400

PHOSPHOLIPASE A 2

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[38] Purification and Characterization of Cytosolic Phospholipase A 2 Activities from Canine Myocardium and Sheep Platelets B y STANLEY L . H A Z E N , LORI A .

LOEa,

a n d RICHARD W . GROSS

Introduction Activation of intracellular phospholipases plays a critical role in the generation of biologically active metabolites such as eicosanoids and platelet-activating factor. Although many phospholipases are activated during signal transduction (e.g.,phospholipases A2, C, and D), recent studies have demonstrated the importance of phospholipase A2 activation in the generation of the majority of arachidonic acid mass released during cellular stimulation and in the initiation of platelet-activating factor synthesis.l-4 Furthermore, the concomitant accumulation of unsaturated fatty acids and lysophospholipids during myocardial ischemia has underscored the importance of accelerated phospholipid metabolism mediated by phospholipase A2 as a primary determinant of the pathophysiologic sequelae of myocardial ischemia. 5-7 Although it has traditionally been assumed that intracellular phospholipase A2activities are mechanistically and structurally related to the classic low molecular weight phospholipases A2, recent work has identified several new types of intracellular phospholipases A2 which have kinetic characteristics (e.g., calcium sensitivity, substrate selectivity) which implicate them as the likely enzymic mediators of signal transduction in mammalian cells. In this chapter, we describe the purification and characterization of platelet8 and myocardial9'~° cytosolic phospholipases A2 as examples of I A. D. Purdon and J. B. Smith, J. Biol. Chem. 260, 12700 (1985). z M. J. Broekman, J. Lipid Res. 27, 884 (1986). 3 R. L. Wykle, B. Malone, and F. Snyder, J. Biol. Chem. 255, 10256 (1980). 4 j. D. Hanahan, Annu. Reo. Biochem. 55, 483 (1986). s K. R. Chien, A. Han, A. Sen, L. M. Buja, a n d J . T. Willerson, Circ. Res. 54, 313 (1984). 6 p. B. Corr, R. W. Gross, and B. E. Sobel, Circ. Res. 55, 135 (1984). 7 R. W. Gross, in "Pathobiology of Cardiovascular Injury" (H. L. Stone and W. B. Weglicki, eds.), p. 287. Martinus Nijhoff, Boston, Massachusetts, 1985. 8 L. A. Loeb and R. W. Gross, J. Biol. Chem. 261, 10467 (1986). 9 R. A. Wolf and R. W. Gross, J. Biol. Chem. 260, 7295 (1985). l0 S. L. Hazen, R. J. Stuppy, and R. W. Gross, J. Biol. Chem. 265, 10622 (1990).

METHODSIN'ENZYMOLOGY,VOL. 197

Copyright© 1991by AcademicPress. Inc. All rightsof reproductionin any formreserved.

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intracellular phospholipases A 2 which have markedly different kinetic and physical characteristics in comparison to the venom and pancreatic enzymes.11,12

Phospholipase A 2 Assays Platclctphospholipasc A 2 is assayed in the presence of 65 m M Tris-Cl (pH 7.2), 10 m M CaCl2, whereas myocardial phospholipasc is assayed in the presence of 100 m M Tris-Cl (pH 7.0), 4 m M E G T A . Routine assays of platclct or myocardial phospholipasc A2 activitiesarc performed by incubation of platclctsonicates,myocardial cytosol, or column chromatographic fractions (25-100 ~I) with 2/~M radiolabclcd lipid(600 Ci/mol) in the appropriate assay buffer at a finalvolume of 250/~I for 5 rain at 37° in I0 × 75 m m borosilicatetest tubes. Reactions arc initiatedby injection of cthanolic stock solutions (5-10 /~I)containing the appropriate amount of radiolabclcd phospholipid. For routine assays, reactions are quenched with 100 ~I butanol, vortcxed, centrifuged at 2000 gm~ for 2 rain,and separated by thin-layerchromatography (TLC) on silicaG channeled plates in a petroleum ether/ethylether/ acetic acid (80:20: I, v/v) tank. To identify regions containing radiolabclcd fatty acid, unlabeled olcic acid standard is spotted on each channel and, following chromatography, is visualizedby brief12 staining.Regions corresponding to fatty acid (Rf 0.7) arc scraped into vials and quantified by scintillationspectrometry afteraddition of fluor.This procedure yields near quantitative recovery of fatty acid. W h e n phospholipid, lysophospholipid, and fatty acid products arc to bc isolated and quantified simultaneously, reactions arc quenched with 200 /~l butanol, vortcxcd, and centrifuged, and products arc isolated by T L C utilizing a chloroform/acetone/methanol/acetic acid/water (6 :8 :2 :2 : I, v/v) solvent system, which resolves lysophosphatidylcholine (Rf 0.15), phosphatidylcholinc and lysophosphatidylcthanolamine (Rf 0.40), phosphatidylethanolamine (Rf 0.65), and fatty acid (Rf 1,0).Under these conditions, analyses demonstrated that recoveries of 99% of fatty acid, 95% ofphosphatidylcholinc, 87% of phosphatidylcthanolaminc, 84% of lysophosphatidylcholinc, and 74% of lysophosphatidylcthanolamine are routinely achieved. 11 H. M. Verheij, A. J. Slotboom, and G. H. de Haas, Rev. Physiol. Biochem. Pharmacol. 91, 91 (1981). 12 E. A. Dennis, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 16, p. 307. Academic Press, New York, 1983.

