylethanolamine exclusively from doubly-labeled 1-[9,10-3HE]palmi toyl-2-[1-~4C]linoleoylphosphatidylethanolamine. 9 The enzyme requires Ca 2+ for activity, although this can be replaced by Sr 2+ to give about 30% of the activity measured in the presence of Ca 2+ . The Ca 2+ requirement of the enzyme is not mediated by calmodulin. 25The specific activity of the enzyme obtained after rapid immunoaffinity purification amounts to 28 or 50 U/mg, depending on whether protein is measured by the Lowry procedure or by amino acid analysis, respectively. 24 These specific activities are obtained in routine assays using 0.2 mM phosphatidylethanolamine. Kinetic analyses with this substrate indicate a K m of 1.2 mM and an apparent Vmax of 350 U/mg. Inhibitors. The enzyme is inhibited by p-bromophenacyl bromide, and Ca 2+ protects against this inhibition. 25 Neither diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride nor the thiol reagents N-ethylmaleimide, iodoacetamide, or 5,5'-dithiobis(2-nitrobenzoic acid) inhibit enzyme activity. 9 25j. M. de Winter, J. Korpancova, and H. van den Bosch, Arch. Biochem. Biophys. 234, 243 (1984).
[35] A s s a y a n d P u r i f i c a t i o n o f P h o s p h o l i p a s e A 2 f r o m H u m a n S y n o v i a l F l u i d in R h e u m a t o i d Arthritis By RUTH M. KRAMEg and R. BLAKE PEPINSKY Introduction Phospholipases A 2 (PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) are a diverse family of enzymes that hydrolyze the sn-2 fatty acyl ester bond of phosphoglycerides, liberating free fatty acids and lysophospholipids. PLA2 from mammalian pancreas and snake venoms are abundant and METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
MITOCHONDRIAL PHOSPHOLIPASE A 2
365
a fluoroketone,21 or phosphonate 22 instead of an ester bond at the sn-2 position are also reversible inhibitors. Cobra venom phospholipase A 2 is completely inactivated by p-bromophenacyl bromide, which covalently modifies the active site histidine. 2a The sponge metabolite manoalide 24and its synthetic analog manoalogue 25 cause a partial irreversible inactivation of the enzyme by modification of lysine residues. Acknowledgments Support for this work was providedby the NationalInstitutes of Health (GM-20,501)and the National Science Foundation (DMB 89-17392). 21W. Yuan, R. J. Berman, and M. H. Gelb, J. Am. Chem. Soc. 1119,8071 (1987). W. Yuan and M. H. Gelb, J. Am. Chem. Soc. 110, 2665 (1988). 23M. F. Roberts, R. A. Deems, T. C. Mincey, and E. A. Dennis, J. Biol. Chem. 252, 2405 (1977). z4D. Lombardoand E. A. Dennis, J. Biol. Chem. 260, 7234 (1985). 2~L. J. Reynolds, B. P. Morgan, G. A. Hite, E. D. Mihelich, and E. A. Dennis, J. Am. Chem. Soc. 110, 5172 (1988).
[34] P h o s p h o l i p a s e A 2 f r o m R a t L i v e r M i t o c h o n d r i a B y H. VAN DEN BOSCH, J. G. N. DE JONG, and A. J. AARSMAN
Introduction Phospholipases A2 (EC 3.1.1.4) are abundantly present in pancreatic juice and snake venoms, and detailed insight into the structure and mechanism of these extracellular enzymes is available. 1 Intracellular forms of phospholipase A 2 have been reported for a great variety of cell types, with the enzymes occurring in both soluble form and in association with the membranes of different subcellular organelles. 2 A long standing question concerns the structural relationships between the enzymes that are present in different compartments of a given cell, between the intracellular phospholipases A2 from different cells and tissues, and between these cellular phospholipases A2 and the extracellular ones. One approach to these problems is to purify cellular phospholipases A2 for structural and enzymological characterization. This chapter deals with various aspects of the I H. M. Verheij, A. J. Slotboom, and G. H. de Haas, Rev. Physiol. Biochem. Pharmacol. 91, 91 (1981). 2 H. van den Bosch , Biochim. Biophys. Acta 604, 191 (1980).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991by AcademicPress, Inc, All rights of reproduction in any form reserved.
