Rapid activation of the non-toxic basic isoform of phospholipase A2 from Naja mossambica mossambica (spitting cobra) by long-chain fatty acylation

0041-0101/90 f3.00 + .00 ® 1990 Perpmon Prep Pk

Taxirom Vol . 28, No. 8, pp . 953-%1, 1990. Printed in Gral Britain .

RAPID ACTIVATION OF THE NON-TOXIC BASIC ISOFORM OF PHOSPHOLIPASE A2 FROM NAJA MOSSAMBICA MOSSAMBICA (SPITTING COBRA) BY LONG-CHAIN FATTY ACYLATION SÀLAH CHETTIBI, l FIONA LYALL2

and ANTHONY J .

LAWRENCE' *

'Department of Cell Biology, University of Glasgow, Glasgow G128QQ, Scotland, U .K . and 'MRC Blood Pressure Unit, Western Infirmary, Glasgow GI 16NT, Scotland, U.K . (Acceptedfor publication 4 January 1990) S . CHETTmi, F. LYALL and A. J . LAWRENCE . Rapid activation of the non-toxic basic isoform of phospholipase A2 from Naja mossambica mossambica (spitting cobra) by long-chain fatty acylation. Toxicon 28, 95961, 1990.-Purified phospholipase A2 from Naja naja (common Indian cobra) venom, a basic nontoxic isoform and a toxic isoform from Naja mossambica mossambica venom were treated with an equimolar amount of oleoyl imidazolide, a known activator of bee venom phospholipase A2. The ability of the first two enzymes to induce erythrocyte lysis was strongly activated while hydrolysis of dioctanoyl phosphatidyl choline was only weakly activated. The toxic enzyme showed little change in either assay. The susceptible enzyme from Naja mossambica mossambica venom reacted very much more rapidly with oleoyl imidazolide than did the bee venom enzyme and oleic acid was shown to be a relatively weak antagonist. Activation had very little effect on the metal ion dependence of these enzymes. Although activation was a progressive response having the characteristics of a chemical reaction which could not be mimicked by free fatty acids, the adduct did not appear to be stable under acidic conditions . This evidence suggested that primary amino groups were not the target for acylation .

INTRODUCTION

A2 enzymes are a family of small proteins that exhibit very complex kinetic behaviour. In part this can be attributed to the extreme variety of chemical and physicochemical properties presented by their substrates and to the fact that the enzymes act at a lipid/water interface. There is evidence that different aspects of the enzymic behavior can be regulated independently and ROSENBERG (1988) has recently reviewed the factors which determine enzymic and toxic activity in closely related venom enzymes. A good example of differential regulation is provided by the phospholipase A2 from honey bee (Apis mellifera) venom which can be activated towards some, but not all substrates either by free long-chain fatty acids or else by weakly activated derivatives of these acids, of which the best example is oleoyl imidazolide (DRAINAS and LAWRENCE, 1978 ; CAMERO-DIAZ et al ., PHOSPHOLIPASE

*To whom correspondence should be addressed .

