p-Bromophenacyl bromide prevents cumene hydroperoxide-induced mitochondrial permeability transition by inhibiting pyridine nucleotide oxidation

Mitochondrial permeability transition is commonly characterized as a Ca2+ -dependent non-specific increase in inner membrane permeability that results in swelling of mitochondria and their de-energization. In the present study, the effect of different inhibitors of phospholipase A2--p-bromophenacyl bromide, dibucaine, and aristolochic acid--on hydroperoxide-induced permeability transitions in rat liver mitochondria was tested. p-Bromophenacyl bromide completely prevented the hydroperoxide-induced mitochondrial permeability transition while the effects of dibucaine or aristolochic acid were negligible. Organic hydroperoxides added to mitochondria undergo reduction to corresponding alcohols by mitochondrial glutathione peroxidase. This reduction occurs at the expense of GSH which, in turn, can be reduced by glutathione reductase via oxidation of mitochondrial pyridine nucleotides. The latter is considered a prerequisite step for mitochondrial permeability transition. Among all the inhibitors tested, only p-bromophenacyl bromide completely prevented hydroperoxide-induced oxidation of mitochondrial pyridine nucleotides. Interestingly, p-bromophenacyl bromide had no affect on mitochondrial glutathione peroxidase, but reacted with mitochondrial glutathione that prevented pyridine nucleotides from being oxidized. Our data suggest that p-bromophenacyl bromide prevents hydroperoxide-induced deterioration of mitochondria via interaction with glutathione rather than through inhibition of phospholipase A2.     

1H-NMR and protection studies of interactions between ligands and bovine pancreatic phospholipase A2

A number of long-chain amines and naphthylamine sulfonates have been studied for their ability to inhibit bovine pancreatic phospholipase A2 (PLA2) and to protect PLA2 against alkylation of the active site histidine by p-bromophenacyl bromide. Their areas of interaction on the enzyme were further delineated using observations of chemical shift changes of assigned aromatic signals in the 1H-NMR spectrum of PLA2, while the bound conformations of two amine inhibitors were revealed using transferred nuclear Overhauser effects. The alkyl amines bind rather non-specifically on the surface of the enzyme, over the active site cleft and the interface recognition site.     

Tumor promoter 12-O-tetradecanoylphorbol-13-acetate-induced insulin secretion: Inhibition by phospholipase A2- and lipoxygenase-inhibitors

In isolated pancreatic islets of rats, an insulin secretion was induced by a tumor promoter 12-o-tetradecanoylphorbol-13-acetate in a low glucose medium. The insulin secretion was inhibited by p-bromophenacyl bromide, mepacrine, nordihydroguaiaretic acid and 1-phenyl-3-pyrazolidinone but not by indomethacin. When the insulin secretion was suppressed by p-bromophenacyl bromide, the secretion was partially but not significantly restored by lysophosphatidyl choline and was fully restored by the simultaneous addition of arachidonic acid and lysophosphatidyl choline. These results suggest that activation of phospholipase A₂ and a lipoxygenase product(s) play important roles in the 12-o-tetradecanoylphorbol-13-acetate-induced insulin secretion.     

Investigations on the mechanism of action of crotoxin

Crotoxin, the major toxic protein of Crotalus durissus terrificus, is composed of a basic phospholipase, component-B, and of an acidic subunit, component-A. The crotoxin complex is insensitive to an active site directed reagent, p-bromophenacyl bromide, while its isolated enzymatic component-B is rapidly and irreversibly inactivated. We observed that crotoxin possesses an intrinsic phospholipase A2 activity on monodispersed substrates, indicating that the active site of component-B is not masked by component-A in the complex. The inactivation of component-B by p-bromophenacyl bromide follows pseudo-first order kinetics, with a rate constant proportional to the concentration of phospholipase, as expected for a reaction of second order with respect to the protein. On the basis of a detailed kinetic analysis of this reaction, and of physico-chemical studies of component-B in various experimental conditions, we propose that (1) an equilibrium exists between reactive dimers of component-B and preponderant but non-reactive monomer; (2) component-A protects component-B against inactivation by p-bromophenacyl bromide by preventing the formation of reactive dimers. When comparing the reactivity of component-B with that of other phospholipases, we observed that the enzymes which have not been shown to produce dimers all react with p-bromophenacyl bromide with similar low rates of reaction, while phospholipases which have been reported to form dimers react much more rapidly.     