366
PHOSPHOLIPASEA 2
[34]
purification and properties of rat liver mitochondrial phospholipase A2 as an example of a low abundancy cellular phospholipase A 2. Rat liver mitochondria are one of the first subcellular organelles reported to contain phospholipase A2, 3 and partial purifications of the enzyme have been described. 4,5 Assay Method Principle. E n z y m e activity can be assayed by measuring the release of a radioactive fatty acid from the sn-2 position of radiolabeled phospholipids. The released fatty acid can be conveniently extracted by a modified Dole extraction procedure, 6 thus avoiding cumbersome thin-layer chromatographic (TLC) techniques. H o w e v e r , depending on the nature and unsaturation of the phospholipid substrate, the heptane layer of the Dole extraction procedure may contain up to 25% of the substrate. This can easily be removed by chromatographing an aliquot of the heptane layer over a minicolumn of 300 mg silica gel in a cotton wool-plugged Pasteur pipette. N u m e r o u s assay methods for cellular phospholipases AE have been described and reviewed. 6,7 Many of these, including the method described here, cannot be applied indiscriminately for the measurement of phospholipase A2 activity in crude (sub)cellular fractions because the consecutive action of phospholipase AI and lysophospholipase may also release the fatty acid from the sn-2 position of phospholipids. H o w e v e r , rat liver mitochondria do not contain phospholipase A1 or lysophospholipases to any appreciable extent. 8'9 Although different radiolabeled phospholipids are in use for measurement of cellular phospholipases A2, we prefer phosphatidylethanolamine. This substrate, especially in the presence of Ca a÷ , easily associates with biomembranes and can reach the membrane-associated phospholipases, presumably through lateral diffusion.l° These properties make this phospholipid the substrate of choice to compare the activity
3 p. BjCrnstad, J. Lipid Res. 7, 612 (1960). 4 M. Waite and P. Sisson, Biochemistry 10, 2377 (1971). 5 y. Natori, K. Karasawa, H. Arai, Y. Tamori-Natori, and S. Nojima, J. Biochem. (Tokyo) 93, 631 (1983). 6 H. van den Bosch and A. J. Aarsman, Agents Actions 9, 382 (1979). 7 L. J. Reynolds, W. N. Washburn, R. A. Deems, and E. A. Dennis, this volume [1]. s G. L. Scherphof, M. Waite, and L. L. M. van Deenen, Biochim. Biophys. Acta 125, 406 (1966). 9 j. M. de Winter, G. M. Vianen, and H. van den Bosch, Biochim. Biophys. Acta 712, 332 (1982). lo H. B. M. Lenting, F. W. Neys, and H. van den Bosch, Biochim. Biophys. Acta 917~ 178 (1987).
[34]
MITOCHONDRIAL PHOSPHOLIPASE A 2
367
of membrane-associated phospholipases A2 before and after solubilization, while avoiding the use of detergents.
Reagents 1-Acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphoethanolamine, 1 mM, sonicate in water H Tris-HC1 buffer, 0.5 M, pH 8.5 CaCI z, 0.1 M Dole's extraction medium 12 (2-propanol, n-heptane, 1 N H2SO4, 40 : 10 : l, v/v) n-Heptane Silica gel (Silic AR cc-4, Mallinckrodt, St. Louis, MO) 13 Procedure. The standard incubation mixture contains 0.2 mM substrate, 10 m M CaC1 z, and varying amounts of enzyme preparations in 0.5 ml of 0.1 M Tris-HC1 (pH 8.5). After a 30-min incubation at 37°, the reaction is stopped by addition of 2.5 ml of Dole's extraction medium, followed by addition of 1.5 ml each of n-heptane and distilled water with vortexing for 15 sec after each addition. An aliquot of 1.0 ml of the upper heptane phase (total volume 2.0 ml) is chromatographed over a 300-mg silica gel minicolumn in a Pasteur pipette. The heptane eluate and a 1.0ml diethyl ether wash are collected directly in scintillation vials containing 3.7 ml emulsifier (Packard) scintillation fluid for radioactivity measurements. Blanks containing no enzyme are included in each series to correct for nonenzymatic hydrolysis.