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1985). Whilst the free fatty acids act directly, the derivatives modify the protein to produce an adduct which is stable to a wide variety of conditions including the presence of the powerful fatty acid sequestering agent, serum albumin . The enzyme was fully activated by a single molar equivalent of the acylating agent suggesting that a highly specific site was involved, although the alternative model, that acylation of any one of several susceptible sites on the protein could produce the activation response has not been ruled out. VAN Dm WEILE et al. (1988a,b) have devised methods for long-chain fatty acylation of mammalian pancreatic phospholipase A2 enzymes at specific lysine residues which do not take advantage of the intrinsic selectivity of the site and have demonstrated that this modification also activates the enzyme towards erythrocyte lysis. DRAINAS (1978) proposed that acylation of bee venom phospholipaseA2 occurred at a specific fatty acid binding site and that the target was also the residue which interacted with the carboxylate group of the free fatty acids. The high selectivity shown by this site towards long-chain fatty acid derivatives in comparison with their short-chain analogues, indicated that hydrophobic interactions were the primary determinant of reactivity . There is no clear indication that fatty acid binding sites of other proteins can be selectively acylated by such derivatives and this therefore appeared to be an idiosyncratic property of this enzyme. The model had two problematic features; there was evidence that both fatty acids and their derivatives could occupy the site simultaneously (LAWRENCE and MOORES, 1975) and the pH dependence of fatty acid binding and the rate of acylation did not show the opposing characteristic expected if a single ionizing group were involved . This raises the question of whether a single site is involved and whether the free fatty acids or the bound acyl residues are the true functional activators. Answers to both questions might come from work with other enzymes. If fatty acid binding sites do not automatically possess high reactivity towards long-chain acylating agents, then the frequent occurrence of this feature would indicate that it has a functional role . In this work we investigated the activation of two basic isoforms of the enzyme from Naja mossambica mossambica, one of which is much more highly toxic than the other (DUFrON et al ., 1983 and KIM and EvANs, 1987). To our surprise, the toxic isoform showed no response to oleoyl imidazolide, whilst the non-toxic form was very strongly activated and reacted with the reagent at least 20 times faster than the bee venom enzyme . The faster rate should increase the specificity of group substitution, making this enzyme a most suitable protein for further investigation. MATERIALS AND METHODS Kinetic measurements were carried out by conductimetric assays essentially as described elsewhere (LAWRENCE, 1971 ; DRmNAs and LAWRENCE, 1981 ; CANmRo-DiAz et al., 1985), but using an eight-cell system with digital processing of the results by microcomputer (LAWRENCE and CHE117aI, unpublished observations) . Enzyme activity assays were carried out by measuring the hydrolysis of 0.6 mM dioctanoyl phosphatidyl choline in 2 ml of 10 mM triethanolamine buffer, pH 8 .0, at 37°C, which has a detection sensitivity limit of ca 1 ng of bee venom phospholipase A,. Activation was measured in a modified version of the erythrocyte leakage assay using rabbit erythrocytes which were suspended at 0 .3% v/v in 2 ml isotonic sucrose medium buffered at pH 7 .4 with 10 mM morpholinopropane sulphonic acid/Na' (MOPS buffer), in the presence of 10 pM bovine serum albumin at 37°C. Conductance changes were used as a measurement of electrolyte leakage (LAWRENCE, 1975) . Under these standardized conditions the conductance change produced when the cells are completely lysed is ca 2% f 0 .1 % . A standard leakage curve using valinomycin-induced leakage showed that at least 90% of the conductance response for these cells corresponded to the efflux of potassium ions and counter-ions, (LAWRENCE et al., 1975) . Enzymes, buffers, fatty acids and other reagents were from Sigma Chemical Co . Ltd ., Poole, Dorset, U .K . Oleoyl imidazolide was prepared from oleic acid and carbonyl diimidazolide as described by DRAINAs and LAWRENCE (1978) and used as a solution in acetone. Modification of the enzymes was carried out by adding an

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equimolar concentration of the oleoyl imidazolide to the enzyme solution at pH 8 .0 . Glycerophosphoryl choline was prepared from egg lecithin by a novel method designed to minimize contamination with cadmium ions (CH=I, DRALNAs and LAWRENCE, unpublished work) in which the hydrolysis was accomplished by a strong base anion exchange resin in methanolic solution and the product purified by solvent extraction techniques without foaming the cadmium chloride adduct. Dioctanoyl phosphatidyl choline was prepared from glycerophosphoryl choline free base by the method described by PATEL et al. (1979) for the cadmium chloride adduct. The product was pure as determined by thin layer chromatography on silica gel . Proteins were analysed by electrophoresis using the propionic acid/urea/polyacrylamide gel procedure described by CHErnsl and LAWRENCE (1989) . RESULTS

The phospholipase AZ enzymes from Naja naja, honey bee, and the two basic (toxic and the non-toxic) isoforms from Naja mossambica mossambica, were tested for catalytic activity using dioctanoyl phosphatidyl choline substrate and also for lytic activity in the erythrocyte leakage assay (Table 1). Both types of activity were totally inhibited by EDTA, indicating that the lytic activity requires catalytic action in all cases. The enzymes were then incubated with a molar equivalent of oleoyl imidazolide and the activities compared in the cell leakage assay. Figure 1 shows the degree of activation of these enzymes after 120 min of incubation ; after this time all activities remained at a stable level and none showed any response to a further addition of oleoyl imidazolide. The enzymes from Naja naja venom, bee venom and the less toxic basic isoform from Naja mossambica mossambica, were strongly activated in the lysis assay and weakly activated in the hydrolysis assay, but the toxic isoform from Naja mossambica mossambica, was partially inhibited in both cases (Fig. 3). Although the enzymes that responded to oleoyl imidazolide showed very similar activation factors, the p18 .8 isoform from Naja mossambica mossambica was the most suitable subject for activation studies because it has a high residual activity and gives the largest response to activation of any of these enzymes in the rabbit erythrocyte. This experiment also revealed that the enzymes activated at very different rates with the responsive isoform from Naja mossambica mossambica reaching full activation at the earliest measured time . Figure 2 gives a comparison of the activation rate of this enzyme with that of the bee venom enzyme and also shows how this rate is affected by the presence of an equimolar concentration of free oleic acid. It was necessary to dilute both the enzyme and the oleoyl imidazolide two-fold with respect to the concentrations used for the bee venom enzyme in order to obtain suitable rates for measurement. After correction for the concentration difference it is clear that the TABLE 1 . COMPARISON OF THE HYDROLYTIC AND LYTIC ACTIVITIES OF VENOM PHOSPHOLIPASE (PLA 2) ENZYMES