Preferential activation of phospholipase A2 by low concentrations of phosphatidic acid with long-chain fatty acids in rabbit platelets.

The role of phosphatidic acid (PA) in the signal transduction system of platelets was studied using 1-stearoyl 2-arachidonoyl PA (PASA). When PASA was added to rabbit platelets, aggregation occurred. BW755C, a dual inhibitor of cyclooxygenase and lipoxygenase, as well as p-bromophenacyl bromide and mepacrine, inhibitors of phospholipase A2, inhibited the aggregation induced by low concentrations of PASA, but not that induced by high concentrations. PASA also stimulated, in a dose-dependent manner, arachidonic acid liberation, lysophosphatidylcholine and diacylglycerol formation, and mobilization of intracellular Ca2+; all of which were dependent on the presence of Ca2+ in the outer medium. The arachidonic acid liberation was inhibited by p-bromophenacyl bromide or mepacrine, while diacylglycerol formation by low concentrations of PASA was inhibited by BW755C. With platelet membrane fractions or with the platelets made permeable to Ca2+ by pretreatment with ionomycin, PASA caused arachidonic acid liberation in the presence of Ca2+. Furthermore, PASA enhanced the activity of phospholipase A2 partially purified from platelet cytosol acting on 1-palmitoyl-2-[14C]arachidonoyl-glycerophosphoethanolamine. These results provide evidence that PASA preferentially potentiates the activation of phospholipase A2 in cooperation with Ca2+, suggesting that PA acts as a positive feedback regulator to potentiate the activation of phospholipase A2 and contributes to the amplification of platelet activation.     

Phospholipase A of Guinea Pig Spermatozoa: Its Preliminary Characterization and Possible Involvement in the Acrosome Reaction

Phospholipase A activity was demonstrated in guinea pig spermatozoa using [U-¹⁴C] phosphatidyl choline as a substrate. The activity had a neutral pH optimum, was stimulated by Ca²⁺ and low concentrations of detergent, and. was inhibited by EDTA, mepacrine and p-bromophenacyl bromide. Appropriate concentrations of mepacrine and p-bromophenacyl bromide inhibited the acrosome reactions of capacitated spermatozoa without interfering with their motility. These results support the notion that phospholipase A is involved in the acrosome reaction of mammalian spermatozoa.     

Histidine and lysine residues and the activity of phospholipase A2 from the venom of Bitis gabonica.

Chemical modification of phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) from the venom of gaboon adder (Bitis gabonica) showed that histidine and lysine residues are essential for enzyme activity. Treatment with p-bromophenacyl bromide or pyridoxal 5'-phosphate resulted in the specific covalent modification of one histidine or a total of one lysine residue per molecule of enzyme, respectively, with a concomitant loss of enzyme activity. Competitive protection against modification and inactivation was afforded by the presence of Ca2+ and/or micellar concentrations of substrate analogue, lysophosphatidylcholine. Neither modification caused any significant conformational change, as judged from circular dichroic properties. Amino acid analyses and the alignment of peptides from cyanogen bromide and proteolytic cleavage of modified enzyme preparations delineated His-45 as the only residue modified by p-bromophenacyl bromide. However, pyridoxal 5'-phosphate was shown to have reacted not with a single lysine but with four different ones (residues 11, 33, 58 and 111) in such a manner that an overall stoichiometry of one modified lysine residue/molecule enzyme resulted. Apparently, the essential function of lysine could be fulfilled by any one out of these four residues.     