Enzyme Purification
Isolation of Mitochondria. Crude mitochondrial fractions are prepared from 10 or 20% (w/v) rat liver homogenates in 0.25 M sucrose, 1 mM EDTA, 1 m M phenylmethanesulfonyl fluoride, and 20 mM Tris-HCl (pH 7.4). The homogenate is first spun for 10 min at 1000 g and then for 10 min at 8000 g. The final pellet is resuspended in 50 mM Tris-HC1 (pH 8.0) at II This substrate can be prepared biosynthetically from [1-14C]linoleic acid and l-acyllysophosphatidylethanolamine using rat liver microsomes [H. van den Bosch, A. J. Aarsman, and L. L. M. van Deenen, Biochim. Biophys. Acta 348, 197 (1974)]. Alternatively, commercially available preparations can be used. Routinely, the labeled substrates are diluted with either rat liver or egg yolk phosphatidylethanolamine to give a final specific activity of 300 dpm/nmol. Stock sonicates can be stored frozen and can be reused after thawing and brief resonication. t2 V. P. Dole, J. Clin. Invest. 35, 150 (1956). 13 This type of silica gel gives optimal results with respect to fatty acid recovery and removal of excess substrate from the heptane layer.
368
PHOSPHOLIPASE A 2
[34]
a protein concentration of approximately 20 mg/ml. All these and further operations are carried out at 0-4 ° . Solubilization. The resuspended mitochondria are poured into 10 times their volume of ice-cold acetone containing 0.125 ml of concentrated ammonia per liter acetone. 4 After stirring for 5 min, the mixture is centrifuged for 5 min at 16,000 g. The supernatant is decanted, and the pellet is briefly dried under a stream of nitrogen and resuspended in extraction buffer consisting of 20 mM Tris-HC1 buffer (pH 7.4) containing 2 mM EDTA, 1 M KC1, and 10% (v/v) glycerol, 14 using a volume equivalent to one-half that of the original mitochondrial suspension. The suspension is stirred for 1 hr and then centrifuged for 20 min at 25,000 g to yield a clear extract. This can be stored frozen without any appreciable loss of activity. Removal of the salt by dialysis leads to the formation of protein precipitates that contain all phospholipase A2 activity. This excludes ion-exchange chromatography at this stage, and gel filtration in buffers containing 1 M KC1 is used as the next purification step. 15The drawback of the low capacity of gel filtration as a first purification step is compensated by an unusually high purification factor due to the low molecular weight of the enzyme. Gel Filtration. The extract (24 ml, 215 mg protein, 1600 mU) is filtered over an AcA 54 column (3.5 x 145 cm), equilibrated and eluted with extraction buffer, at a flow rate of 40 ml/hr. Fractions of 20 ml are collected (Fig. 1). Enzyme after AcA 54 chromatography is stable when stored at 4° (less than 25% activity loss in 6 months) and forms a convenient starting material for further purification. Hydroxyapatite Chromatography. Pooled fractions from the gel-filtration step (240 ml) are concentrated 2-fold in a dialysis bag put in 300 g Aquacide F (Calbiochem, La Jolla, CA) and dialyzed overnight against 10 volumes of 3 mM potassium phosphate (pH 7.4), 0.2 M KCI, and 10% (v/v) glycerol. The dialyzate is pumped onto a hydroxyapatite column (1.8 × 4.5 cm) at a flow rate of 15 ml/hr. The column is rinsed with 1 bed volume of buffer and then eluted with a gradient consisting of 4 bed volumes each of 3 and 300 mM potassium phosphate (pH 7.4) in 0.2 M KCI and 10% (v/v) glycerol. Fractions of 4 ml are collected. Phospholipase A2 activity elutes at 100 mM phosphate, somewhat retarded with respect to the main protein peak. Matrex Gel Blue A Chromatography. Pooled fractions from the previous step (48 ml) are dialyzed against 50 volumes of 20 mM Tris-HCl (pH 7.4), 0.2 M KC1, and 10% (v/v) glycerol. The dialyzate is pumped onto a 14 Initially 10 m M 2-mercaptoethanol was included in this buffer, but this can be omitted. 15 For unknown reasons AcA 54 gives considerably higher recoveries than Sephadex G-75. When the AcA column is eluted with a buffer containing 0.15 rather than 1 M KCI, phospholipase A2 activity elutes in the void volume protein peak without much increase in specific activity.