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PHOSPHOLIPASEA2

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Miscellaneous Procedures and Sources of Materials

Synthesis of high specific activity radiolabeled plasmenylcholine molecular species (sn-2 radiolabeled with either palmitic, oleic, or arachidonic acids) is achieved by the dicyclohexylcarbodiimide-mediated synthesis of radiolabeled fatty acid anhydride and the subsequent N,N-dimethyl-4aminopyridine-catalyzed condensation of radiolabeled anhydride with homogeneous 1-O-(Z)-hexadec- 1'-enyl-sn-glycero-3-phosphocholine.13 Syntheses of sn-2-radiolabeled diacyl- and 1-O-alkyl-2-acylglycerophosphocholine molecular species are achieved similarly utilizing either 1-hexadecanoyl-sn-glycero-3-phosphocholine or 1-O-hexadecyl-sn-glycero-3-phosphocholine and the appropriate radiolabeled fatty acid as starting materials. Protein content is determined utilizing the Bio-Rad (Richmond, CA) protein assay kit or QuantiGold (Diversified Biotech, Newton, MA) with bovine serum albumin (BSA) as standard. Oleic, palmitic, and arachidonic acids are purchased from Nu Chek Prep (Elysian, MN). Bovine heart lecithin is obtained from Avanti Polar Lipids (Birmingham, AL). Radiolabeled reagents are purchased from New England Nuclear (Boston, MA). Chromatography resins and fast protein liquid chromatography (FPLC) columns are purchased from PharmaciaLKB (Piscataway, N J) and high-performance liquid chromatography (HPLC) columns are obtained from P. J. Cobert (St. Louis, MO). All other materials are obtained from Sigma (St. Louis, MO). Purification of Canine Myocardial Cytosolic Phospholipase A2 Early studies demonstrated that the cytosolic fraction possesses the major measurable phospholipase A2 activity in canine myocardial homogenates and that this activity is calcium independent and selective for plasmalogen substrate.9 To gain insight into the physical and kinetic characteristics of the enzyme catalyzing this novel phospholipase A 2 activity, the enzyme was purified to homogeneity utilizing sequential column chromatographies, l0 Fresh ventricular tissue is obtained from mongrel dogs (25-35 kg), fed ad libitum) after intravenous injection of sodium pentothal (40 mg/kg). The heart is surgically removed and placed in homogenization buffer at 0° (0.25 M sucrose, 10 mM imidazole, 10 mM KC1, 10 mM potassium phosphate, pH 7.8, adjusted with HCl). All subsequent procedures are performed at 0°-4 °. Ventricular tissue (100-150 g) is rapidly isolated, trimmed of fat, rinsed, weighed, placed in fresh homogenization buffer (3 : 1, v/w), 13R. A. Wolf and R. W. Gross, J. Lipid Res. 26, 629 (1985).