Inhibition studies on the membrane-associated phospholipase A2 in vitro and prostaglandin E2 production in vivo of the macrophage-like P388D1 cell. Effects of manoalide, 7,7-dimethyl-5,8-eicosadienoic acid, and p-bromophenacyl bromide

In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostaglandin production in the intact cell from which it is derived.     

Assessment of a role for phospholipase A2 and arachidonic acid metabolism in human lymphocyte natural cytotoxicity

The reagents quinacrine, hydrocortisone, and dexamethasone have been assumed to affect phospholipase A2 (PA2) when they reduce natural killer (NK) activity. However, these reagents did not reduce lymphocyte incorporation of [14C]arachidonate, which implies that they are not acting as PA2 inhibitors in this lymphocyte system. However, p-bromophenacyl bromide (BPB), which is an active site inhibitor of PA2, irreversibly abrogated NK activity of pretreated lymphocytes, disrupted target cell binding, and reduced [14C]arachidonic acid incorporation by 70-80% as compared to controls. Other observations contrary to expectations for PA2 inhibitors were: (1) quinacrine inhibited NK lysis when lymphocytes were pretreated and (2) the glucocorticoids only inhibited NK activity when continuously present in the assay. Furthermore, NK inhibition by hydrocortisone did not require protein synthesis. The lipoxygenase inhibitors, nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosotetraynoic acid (ETYA), and hydroxyphenylretinamide, and not cycloxygenase inhibitors, reduced NK activity. These data suggest that arachidonate must be metabolized through the 5-lipoxygenase pathway in order to function in NK.     

Involvement of phospholipase A2 activation in anthrax lethal toxin-induced cytotoxicity

The molecular mechanism of cytotoxic effect exerted by the lethal toxin (LeTx) of Bacillus anthracis is not well understood. In the present study, using primary culture of mouse peritoneal macrophages, we have investigated possible cytotoxic mechanisms. LeTx was not found to induce high levels of nitric oxide (NO) production for NO-mediated toxicity. Fragmentation of DNA, a biochemical marker of apoptosis, was not observed in LeTx-treated cells. Pretreatment of cells with antioxidants such as melatonin and dehydroepiandrosterone (DHEA) did not protect the LeTx-induced cytotoxicity. However, addition of phospholipase A2 (PLA2) inhibitors (quinacrine, p-bromophenacyl bromide, manoalide, butacaine) to the culture medium resulted in the inhibition of cytotoxicity of LeTx in a dose-dependent manner. LeTx-induced cytotoxicity was also inhibited by the tyrosine-specific protein kinase inhibitor genistein, but not by the protein kinase C inhibitors staurosporine or H-7. The results of these studies indicate a role for PLA2 and protein kinase in the cytotoxic mechanism of macrophages by anthrax lethal toxin.     

Chemical modification of the histidine residue in phospholipase A2 (Naja naja naja). A case of half-site reactivity.

