C o b r a V e n o m P h o s p h o l i p a s e A2: Naja naja naja
By LAURE J. REYNOLDS and EDWARD A. DENNIS Introduction 1,2-Diacyl-sn-glycero-3-phosphatide + H20--~ 1-acyl-sn-glycero-3-phosphatide + fatty acid
Phospholipase A 2 (PLA2; EC 3.1. !.4) is a major component of most snake venoms and can be purified in large quantities from this source. Purification of the enzyme is complicated by the presence of multiple isozymes in many species. The venom from the Indian cobra (Naja naja naja) contains as many as 14 PLA2 isozymes. 1'2 This chapter describes the purification of an acidic phospholipase A2 from this source) This purification is less harsh than a procedure previously published in this series4 and yields a purer enzyme preparation.
Principle. Phospholipase AE catalyzes the hydrolysis of the fatty acid in the sn-2 position of phospholipids. The reaction is followed by titrating the fatty acid that is released using a pH-stat apparatus. The substrate for this reaction is egg phosphatidylcholine (PC), which is solubilized in Triton X-100 mixed micelles. Reagents Egg phosphatidylcholine (purified from egg yolks by the method of Singleton et al.5), 200 mM in CHC13 Triton X-100, 200 mM CaC12, 200 mM KOH, 5 mM Procedure. 4'6The standard assay mixture contains 5 mM egg phosphatidylcholine, 20 mM Triton X-100, and 10 mM CaC12. The appropriate volume of egg phosphatidylcholine solution is measured into a homogenil M. K. Bhat a n d T. V. Gowda, Toxicon 27, 861 (1989). 2 j. Shiloah, C. Klibansky, and A. deVries, Toxicon 11, 481 (1973). 3 T. L. Hazlett a n d E. A. Dennis, Toxicon 23, 457 (1985). 4 R. A. D e e m s and E. A. Dennis, this series, Vol. 71 . 5 W. S. Singleton, M. S. Gray, M. L. Brown, and J. L. White, J. Am. Oil Chem. Soc. 42, 53 (1965). 6 E. A. Dennis, J. Lipid Res. 14, 152 (1973).
METHODS IN ENZYMOLOGY, VOL. 197
Copyright © 1991by AcademicPress, Inc. All rights of reproduction in any form reserved.
zation tube. The sample is dried first under a stream of nitrogen then under vacuum until all the chloroform has evaporated. The appropriate amounts of Triton X-100 and C a C l 2 a r e added, and the solution is brought to the final volume by addition of deionized water. The phospholipid is solubilized by heating to 40o-50 ° followed by vortexing. The enzymatic reaction is followed using a Radiometer pH-stat apparatus (Westlake, OH) equipped with a 0.25-ml burette. Two milliliters of assay mix is brought to pH 8.0 and 40° under a stream of nitrogen. The reaction is initiated by addition of enzyme, and the fatty acids released are titrated with 5 mM KOH. Further details and comments on the assay procedure have been discussed previously in this series. 4 Comments. The assay is also routinely performed with commercially available dipalmitoylphosphatidylcholine as a substrate. With this substrate, micelles are prepared in the same manner, except the solution is heated to 60° before vortexing. Assays with dipalmitoylphosphatidylcholine give comparable rates to the egg PC assays.
Purification Procedure The purification scheme used here was first described by Hazlett and Dennis. 3 The PLA2 is quite stable, and purification can be carded out at room temperature. Protein concentrations are determined by the method of Lowry et al. 7 using the correction factor for Naja naja naja phospholipase A 2 which was determined by Darke et al. 8 The Lowry assay overestimates the protein concentration for this enzyme when bovine serum albumin is used as a standard. Therefore, protein values obtained by this method must be multiplied by a factor of 0.66 to obtain the correct value. Although this correction is valid for the purified PLA2 only and not necessarily for the crude venom, for consistency, the correction has been applied to all protein values reported in Table I. Caution: When handling the lyophilized venom, care should be taken not to disperse or inhale the powder. Use of gloves and a mask is recommended at this stage. Non-PLA2 column fractions which may contain toxins are treated with sodium hypochlorite or strong acid prior to disposal. After use, column packing materials, which may retain toxins, are usually disposed of and not reused. Step 1. Deionized water (500 ml) is added to a flask containing approxi7 0 . H. Lowry, N. J. Rosenbrough, A. L. Fair, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 8 p. L. Darke, A. A. Jarvis, R. A. Deems, and E. A. Dennis, Biochim. Biophys. Acta 626, 154 (1980).