Enzyme Bee venom PLA2 pI 9.7 isoform of Naja mossambica mossambica PLA 2 pl 8 .8 isoform of Naja mossambica mossambica PLA 2 Naja naja (common Indian cobra) PLA2

Hydrolysis of dioctanoyl phosphatidyl choline (pmoles/mg/min)

Electrolyte leakage from rabbit erythrocytes (%/min)

2160± 80 1284± 95 7133±115 5440± 80

3 .62±0 .04 20 .05±0 .07 13 .1 ±0 .14 2.57±0 .014

The enzyme concentrations were 0.125, 0 .05, 0 .125 and 0 .125 pg/ml for bee venom, pI 8 .8 isoform of Naja mossambica mossambica, pl 9.7 isoform of Naja mossambica mossambica and Naja naja venom respectively for the hydrolysis assay and lug/id in the electrolyte leakage assay. The assays were carried out as described in Materials and Methods and results are shown as means ±S .D .

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FIG . 1 . ACTIVATION OF VENOM PHOSPHOLIPASE A2 INDUCED LEAKAGE FROM RABBIT ERYTHROCYTES BY OLEOYL IMIDAZOLIDE . One hundred microliter aliquots of solutions of purified venom phospholipase A2 enzymes

(I mg/ml in 10 mM triethanolamine/Cl buffer pH 8 .0) were mixed with 2 pl of an 0.2% w.v solution of oleoyl imidazolide in acetone and incubated for 2 hr at 37°C . Two microliter samples were tested for the ability to promote leakage from rabbit erythrocytes suspended in isotonic sucrose medium buffered at pH 7.4 with 10 mM MOPS/Na and in the presence of 10 pM bovine serum albumin. The conductance change was used as an indirect measure of erythrocyte leakage and the change produced by total lysis is ca 2% of the initial value. p Untreated controls ; enzyme treated with oleoyl imidazolide. A, Bee venom phospholipase A2 ; B, non-toxic isoform of phospholipase A 2 from Naja mossambica mossambica (p18.8); C, toxic isoform of phospholipase A2 from Naja mossambica mossambica (p19 .7); D, phospholipase A2 from Naja raja venom.

difference in reactivity of these two enzymes is > 20-fold. The presence of an equimolar concentration of oleic acid decreased the rate less than 1 .5-fold indicating that it bound more weakly to this site than did oleoyl imidazolide, it also lowered the maximum rate attained in the assay, which is difficult to reconcile with the fact that no effects of fatty acid are ever seen when albumin is present with the erythrocytes before fatty acid is added. To test the possibility that the activation phenomena might be due to alteration of the metal ion binding sites of the enzymes, activity measurements were made in the presence of the transition metal ion chelator o-phenanthroline and also at elevated calcium levels. We are aware that the satisfactory study of these enzymes requires the use of a metal ion buffer as the only way to control calcium levels whilst reducing transition metal ion contamination to negligible levels and have established that nitrilotriacetic acid is an ideal suitable buffer compound for this purpose (CHE1-mi and LAWRENCE, unpublished work). Results obtained with bee venom enzyme showed that the effective Kd for calcium was ca 5 juM and that the level of calcium in our double-distilled water was ca 8-10 MM . Figure 3 illustrates the important features of the ion dependence of these enzymes . The three phospholipase A2 enzymes from bee venom, non-toxic isoform from Naja mossambica mossambica, and toxic isoform from Naja mossambica mossambica were all activated by o-phenanthroline or by excess calcium indicating that they have very similar affinity for