Reaction of phospholipase A2 (Naja naja naja) with p-bromophenacyl bromidine leads to almost complete loss of enzymatic activity. The rate of inactivation is pH-dependent with pKa equals 6.9 for the ionizing residue. p-Bromophenacyl bromide modifies 0.5 mol of histidine/mol of enzyme as judged by amino acid analysis and incorporation studies with 14C-labeled reagent. The rate of inactivation is affected by various cations; a saturating concentration of Ca2+ decreases the rate 5-fold, while Mn2+ increases the rate by a factor of 2. Triton X-100, which by itself has little affinity for the enzyme, protects against inactivation, presumably by sequestering p-bromophenacyl bromide into the apolar micellar core. The mixed micelle system of Triton X-100, dipalmitoyl phosphatidylcholine, and Ba2+ offers the best protection, lowering the inactivation rate by at least 50-fold. This suggests an active site role for the histidine residue. Ethoxyformic anhydride also modifies phospholipase A2, by acylation of the two amino groups, a tyrosine, and 0.5 mol of histidine/mol of enzyme without totally inactivating the enzyme. Removal of the ethoxyformyl group from the histidine does not reactivate the enzyme. Thus, modification of 0.5 mol of histidine with this reagent is not responsible for the 85% loss of activity seen. Ethoxyformylated enzyme, with 0.5 mol of acylated histidine/mol of enzyme, can be further inactivated by treatment with p-bromophenacyl bromide. The resulting derivative contains 0.4 mol of the 14C-labeled p-bromophenacyl group. Other modifiable groups do not show this half-residue reactivity. For example, oxidation of phospholipase A2 with N-bromosuccinimide leads to rapid destruction of 1.0 tryptophan residue and 5% residual activity. The results of these chemical modification experiments can be interpreted in terms of a model in which the active species of enzyme interacting with mixed micelles is a dimer (or possibly higher order aggregate). The dimer, though composed of identical subunits, is asymmetric; the histidine of one subunit is accessible to ethoxyformic anhydride, while the other histidine is near a hydrophobic region of the enzyme and is chemically reactive toward p-bromophenacyl bromide.     

Interaction of Trimeresurus flavoviridis phospholipase A2 and its fragment with calcium ion

Dimeric T. flavoviridis phospholipase A2 has been studied in terms of the interaction with essential Ca2+ by equilibrium gel filtration, ultraviolet difference spectroscopy, fluorescence measurements, and chemical modifications with p-bromophenacyl bromide. The subunit bound to Ca2+ with a 1:1 molar ratio and no cooperative binding was observed. The hypochromic effect produced upon the binding of Ca2+ is due to perturbation of (a) specific tryptophan residue(s) located in the vicinity of the active site and appears to be characteristic of this enzyme. On the basis of the pH dependence of the dissociation constants, it has been found that the alpha-amino group (pKa 8.7) controls the binding of Ca2+. Deprotonation of the alpha-amino group is possibly accompanied by conformational transition to the active form which is able to bind Ca2+. This is in contrast to the case of bovine pancreatic phospholipase A2 in which Asp-49 (pKa 5.2) is responsible for the metal ion binding (Fleer et al. (1981) Eur. J. Biochem. 113, 283-288). Des-octapeptide(1-8)-phospholipase A2 (L-fragment) was found to be capable of binding Ca2+ under the control of a group with a pKa of 7.6. This pKa value was similar to an apparent pKa of 7.5 determined for the histidine residue in the active site of the native enzyme by way of p-bromophenacyl bromide modification. It appears that the N-terminal (octapeptide) sequence affects the binding mode of Ca2+, possibly because of conformational transition arising from its removal. The reinvestigation showed that the N-terminal octapeptide sequence is Gly-Leu-Trp-Gln-Phe-Glu-Asn-Met.     

Enzymatic N-glycosylation and O-glycosylation of synthetic peptide acceptors by dolichol-linked sugar derivatives in yeast.

Treatment of taipoxin with p-bromophenacyl bromide resulted in modification of single histidine residues in the alpha and beta subunits. The modification decreased the neurotoxicity (lethality) 350-fold, but the inhibitory action on high-affinity choline transport was reduced only threefold. The phospholipase activity and Ca2+-association constants for taipoxin and its subunits were determined. A model for the neurotoxicity of taipoxin indicates the alpha subunit as the ultimate cause of the disruption of synaptic transmission.     

Preliminary X-ray crystallographic studies of a Lys49-phospholipase A2 homologue from Bothrops pirajai venom complexed with p-bromo-phenacyl bromide and alpha-tocopherol inhibitors

PrTX-I, a non-catalytic and myotoxic Lys49-PLA(2) from Bothrops pirajai venom has been crystallized alone and in complex with bromophenacyl bromide (BPB), alpha tocopherol and alpha tocopherol acetate inhibitors. These crystals have shown to diffract X-rays between 2.34 and 1.65 A resolution. All complexes crystals are isomorphous and belong to the space group P2(1) whereas native PrTX-I crystals belong to the P3(1)21.     