[34]
MITOCHONDRIAL PHOSPHOLIPASE A 2 I
I
I
I
I
I
I
I
[
I
I
369
q-! /
q12~
0
~:2o 40 8O IO0
2 "~ I
15 20 25 30 35 40 45 50 55 6 0 6 5 FRACTION NUMBER
FIG. 1. AcA 54 chromatography of phospholipase A 2 solubilized from rat liver mitochondria. For details, see text. The eluent containing phospholipase A 2represents a stable enzyme preparation that can be used for further purification by either hydroxyapatite and Matrex gel Blue A chromatography, ligand affinity chromatography, or immunoaffinity chromatography. [Reproduced with permission from J. G. N. de Jong, H. Amesz, A. J. Aarsman, H. B. M. Lenting, and H. van den Bosch, Eur. J. Biochem. 164, 129 (1987).]
Matrex gel Blue A column (0.8 × 7 cm) at a flow rate of 10 ml/hr. The column is previously treated with 0.5 N NaOH/8 M urea according to the instructions of the supplier (Amicon, Danvers, MA) and equilibrated with the above dialysis buffer. After sample application the column is rinsed with 2 bed volumes of the same buffer and then eluted with a linear gradient consisting of 7 bed volumes each of 0.2 and 1.0 M KC1 in 20 mM TrisHC1 (pH 7.4) and 10% (v/v) glycerol. Fractions of 2.3 ml are collected. Phospholipase A2 elutes at 0.75 M KC1. Table I summarizes the purification procedure.
TABLE I PURIFICATION
OF
PHOSPHOLIPASE
A 2 FROM
RAT
LIVER
MITOCHONDRIA
Step
Total protein a (rag)
Total activity b (mU)
Specific activity (mU/mg)
Recovery (%)
Purification (-fold)
Mitochondria Extract AcA 54 Hydroxyapatite Matrex gel Blue A
1343 216 4.5 0.47 0.095
2283 1598 1440 1233 822
1.7 7.4 320 2630 8650
100 71 63 54 36
4 188 1550 5090
a Protein is measured according to O. H. Lowry, N. J. Rosebrough, A. L. Fan', and R. J. Randall, J. Biol. Chem. 193, 265 (1951). Dilute samples, namely, those from A c A 54 fraction on, were first precipitated with trichloroacetic acid (7%, w/v, final) in the presence of 0.02% (w/v) sodium deoxycholate. b One milliunit (mU) represents the release of 1 nmol fatty acid/rain.
370
PHOSPHOLIPASEAz
[34]
OH
I TosCL Tos-O- (CH2)10- OH [ 1. POCL3 2. ChoLine TosyLate
O II + Tos- O- (CH2)10- O- ?- O-CH 2 -CH 2 -N (CH3)3 OE3
l
÷
CH2-O- P - O - C H 2- CH2-N (CH3)3 FIG. 2. Schematic representation of the synthesis of a ligand affinity adsorbent for phospholipase A2. For details, see text. [Reproduced with permission from A. J. Aarsman, F. Neys, and H. van den Bosch, Biochim. Biophys. Acta 792, 363 (1984).]
Affinity Purification of Phospholipase A 2 Alternative, single-step, purification procedures for rat liver mitochondrial phospholipase A 2 starting from the stable preparation obtained after AcA 54 gel filtration rely on affinity chromatography and can be distinguished in ligand affinity chromatography and immunoaffinity chromatography. The leading thought behind the development of the ligand affinity adsorbent represented in Fig. 2 has been that phospholipase A 2 is able to hydrolyze glycolphosphatidylcholines 16 and to bind n-alkylphosphocholines in a Ca2÷-dependent manner. 17 Thus, the e n z y m e binds to immobilized decane-l-O-phosphocholine in the presence of Ca 2+ and can be eluted in the presence of EDTA. TM Synthesis o f Ligand Affinity Adsorbent. The synthesis (Fig. 2) starts with the preparation of the monotosylated derivative of 1,10-decanediol. 16G. n. de Haas, N. M. Postema, W. Nieuwenhuizen, and L. L. M. van Deenen, Biochim. Biophys. Acta 159, 103 (1968). 17M. C. E. van Dam-Mieras, A. J. Slotboom, W. A. Pieterson, and G. H. de Haas, Biochemistry 14, 5387 (1975). 18A. J. Aarsman, F. Neys, and H. van den Bosch, Biochim. Biophys. Acta 792, 363 (1984).