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CYTOSOLIC PHOSPHOLIPASES A 2

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and rapidly minced (0.2 x 0.4 cm pieces) with a sharp pair of scissors. Homogenization is performed with 3 strokes of a loose-fitting Potter-E1vehjem apparatus operated at 2000 rpm. Nuclei and cellular debris are removed from the homogenate by centrifugation at 1000 gm~xfor 10 min. The resultant supernatant is centrifuged at 10,000 gmaxfor 10 min, and the mitochondrial pellet (which does not contain substantial phospholipase activity) is discarded. The supernatant is subsequently centrifuged at 100,000 gmax for 60 min, yielding a microsomal fraction (pellet) and a cytosolic fraction (supernatant). Myocardial cytosol is filtered through glass wool, dialyzed twice (8 hr each) against 50 volumes of buffer 1 (15 mM imidazole, 5 mM potassium phosphate, 10% glycerol, pH 7.8), and applied to a preequilibrated DEAESephacel column (5 x 7 cm) at 3 ml/min. After washing with buffer 1 [containing I mM dithiothreitol (DTT)], phospholipase A 2activity is eluted by direct application of 100 mM NaC1 in elution buffer (10 mM imidazole, 10 mM KC1, 10% glycerol, 1 mM DTT, pH 8.0). Fractions possessing phospholipase A2 activity are pooled and dialyzed against 20 liters of buffer 2 (I0 mM imidazole, 10 mM KCI, 25% glycerol, 1 mM DTT, pH 8.0) overnight. The dialyzed DEAE-Sephacel eluate is subsequently loaded at 1.8 ml/min onto a Polybuffer Exchanger-94 chromatofocusing column (1.6 x 30 cm) previously equilibrated with buffer 2. Application ofa polybuffer (PB) mixture (10% PB96, 5% PB74, 25% glycerol, 1 mM DTT, pH 7.1) results in elution of a sharply focused peak of phospholipase A2 activity with an apparent isoelectric point of 7.55 (Fig. 1A). Chromatofocusing fractions containing phospholipase A2 activity are immediately applied to a 1 x 1 cm column of N6-[(6-aminohexyl)carbamoylmethyl]ATP-agarose previously equilibrated with buffer 3 (10 mM imidazole, 25% glycerol, 1 mM DTT, pH 8.3) at 2 ml/min. The column is washed with 20 ml of buffer 3 alone, with 5 ml of buffer 3 containing 10 mM adenosine, with 15 ml of buffer 3 containing 10 mM AMP, and once again with 5 ml of buffer 3 alone (to remove all UV-absorbing AMP). Application of buffer 3 containing 1 mM ATP results in the quantitative elution of phospholipase A2 activity from the ATP-agarose matrix (Fig. 1B). Phospholipase A 2activity in the ATP-agarose eluate is purified 52,000fold (from canine myocardial cytosol) and is moderately stable when stored at 00-4 ° (tl/2 5-7 days) or stable indefinitely (6 months) in liquid nitrogen. Phospholipase A2 is further purified by the direct application of the ATP-agarose eluate to an HR 5/5 Mono Q column previously equilibrated with buffer 4 (20 mM imidazole, 25% glycerol, 1 mM DTT, pH 8.3). Application of a nonlinear salt gradient (0-450 mM NaCI) results in the elution of phospholipase A2 activity (Fig. 2A). Mono Q active fractions are identified and immediately loaded onto a Koken hydroxylapatite HPLC

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FIG. 1. Chromatofocusing and ATP affinity chromatographies of canine myocardial cytosolic phospholipase A2. Eluate from the DEAE-Sephacel column was further purified utilizing sequential chromatofocusing (A) and ATP-agarose (B) chromatographies as described in the text. (A) Fatty acid release from l-O-(Z)-hexadec-l'-enyl-2-[9,10-3H]octadec-9'-enoyl sn-glycero-3-phosphocholine (0); UV absorbance at 280 nm (--); pH (11). (B) Fatty acid release from 1 - O - ( Z ) - h e x a d e c - l ' - e n y l - 2 - [ 9 , 1 0 - 3 H ] o c t a d e c - 9 ' - e n o y l - s n - g l y c e r o - 3 - p h o s p h o choline (O).

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Purification and characterization of cytosolic phospholipase A2 activities from canine myocardium and sheep platelets.

400 PHOSPHOLIPASE A 2 [38] [38] Purification and Characterization of Cytosolic Phospholipase A 2 Activities from Canine Myocardium and Sheep Platel...
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