Phospholipase A2-mediated superoxide production of murine peritoneal macrophages induced by chrysotile stimulation

In the present study, we investigated how chrysotile-stimulated macrophages generate superoxide using murine peritoneal macrophages, with special attention to the modulatory role of phospholipase A(2) (PLA(2)). We examined the effects of the following inhibitors and antagonists for signaling molecules on the superoxide anion (O(-)(2)) production of chrysotile-stimulated macrophages: p-bromophenacyl bromide (pBPB) and mepacrine for PLA(2); islet-activating protein (IAP) for G-protein; H-7 for protein kinase C (PKC); AA861 for 5-lipoxygenase (5-LO); indomethacin for cyclo-oxygenase (COX); ETYA for both 5-LO and COX; hexanolamine PAF for platelet-activating factor (PAF). The PLA(2) and PKC inhibitors effectively inhibited the chrysotile-induced superoxide anion production of macrophages, but not the G-protein inhibitor, the 5-LO and COX inhibitors, and the PAF antagonist. We also examined the effects of the PLA(2) inhibitors on macrophages stimulated by phorbol 12-myristate 13-acetate (PMA) which directly activates PKC. The two structurally different PLA(2) inhibitors showed differential effects on the PMA-induced superoxide generation: pBPB inhibited it but mepacrine did not. These results suggested that (1) PLA(2) and PKC modulate the chrysotile-induced O(2) production, and (2) two different kinds of PLA(2) work upstream and downstream of PKC, but (3) G-protein, 5-LO and COX metabolites, and PAF have no modulatory role in the reaction.     

Characterization and inhibitor sensitivity of human sperm phospholipase A2: evidence against pivotal involvement of phospholipase A2 in the acrosome reaction

The kinetic properties and inhibitor sensitivity of human sperm phospholipase A2 (PLA2; EC 3.1.1.4) were studied. Phospholipase activity was isolated from human spermatozoa by acid extraction. Hydrolysis of dipalmitoyl phosphatidylcholine was specific to the sn-2 position. Activity was sensitive to product inhibition (60% inhibition by 0.1 mM lysophosphatidylcholine). The effects of Ca2+ and sodium deoxycholate on enzyme activity were biphasic; maximal activities were observed at 0.5 mM concentration of each agent. PLA2 was stimulated (135%) by 3% dimethylsulfoxide and was inhibited by elevated ionic strength (approximately 70% inhibition with either 0.2 M NaCl or 0.2 M KCl). Two molecular forms of PLA2 were kinetically distinguishable, one with an apparent Michaelis constant and maximal reaction velocity of 3.0 microM and 0.64 mlU/mg protein and the other with respective constants of 630 microM and 32.0 mlU/mg protein. Both forms of the enzyme were Ca2+ dependent and heat stable; however, the low-Km activity was less resistant to 60 degrees C preincubation at pH 7.5 (28% inactivation of low-Km activity after 45 min, as compared to no effect on high-Km activity). Quinacrine was a noncompetitive PLA2 inhibitor with Kis for low- and high-Km activities of 0.42 mM and 0.49 mM, respectively. Trifluoperazine (calmodulin antagonist) inhibited the high-Km activity noncompetitively (Ki = 87 microM) and the low-Km activity by a mechanism consistent with the removal of a nonessential activator. Dissociation and rate constants for inactivation of low- and high-Km activities by p-bromophenacyl bromide were 0.28 mM and 0.032 min-1, and 0.73 mM and 0.066 min-1, respectively. PLA2 was inhibited by p-nitrophenyl-p'-guanidinobenzoate, at higher concentrations (10(-4)-10(-3) M) than required to inhibit trypsinlike proteinases; p-aminobenzamidine, another potent trypsin/acrosin inhibitor, stimulated (approximately 40%) PLA2 at concentrations from 2-5 mM but inhibited PLA2 (40-50%) at a concentration of 10 mM. MnCl2 (5mM) inhibited low- and high-Km PLA2 activities by 77% and 76%, respectively. Quinacrine (0.4 mM), trifluoperazine (20 microM), p-bromophenacyl bromide (20 microM), and MnCl2 (5 mM) were tested as inhibitors of the ionophore A23187-induced human acrosome reaction. Inhibition was noted only with quinacrine (32%) and MnCl2 (93%). The effect of MnCl2 was restricted to an interaction with A23187, rather than with PLA2; p-Bromophenacyl bromide inhibited (P less than 0.05) PLA2 (29%) when added to intact spermatozoa but had no effect on the acrosome reaction. PLA2 inhibition was poorly correlated with the acrosome reaction.(ABSTRACT TRUNCATED AT 400 WORDS)     