COBRA VENOM PHOSPHOLIPASE A 2
TABLE I PURIFICATION OF PHOSPHOLIPASE A 2 FROM COBRA VENOMa
Step 1. 2. 3. 4.
Soluble venom Affi-Gel Blue DE-11 SP-Sephadex
Total protein (mg)
Total activity (103 units)
Specific activity (units/mg)
5285 374 220 163
872 552 416 356
165 1480 1890 2180
1.0 9.0 11.5 13.2
Reprinted from Hazlett and Dennis 3 with permission.
mately 10 g (dry weight) of lyophilized venom [Naja naja naja (Pakistan), obtained from Miami Serpentarium Laboratories, Punta Gorda, FL]. Most of the venom is dissolved by swirling the mixture gently for several minutes. Much of the venom material remains insoluble and is removed by centrifugation of the mixture for 15 min at approximately 8000 g. The supernatant is diluted 1 : 1 with 50 mM ammonium acetate (pH 6.0). The pH is checked with pH paper and adjusted to pH 6.0 with acetic acid if necessary. Step 2. A 200-ml volume of Affi-Gel Blue [Bio-Rad (Richmond, CA), 100-200 mesh, 75-150/zm] is suspended in approximately 100 ml of 20 mM ammonium carbonate (pH 10.5). This buffer is prepared by bringing a solution of ammonium carbonate to pH 10.5 with concentrated ammonium hydroxide, then diluting to the final volume with deionized water. The suspension is poured into a 5 cm diameter column and washed with the same buffer (-800 ml) at a rate of 2.5-3 ml/min to remove any free blue dye. The height of the packed column is 11.5 cm. The column is equilibrated with 50 m M ammonium acetate (pH 6.0) until the pH returns to about 6.0. After equilibration of the column, the sample is loaded. The column effluent is monitored at 280 nm. A large protein peak elutes in the flowthrough fraction. When loading is complete, the column is washed with 50 mM ammonium acetate (pH 6.0) until the absorbance returns to baseline. At this point, the second buffer, 50 mM ammonium bicarbonate (pH 8.0), is started. A small peak, which is sometimes barely observable, starts to elute after 250-300 ml of this buffer. When the absorbance again returns to baseline, the final buffer, 20 mM ammonium carbonate (pH I0.5), is started. This buffer elutes a large protein peak which contains 63% of the initial activity. The protein peak is pooled, neutralized to pH 7.5, and lyophilized, leaving a salt-free protein powder. We typically encounter two problems with the lyophilization. First, the sample usually foams
PHOSPHOLIPASE A 2
when initially placed under vacuum and should be monitored carefully at this stage. This problem is minimized by having the sample well frozen before starting lyophilization. Second, during later stages of lyophilization the sample often thaws and must be diluted with water and then refrozen. Step 3. The enzyme is next passed through a DEAE-cellulose column (Whatman DE-11 or DE-23, 4.2 × 30 cm). The resin is prepared as recommended by the manufacturer. Approximately 600 ml (settled volume) of prepared resin is stirred into I liter of 50 mM sodium phosphate (pH 7.5) containing 0.3 M NaCI. The pH of the slurry is readjusted to 7.5 with HC1. The excess buffer is decanted, and the column is packed with the remaining slurry. The column is equilibrated with 5 mM sodium phosphate (pH 7.5) at a rate of 2.8 ml/min. The lyophilized protein is dissolved in 200 ml of the same buffer and loaded onto the column. When loading is complete the column is washed with 5 mM sodium phosphate (pH 7.5). After the absorbance returns to baseline, a second buffer is started which is composed of 0. I M NaC1 in 5 mM sodium phosphate (pH 7.5). When the absorbance returns to the baseline, the third buffer (0.3 M NaC1 in 5 mM sodium phosphate, pH 7.5) is started. The majority of the loaded protein is eluted by this high-salt buffer. The peak is pooled and dialyzed in Spectrapor 1 tubing (Spectrum Medical Industries, Los Angeles, CA) 6000-8000 MW cutoff) against 10 mM sodium phosphate (pH 6.0). Step 4. SP-Sephadex C-25 (25 g) is swollen in I0 mM sodium phosphate oH 6.0) and poured into a 2.4 cm diameter column to a final bed height ,f 35 cm. The column is equilibrated in the same buffer, at a flow rate of ml/min, before loading the dialyzed protein. The phospholipase A2 passes :hrough the column while a minor contaminant is retained. After loading, the column is washed with 10 mM sodium phosphate (pH 6.0) until baseline absorbance is again reached. The flow-through fractions that contain PLA 2 are pooled, separated into 40-ml aliquots, and stored at - 20°. This protein preparation is stable for several years.