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FIG . 2 . THE TIME-COURSE OF ACTIVATION OF PHOSPHOLwASE A2 ENZYMES BY OLEOYL IMIDAZOLIDE. Solutions of purified phospholipase A, enzymes prepared as in Fig. 1, were treated with oleoyl imidazolide, samples withdrawn at time intervals and activity measured in the rabbit erythrocyte leakage assay in the presence of 10 uM bovine serum albumin . The non-toxic p18 .8 isoform of phospholipase A, from Naja mossambica mossambica (20 jig in 100 Pl) was treated with 0.4 ug of oleoylimidazolide in the absence and presence of 0.4 ug oleic acid . The phospholipase A, from the honey bee venom (100 jig in 100 ul) was treated with 2 jig of oleoyl imidazolide only. p-p the non-toxic isoform of phospholipase A, from Naja mossambica mossambica (pI8 .8) in the presence of oleoyl imidazolide; 0-19 the non-toxic isoform of phospholipase A, from Naja mossambica mossambica (p78 .8) in the presence of oleoyl imidazolide and oleic acid ; 0-0 phospholipascA, from bee venom in the presence of oleoyl imidazolide only . Each value is the mean of three samples t S.D . The inset shows the time course for bee venom phospholipase A, plotted on an extended time-scale allowing the reaction to approach completion ; on this scale S.D . values were too small to be shown.

calcium and that contaminating transition metal ions, probably copper or zinc, were powerful competitors for calcium (Tu, 1977). It is, however, of great interest that the lytic actions of these enzymes are almost insensitive to o-phenanthroline . The lysis assays required the presence of albumin, and in order to see whether or not this could affect residual activity, the response of normal and activated enzyme to albumin were tested in the activity assay. In all cases albumin changed the shape of the reaction progress curves indicating that it lowered the affinity of the enzyme for the substrate. It had differential effects on the normal and activated forms of these enzymes, but no consistent pattern emerged (data not shown) . Samples of activated and normal enzymes were subjected to propionic acid/urea/ polyacrylamide gel electrophoresis (Fig. 4). The non-toxic isofotm of the Naja mossambica

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THE EFFECT OF CALCIUM AND o-PHENANTHROLINE ON NATIVE AND MODIFIED FORMS OF VENOM PHOSPHOLIPASE A, ENZYMES.

Two microliters of 0.1 mg/ml solutions from native bee venom; the toxic isoform from Naja mossambica mossambica (p19 .7) and 1 pl of 0.1 mg/ml of the non-toxic isoform from Naja mossambica mossambica (p18 .8) phospholipase A, and phospholipase A, treated with oleoyl imidazole (as in Fig. 1), were assayed by conductimetric estimation of the hydrolysis of dioctanoyl phosphatidyl choline (0 .5 mM) in 2 ml of 10 mM triethanolamine/Cl - buffer, pH 8 .0 at 37°C. 1, with no additions (controls) ; 2, as 1 but including 100 pM o-phenanthrohne ; 3, as 1 but including 100 pM calcium chloride ; 4, as 1 but including both 100 pM o-phenanthroline and 100 pM calcium chloride . Each value is the mean of three samples t S.D . A, native bee venom phospholipaseA,; B, activated bee venom phospholipase A,; C, native pf 8.8 isoform of phospholipase A, from Naja mossambica mossambica ; D, activated p18.8 isoform of phospholipase A, from Naja mossambica mossambica ; E, native p19.7 isoform of phospholipase A, from Naja mossambica mossambica; F, activated p19.7 isoform of phospholipase A, from Naja mossambica mossambica.

mossambica enzyme was used as a control for resolution by treating it with acetic anhydride in the molar ratios 1 :1, 1 :10 and 1 :100 (channels 9, 11, 12). The higher concentrations (channels 11, 12) produced a step pattern indicating that a loss of a single charged group reduced the mobility by approximately 10% . Full modification (Channel 12) appears to correspond to a loss of 8 charges, as predicted for the 8 primary amino groups known to be present with the protein. Addition of a single molar equivalent (channel 9) did not appear to be enough to modify a single charge equivalent (presumably because of the competing hydrolysis reaction) however, it clearly produced a greater charge than did oleoyl imidazolide (compare channels 9 and 10 with channels 3 and 4).