[34]
MITOCHONDRIAL PHOSPHOLIPASE A 2
371
This is synthesized by adding p-toluenesulfonyl chloride (19.1 g, 100 mmol) in 20 ml pyridine dropwise to a solution of 17.4 g (100 mmol) of 1,10-decanediol in 40 ml pyridine at 0% After 1 hr at 0° and 3 hr at room temperature, the reaction is stopped by addition of ice-cold 1 N HC1, and the products are extracted with 400 ml of diethyl ether. The ether layer is washed successively with 300 ml each of a 5% (w/v) NaHCO3 solution and water. After drying on Na2SO 4 the ether layer is concentrated in vacuo, and the product is purified on a column (4 × 50 cm) of kieselgel 60 (Merck, Darmstadt, FRG) using petroleum ether/diethyl ether (3:7, v/v) as eluent. The monotosyl derivative of 1,10-decanediol is obtained in a yield of 70%. gf values on TLC [Schleicher and Schiill (Dassel, FRG) F 1500 (LS 254) in CHC13] are 0.07, 0.27, and 0.65 for 1,10-decanediol, monotosyl, and ditosyl derivatives, respectively. The tosylated derivatives can be detected under 256-nm UV light. The monotosyl derivative (1.71 g, 5.2 mmol) together with 12.5 mmol pyridine in 30 ml dry dichloromethane is added dropwise to a stirred solution of 843 mg (5.5 mmol) of POCI3 in 20 ml of dry dichloromethane at 0% Stirring is continued for 1 hr at room temperature after which TLC analysis shows the complete disappearance of the monotosyl derivative of decanediol. Pyridine (1 ml, 12.5 mmol) and choline toluene sulfonate (1.9 g, 6.9 mmol) are added, and stirring is continued for 16 hr. 19 After addition of 1 ml each of pyridine and water and stirring for 2 hr, water (50 ml) and methanol (100 ml) are added, and the pH is adjusted to 3 with 1 N HC1. The product is extracted twice with 50 ml CHCI 3, evaporated to dryness, and percolated over a mixed ion-exchange column (Amberlite IRA-45 and IRC-50) in chloroform/methanol/water (5:4:1, v/v). The product is further purified on kieselgel 60 by elution with chloroform/ methanol mixtures. The purified compound, namely, 10-O-p-toluenesulfonyldecane-l-O-phosphocholine, shows an Rf value of 0.33 on TLC (see above) with chloroform/methanol/water (60:40: 10, v/v) as developing solvent and is UV-, phosphor-, and choline-positive. The ligand is coupled to AH Sepharose 4B according to the procedure of Nilsson and Mosbach. 2° Ligand (245 mg, 0.5 mmol) is added to 2 g freeze-dried AH Sepharose 4B, swollen and washed as recommended by the supplier (Pharmacia, Uppsala, Sweden), in l0 ml of 0.2 M NaHCO 3 (pH 10.7) and allowed to react for 20 hr at 37° with gentle agitation. The gel is washed successively with 150-ml portions of water, 0.1 M sodium acetate buffer (pH 4.0), water, 0.5 M NaCl, water, 0.2 M sodium bicarbon-
19 H. Brockerhoff and N. K. W. Ayengar, Lipids 14, 88 (1979). 2o K. Nilsson and K. Mosbach, Biochem. Biophys. Res. Commun. 102, 449 (1981).