Purity. This procedure yields a PLA 2 preparation that displays a single protein band on analytical isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and Ouchterlony double-diffusion precipitation tests. 3 Phospholipase A 2 purified by the previous method4 contains a minor contaminant, not visible on SDS-polyacrylamide gels, which is removed by the third column in this procedure. Stability. Cobra venom phospholipase A 2 contains seven disulfide bonds, which make the enzyme extremely stable. The enzyme retains
COBRAVENOMPHOSPHOLIPASE A 2
activity after incubation in the absence of buffers for 12 hr at 600. 9 Circular dichroism spectra show that the enzyme begins to denature at 80°, although this change is reversed upon cooling. 9 In fact, the enzyme can be heated at 100° for 10 min at pH 3-4 with only 5% loss in activity. 4 Cobra venom phospholipase A2 is also fairly stable to chaotropic salts. The enzyme retains 60% of its activity when assayed in the presence of 6 M guanidine hydrochloride. 4 For complete denaturation, the protein must be subjected to 8 M guanidine hydrochloride at 40 ° in the presence of I0 mM dithiothreitol and 10 m M EDTA. 9 The enzyme is stable at pH 9.51° but is unstable at pH 10.5. 9 Primary Structure. Cobra venom phospholipase A 2 is classified as a type I phospholipase based on its disulfide bond pattern. 9,11It is composed of a single polypeptide chain of 119 residues. The amino acid sequence of the enzyme has been determined. 9 The molecular weight, calculated from the sequence, is 13,348. Analysis of the circular dichroism spectrum of the native protein indicates that it is composed of 42-50% a helix. 9 Physical Properties. The extinction coefficient of the enzyme, ~0.~% x-,278 , is 2.2. 8 The protein is acidic, with an isoelectric point of 5.1.10 The enzyme exhibits a broad activity optimum between pH 7 and 9. l° Below 0.05 mg/ml cobra venom phospholipase A 2 exists as a monomer. ~2Above this concentration the enzyme undergoes a concentration-dependent aggregation and exists as a dimer or higher-order aggregate. Cofactor. Cobra venom phospholipase A 2displays an absolute requirement for the presence of Ca 2÷ for catalysis. The kinetically determined K m for Ca 2÷ is 1 mM 13 whereas the KD for Ca 2÷ has been estimated at 0.15 mM. 1° Specificity. This phospholipase catalyzes the hydrolysis of the fatty acid ester bond at the sn-2 position of L-phospholipids. The enzyme will slowly catalyze the hydrolysis of monomerically dispersed phospholipids, but enzyme activity is much greater ~¢hen hydrolyzing lipids that are aggregated at a lipid-water interface. ~4 The enzyme is sensitive to the nature of the interface and displays greater activity toward detergentmixed micelles than to phospholipid vesicles. The activity of the enzyme in mixed micelles is also sensitive to the phospholipid-detergent ratio. The highest activity is seen at high mole fractions ofphospholipid. Increas9 F. F. Davidson and E. A. Dennis, Biochim. Biophys. Acta 1037, 7 (1990). 10 M. F. Roberts, R. A. Deems, and E. A. Dennis, J. Biol. Chem. 252, 6011 (1977). 11 R. L. Heinrikson, E. T. Kreuger, and P. S. Keim, J. Biol. Chem. 252, 4913 (1977). 12 R. A. Deems and E. A. Dennis, J. Biol. Chem. 250, 9008 (1975). 13 E. A. Dennis, Arch. Biochem. Biophys. 158, 485 (1973). 14 M. F. Roberts, A.-B. Otnaess, C. R. Kensil, and E. A. Dennis, J. Biol. Chem. 253, 1252 (1978).