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FIG . 4 . GEL ELECTROPHORESIS OF THE NATIVE AND MODIFIED VENOM PHOSPHOLIPASE A

2 ENZYMES.

One hundred microliters of 1 mg/ml solutions of native and modified venom phospholipase A, enzymes from bee venom, the non-toxic p18 .8 isoform of phospholipase A, from Naja mossambica mossambica, the toxic p19.7 isoform of phospholipase A, from Naja mossambica mossantbica and Naja naja venom were mixed with 2:l v/v of water containing 50% glycerol with neutral red marker dye and 20 pl was applied to 22 .5% propionic acid/urea/polyacrylamide gel electrophoresis. Channels 1:2, native and modified bee venom phospholipase A, respectively; 3 :4, native and modified enzyme of the non-toxic p18.8 isoform of phospholipase A, from Naja mossambica mossambica respectively; 5:6, native and modified enzyme of the toxic p19.7 isoform of phospholipaseA, from Naja mossambica mossambica respectively ; 7:8, native and modified phospholipase A, from Naja naja venom; 10, the native form of p18.8 isoform of phospholipase A, from Naja mossambica mossambica; 9:11 :12 = the non-toxic isoform of phospholipase A, from Naja mossambica mossambica treated with acetic anhydride in the molar ratios of 1 :1, 1:10 and 1:100 respectively and 13 myoglobin chymotryptic fragments markers (mol . wt = 2510-16950) .

None of the enzymes that were activated by oleoyl imidazolide showed any mobility change on this gel but paradoxically the enzyme which is not activated by the reagent largely failed to migrate on the gel (channel 6). Our interpretation is that it reacted readily and irreversibly and that the modified form aggregated very strongly, but still retained more than 50% of its catalytic activity (Fig. 4). DISCUSSION

The activation of phospholipase A2 enzymes by fatty acylating derivatives typified by oleoyl imidazolide raises several interesting chemical and biological problems . Undoubtedly the most important is whether or not this type of activation has any functional biological role and as yet there is no evidence to indicate that it does. The chemistry of the reaction between the enzyme and the reagents is obscure. Free fatty acid cannot activate these enzymes in the presence of albumin (DRAINAs and LAWRENCE, 1981) thus the progressive nature of activation reflects an underlying chemical modification and not the hydrolysis of the reagent. Our original assumption was that the fatty acid binding site had both strong hydrophobic interaction with the acyl side chain and also charge charge

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interactions with the ionized carboxylate group. Had the site possessed a reactive aminogroup the expectation was that the free fatty acids would bind to it much more strongly than their non-charged derivatives. Here we show that oleic acid does not compete strongly with the imidazolide at neutral pH. LYALL (1984) studied the rate of activation of the bee venom enzyme by oleoyl imidazolide and proposed that it was subject to an apparent pKa of ca 6.5. This, together with our current electrophoresis data indicates that a primary amino-group is not directly involved in the activation reaction and that a novel type of linkage may be present. The work of VAN DER WEILE et al. (1988a,b) showed that the porcine or bovine pancreatic enzyme could be activated by the long-chain fatty acylation of lysine residues 10 or 116. Even though these residues lie near the substrate binding surface, there is no indication that they have elevated reactivity towards longchain acylating agents . These workers propose that the long-acyl residue increases the hydrophobicity of the site and aids penetration of the lipid surface. We have produced arguments based on cross-linking studies and stability measurements (LAWRENCE and MooRES, 1975 ; CAMERo-DIAz et al., 1985) that modification of the bee venom enzyme changes the protein conformation and that the acyl residue is buried in the protein. On this model both free fatty acids and bound acyl residues act as allosteric modifiers. We believed that a buried site would be more likely to possess high reactivity towards acylating agents than would a surface site and it would therefore be very interesting to compare the structure and properties of enzymes which show this reactivity with those that do not. The former group include those from honey bee, bumble bee, European wasp, Russels viper venom and now Naja naja and the non-toxic basic isoform from Naja mossambica mossambica . The latter group includes the porcine pancreatic enzyme and the toxic isoform from Naja mossambica mossambica . Comparison of the two enzymes from Naja mossambica mossambica, will undoubtedly be a fruitful way to study the chemistry and biology of activation . The full primary sequence of these proteins shows minimal differences (WAITE, 1987). (1) = Naja mossambica mossambica, CM-II (non-toxic form); (2) = Naja mossambica mossambica, CM-III (toxic form). 36 52 55 57 63 69 82 91 119 121 E (1) A G E A E L L E E A L K (2) P E K A G M F K N G F