磷脂酶A2在内毒素致大鼠肺损伤中的作用

大鼠静脉注射大肠杆茵内毒素（３０ｍｇ／ｋｇ）后３ｈ肺血管外水量和支气管肺泡灌洗液中蛋白浓度明显增加,表明发生了通透性肺水肿;同时血清和支气管肺泡灌洗液中磷脂酶Ａ２（ＰＬＡ２）活性升高,且ＰＬＡ,活性的升高与肺血管外水量的增加呈显著正相关。预先给予ＰＬＡ２抑制剂对溴苯酰基溴可抑制内毒素引起的ＰＬＡ２活性升高和通透性肺水肿。提示ＰＬＡ２介导了内毒素引起的肺损伤。     

Antibodies to beta-bungarotoxin and its phospholipase inactive derivative

Antisera were raised against the presynaptic neurotoxin beta-bungarotoxin and against its phospholipase-inactive derivative, modified by reaction with p-bromophenacyl bromide. The cross-reactivity of the antisera to other phospholipase A2 enzymes and polypeptide neurotoxins was examined. The antisera inhibited both the neurotoxic effects of beta-bungarotoxin at the frog motor endplate and the enzymatic activity of the toxin on model phospholipid membranes, although it is unlikely that the catalytic active centre is the locus of any major determinant.     

Does Phospholipase A2 Participate in the Acrosome Reaction of Sea Urchin Sperm?: A Pharmacological Study

We examined whether phospholipase A₂ (PLA₂) is involved in the initiation of the acrosome reaction of sperm of the sea urchin, Strongylocentrotus intermedius , using inhibitors and an activator of this enzyme. Quinacrine and p-bromophenacyl bromide (PBPB) inhibited the egg jelly-induced acrosome reaction at 100 μM, but not the ionomycin-induced one. Depression of egg jelly-induced increase of intracellular free Ca²⁺concentration ([Ca²⁺]_i) by these reagents was expected and examined using fura 2. Quinacrine interfered with the flourescence of fura 2, but PBPB was found to depress at concentrations which could inhibit the acrosome reaction. Furthermore, melittin, which is known to stimulate PLA², caused a [Ca²⁺]_i increase and a formation of acrosomal process-like structure on sperm head. These results suggest that PLA₂ participates in the early step(s) of the acrosome reaction of sea urchin sperm.     

A basic phospholipase A from the venom of the Australian king brown snake (Pseudechis australis) showing diverse activities against membranes

1. A basic phospholipase A (MSPA) was isolated from the venom of the Australian king brown snake, Pseudechis australis. 2. MSPA had an approximate Mr of 13,000 and consisted of a single polypeptide chain of 119 amino acid residues cross-linked by seven disulphide bridges. 3. MSPA exhibited direct haemolytic, anticoagulant and myotoxic activities. 4. Treatment of MSPA with p-bromophenacyl bromide modified a single histidine residue, resulting in complete loss of enzyme activity.