372
PHOSPHOLIPASEA2
[34]
ate, and water. Phosphor determination21 on destructed gels shows the coupling of 1.0-3.2/zmol ligand/ml gel for various preparations. Ligand Affinity Chromatography. The ligand affinity adsorbent is packed into a column (bed 0.5 x 3.5 cm) and equilibrated with 50 mM Tris-HCl (pH 7.4) containing 0.2 M KC1, 10% (v/v) glycerol, 10 mM CaCI2, and 1 mM EDTA. Pooled AcA 54 fractions 22 (see Fig. 1) are dialyzed against this equilibration buffer, and the dialyzate, containing up to 1 mg protein, is loaded on the column at a flow rate of 8 ml/hr. The column is rinsed with equilibration buffer until the eluate is free of material absorbing at 280 nm (approximately 10 bed volumes) and then eluted with the above buffer except that 0.2 M KC1 and 10 mM CaCI2 are replaced by 0.5 M KC1 and 50 mM EDTA, respectively. Fractions of 0.67 ml are collected at a flow rate of 8 ml/hr throughout. Phospholipase A 2 starts to elute after 2 to 5 bed volumes and is recovered in 80 to 100% yield in 10 to 15 bed volumes. Immunoaffinity Chromatography. Starting from pooled AcA 54 fractions the enzyme can be obtained in homogeneous and more concentrated form by immunoaffinity chromatography. Since this method depends on the availability of purified monoclonal antibodies to the enzyme23 it is not described here in detail. A full account of this procedure has been published recently. 24 Properties
Stability. In the presence of 10% (v/v) glycerol the enzyme is stable until AcA 54 purification. Thereafter, the enzyme preparations steadily lose activity on storage, presumably due to adsorption of the enzyme to glass and other surfaces at these low protein concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of stored or dialyzed enzyme solutions deafly shows a decrease of enzyme protein over time in solution. Rinsing of the tube or dialysis bag with SDS solutions shows the presence of adsorbed protein. The enzyme itself is fairly stable and can be treated for 24 hr with 5 M urea without loss of activity provided the urea concentration in the assay mixture remains less than 0.2 M. Purity. Pooled fractions from Matrex gel Blue A, 9 ligand affinity chro2t G. Rouser, S. Fleischer, and A. Yamamoto,Lipids 5, 494 (1970). 22Direct application of the mitochondrialextract to the affinitycolumnresults in partial elution of the phospholipase A2 activityin the break-throughpeak. 23j. G. N. de Jong, H. Amesz, A. J. Aarsman, H. B. M. Lenting,and H. van den Bosch, Eur. J. Biochem. 164, 129 (1987). 24A. J. Aarsman,J. G. N. de Jong, E. Arnoldussen,F. W. Neys, P. D. van Wassenaar,and H. van den Bosch, J. Biol. Chem. 264, 10008 (1989).
[35]
PLA 2 F R O M
HUMAN RHEUMATOID SYNOVIAL FLUID
373
matography, 18and immunoaffinity chromatography 24all give a single band on SDS-PAGE corresponding to a molecular weight of 14,000. A single amino acid sequence is obtained 24 for the N-terminal 24 residues, which indicates that the enzyme belongs to group II phospholipases A 2, lacking Cys-11. Enzymatic Properties. The enzyme is absolutely specific for the acyl ester bond at the sn-2 position of phospholipids, as could be deduced from the release of [~4C]linoleate and the production of [3H]palmitoyllysophosphatidylethanolamine exclusively from doubly-labeled 1-[9,10-3HE]palmi toyl-2-[1-~4C]linoleoylphosphatidylethanolamine. 9 The enzyme requires Ca 2+ for activity, although this can be replaced by Sr 2+ to give about 30% of the activity measured in the presence of Ca 2+ . The Ca 2+ requirement of the enzyme is not mediated by calmodulin. 25The specific activity of the enzyme obtained after rapid immunoaffinity purification amounts to 28 or 50 U/mg, depending on whether protein is measured by the Lowry procedure or by amino acid analysis, respectively. 24 These specific activities are obtained in routine assays using 0.2 mM phosphatidylethanolamine. Kinetic analyses with this substrate indicate a K m of 1.2 mM and an apparent Vmax of 350 U/mg. Inhibitors. The enzyme is inhibited by p-bromophenacyl bromide, and Ca 2+ protects against this inhibition. 25 Neither diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride nor the thiol reagents N-ethylmaleimide, iodoacetamide, or 5,5'-dithiobis(2-nitrobenzoic acid) inhibit enzyme activity. 9 25j. M. de Winter, J. Korpancova, and H. van den Bosch, Arch. Biochem. Biophys. 234, 243 (1984).
[35] A s s a y a n d P u r i f i c a t i o n o f P h o s p h o l i p a s e A 2 f r o m H u m a n S y n o v i a l F l u i d in R h e u m a t o i d Arthritis By RUTH M. KRAMEg and R. BLAKE PEPINSKY Introduction Phospholipases A 2 (PLA2; phosphatide 2-acylhydrolase, EC 3.1.1.4) are a diverse family of enzymes that hydrolyze the sn-2 fatty acyl ester bond of phosphoglycerides, liberating free fatty acids and lysophospholipids. PLA2 from mammalian pancreas and snake venoms are abundant and METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