Clearly, from the point of view of an acylation reaction, lysine residues are of great interest, but the responsive enzyme has 31ess such residues than the non-responsive form . The most attractive feature of the sensitive isoform for this study is that it reacts with oleoyl imidazolide very much faster than the bee venom enzyme . This must mean that the shape of the hydrophobic cleft is such that the carbonyl group of the fatty acid derivative is more favorably positioned for reaction in this enzyme than in the less reactive species. DRAINAs and LAWRENCE (1978) showed that activation of the bee venom enzyme by oleoyl imidazolide was complementary to activation by calcium although activation by oleoyl imidazolide was still very significant at saturating calcium concentration . This is consistent with the model that both types of activators have their primary effect at the enzymesubstrate binding step . An alternative model is that activation by acylating agents in some way decreased the ability of inhibitory ions to compete with calcium. Here we have tested the action of both types of modulators in an assay that shows minimal response to acylation, but in which kinetics are relatively simple to interpret. The results show that all of the enzymes have very high affinity for calcium although in all but the toxic isoform from Naja mossambica mossambica, this can be masked by inhibitory metal ions. Never-

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theless, there is no evidence that the chemically modified proteins either bind calcium more strongly or bind inhibitory metal ions less strongly than untreated controls . In the activation assay however, chelation of transition metal ions has little effect whilst the relative activation produced by the modification falls as calcium concentration increases, supporting the earlier observation that these two models of activation were complementary (DRAINAS and LAWRENCE, 1978) . Acknowledgements-The authors thank Professor A. S. G. CURTIS for helpful discussion . This work was supported by the Algerian Ministry of Higher Education.

REFERENCES CAmERo-DIAz, R. E., ELANSARi, O., LAWRENCE, A. J., LYALL, F . and MCLWD, W. A. (1985) Activation of bee venom phospholipase A, by oleoyl imidazolide produces a thiol- and proteinase-resistant conformation . Biochim. biophys. Acta 830, 52-58. CHETTIRi, S. and LAWRENCE, A. J. (1989) High resolution of honey bee (Apis mellii era) venom peptides by propionic acid/urea polyacrylamide gel electrophoresis after ethanol precipitation . Toxicon 27, 781-787. DRAINAS, D. and LAWRENCE, A. J. (1978) Activation of bee venom phospholipase A, by oleoyl irnidazolide. Ew.

J. Biochem. 91, 131-138. DRAINAs, D. and LAWRENCE, A. J. (1981) Albumin mediates lysis of erythrocytes by bee venom phospholipase A, activated with oleoyl imidazolide. FEBS Lett. 114, 93-97. DuFroN, M. J., EAKER, D. and HIDER, R. C. (1983) Conformational properties of phospholipases A, . Secondarystructure prediction, circular dichroism and relative interface hydrophobicity . Eur. J. Biochem. 137, 537-544. LAWRENCE, A. J. (1971) Conductimetric enzyme assays. Eur. J. Biochem. 48, 277-586 . LAwRENCE, A. J. (1975) Lysolecithin inhibits an action of bee venom phospholipase A, in erythrocyte membrane . FEBS Lett. 58,186-189. LAWRENCE, A. J. and Moop .Es, G. R. (1975) Activation of bee venom phospholipase A, by fatty acids aliphatic anhydrides and gluteraldehyde . FEBS Lett . 49, 287-291. Kim, R. M. and EVANS, H. J. (1987) Structure-function relationships of phospholipases. The anticoagulant region of phospholipases A, . J. biol. Chem. 262, 14402-14407. PATEL, K. M. and MoRxiserr, J. T. (1979) A convenient synthesis of phosphatidylcholines : acylation of snglycero-3-phosphocholine with fatty acid anhydride and 4pyrrolidinopyridine. J. Lipid Res. 20, 674678 . RosENBERG, P. (1988) PhospholipaseA, toxins. In : Neurotoxins in Neurochemistry, pp. 27-42 (OLIVER DOLLY, J., Ed.). Ellis Horwood Limited. Tu, A. T. (1977) Venoms: chemistry and molecular biology. New York: John Wiley and Sons . VAN DER WEILE, F . C., ATsmA, W., DuKMAN, R., ScHREuRs, A. M. M., SLoTeoom, A. J. and DE HAAs, G. H. (1988a) Site-specific e-NH, monoacylation of pancreatic phospholipaseA,. 1 . Preparation and properties . Biochemistry 27, 1683-1688 . VAN DER WEux, F. C ., ATSMA, W., RoELoFsEN, B. et al. (1988b) Site-specific e-NH, monoacylation of pancreatic phospholipase A,. 2. Transformation of soluble phospholipase A, into a highly penetrating "membrane bound" form. Biochemistry 27, 1688-1694 . WArrE, M. (1987) Handbook of Lipid Research, Vol. 5, The Phospholipases . New York and London : Plenum Press.