ing the amount of detergent in the micelle results in lower enzymatic activity due to surface dilution of the phospholipid substrate.13 When a single type of phospholipid is present in an assay, the enzyme shows a preference for phosphatidylcholine over phosphatidylethanolamine over phosphatidylserine.14 In addition to this head group specificity, the enzyme is sensitive to the chain length of the fatty acid. The enzymatic rate increases as the chain length decreases from 16 carbons (palmitate) to 8 carbons) 4 This effect could represent the specificity of the enzyme. However, it may be due to slight changes in the lipid interface caused by the presence of the different fatty acids or to a decrease in product inhibition by the shorter fatty acids. 15 The enzyme can hydrolyze thio ester bonds in the sn-2 position of phospholipids but will not hydrolyze amide bonds such as that in sphingomyelin) 4 The ability to hydrolyze thio ester bonds has been utilized to develop a thiol-based assay for the enzyme) 6 Kinetic Properties. The rate of phospholipase A 2 hydrolysis depends on both the bulk concentration of phospholipid and on the concentration of phospholipid in the interface (mole ratio). Kinetic analysis of phospholipase A 2 activity is very complex and must vary both of these parameters. 17 When the mole ratio of Triton X-100 to egg phosphatidylcholine is held constant at 2 : 1, the apparent K m is estimated to be between 2 and 5 mM with an apparent Vm~xof 2000 units/mg. 6 The rate of egg phosphatidylethanolamine hydrolysis is 15-fold slower than the rate of PC hydrolysis. 15,18 However, the rate of phosphatidylethanolamine hydrolysis increases up to 20-fold with the addition of choline-containing activators such as dodecylphosphorylcholine, sphingomyelin, or phosphatidylcholine) 5A8 Inhibitors. Cobra venom phospholipase A2 is inhibited by Ba 2÷ and Sr 2+, which are competitive inhibitors of the Ca 2+ cofactor. The KI) for both these metals is about 0.6 mM. 1° The enzyme is subject to product inhibition by fatty acids. 15This inhibition can be minimized by utilizing a shorter chain phospholipid, the fatty acid products of which can diffuse out of the micelle, or by adding bovine serum albumin to the assay to extract fatty acids from the micelles. The enzyme is inhibited by aromatic dyes such as Cibacron Blue, which binds the enzyme reversibly with a KI) about 2/zM and K i of 3.5/£M. 19 Phospholipids which contain an amide, 2°
i5 A. Pliickthun and E. A. Dennis, J. Biol. Chem. 260, 11099 (1985). 16 L. Yu and E. A. Dennis, this volume [5). 17 R. A. Deems, B. R. Eaton, and E. A. Dennis, J. Biol. Chem. 250, 9013 (1975). is M. Adamich, M. F. Roberts, and E. A. Dennis, Biochemistry 18, 3308 (1979). 19 R. E. Barden, P. L. Darke, R. A. Deems, and E. A. Dennis, Biochemistry 19, 1621 (1980). 2o L. Yu, R. A. Deems, J. Hajdu, and E. A. Dennis, J. Biol. Chem. 265, 2657 (1990).
MITOCHONDRIAL PHOSPHOLIPASE A 2
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).
 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 220.127.116.11) 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
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