Temperature stability of phospholipase A activity. I. Bee (Apis mellifera) venom phospholipase A2.

Enzymic activity of bee venom phospholipase A₂ has been studied at high temperatures using dispersed egg yolk and sonicated dipalmitoyl and dicaproyl lecithins as substrates. With egg yolk substrate the maximum activity was observed at 65°C and both dipalmitoyl and dicaproyl lecithins gave a maximum at 60°C. At 60°C the enzyme showed loss of activity in the presence of 0·7 M urea. The loss of activity at ca. 60°C could be due to the physical change in the nature of the substrates and/or due to the denaturation of enzyme at this temperature. Preincubation of enzyme at temperatures up to 90°C for 45 min gave the same temperature optimum of 65°C with egg yolk substrate which shows that the heat denaturation, if it occurs, is reversible. O.R.D. studies of pure bee venom phospholipase A₂ showed no change in optical rotation from 10 to 90°C. In spite of the constant optical rotatory dispersion of the protein up to 90°C, sudden loss of enzyme activity was noticed above 60°C with the lecithin substrates.     

Purification and characterization of a phospholipase A2 from Cerastes cerastes (horn viper) snake venom

A single phospholipase A2 has been found in Cerastes cerastes venom, purified to homogeneity by a combination of chromatographic steps involving gel filtration on Sephadex G-50 and ion-exchange chromatography on DEAE-Sephadex A-50. Its mol. wt, its amino acid composition and its partial amino acid sequence have been determined. High homologies between its sequence and those of other Viperid phospholipides A2 have been noticed. The phospholipase was non-lethal to mice up to a dose as high as 25 mg/kg by i.p. and i.v. injection. This non-toxic enzyme exhibited an acidic isoelectric point and hydrolyzed monolayers of different short chain phospholipids. Some kinetic parameters have been studied potentiometric titration (with or without Triton X-100) and the rate of catalysis seemed not to be affected by changes in the physical state of the substrate.     

Phospholipid hydrolysis in serum lipoproteins by a basic phospholipase A2 from Naja nigricollis snake venom and an acidic phospholipase A2 from Naja naja atra snake venom

Apparent Km and Vmax values for PC and PE hydrolysis were determined following exposure of HDL, LDL, and VLDL to a basic phospholipase A2 from N. nigricollis snake venom and an acidic phospholipase A2 from N. nigricollis snake venom and an acidic phospholipase A2 from N. n. atra snake venom. Both enzymes hydrolyzed the lipoprotein phospholipids approximately as fast as they hydrolyzed pure phospholipids in mixed micelles, however, the N. nigricollis enzyme, which has a much stronger anticoagulant effect than the N. n. atra enzyme, had lower apparent Vmax values. These values were highest for phospholipids in VLDL and lowest for HDL, however, the differences between the lipoproteins were relatively small with the N. nigricollis enzyme while the differences were much larger with the N. n. atra enzyme. Fractions of the two enzymes in which varying numbers of lysines were carbamylated showed much larger differences in relative rates of phospholipid hydrolysis in HDL, LDL and VLDL. Triton X-100 eliminates these differences in rates of hydrolysis. These results are discussed in terms of the differences in the organized structure of the lipoprotein classes and in the penetration ability of the phospholipases.     

Quinacrine prevention of intestinal ischaemic mucosal damage is partly mediated through inhibition of intraluminal phospholipase A2

The influence of total ischaemia and revascularization on luminal phospholipid metabolism in the rat small intestine was investigated. Two hours of total ischaemia followed by five minutes of revascularization caused increases in phospholipase A2 activity, and lysophosphalidylcholine content in the gut lumen. The above treatment also resulted in mucosal damage expressed as an increase in N-acetyl-beta-glucosaminidase activity in the lumen. Pretreatment of animals with the phospholipase A2 inhibitor, quinacrine prevented the increases in luminal phospholipase A2 activity and mucosal damage following ischaemia and revascularization. Intraluminal injection of either phospholipase A2 purified from snake venom or Triton X-100 resulted in increased activity of N-acetyl-beta-glucosaminidase in the luminal content. Again, quinacrine pretreatment of animals prevented the increases in mucosal permeability and activity of N-acetyl-beta-glucosaminidase after intraluminal injection of purified phospholipase A2. On the other hand quinacrine pretreatment had no influence on the observed effects of Triton X-100 treatment. These findings suggest that an increase in luminal phospholipase A2 could be involved in mediating the mucosal injury caused by small intestinal ischaemia.     

Effects of beta-bungarotoxin and phospholipase A2 from Naja naja atra snake venom on ATPase activities of synaptic membranes from rat cerebral cortex

Non-neurotoxic phospholipase A2 of Formosan cobra venom possessed higher hydrolytic activity on phosphatidylcholine vesicles and also had higher inhibitory action on Na+-K+-ATpase and Mg2+-ATPase of the rat synaptic membrane than neurotoxic beta-bungarotoxin of Formosan Krait Venom. Na+-K+-ATPase was more susceptible than Mg2+-ATPase to the inhibitory action of toxins, especially in the presence of Triton X-100. The inhibition of ATPases by toxins followed the Michaelis-Menton equation. It is interesting that various phospholipids and ions influenced phospholipase A2 and beta-bungarotoxin inhibition of ATPases. Sphingomyelin antagonized phospholipase A4 more profoundly than beta-bungarotoxin, while egg lecithin had the reverse effect. Both phosphatidylethanolamine and phosphatidylserine protected Na+-K+-ATPase from the inhibitory action of phospholipase A2 but not that of beta-bungarotoxin. High K+ (30 mM) did not affect, while Ca2+ (0.2 mM) decreased, the inhibitory action of phospholipase A2 on Na+-K+-ATPase; in contrast, high K+ reversed, and Ca2+ increased, that of beta-bungarotoxin. These findings imply that phospholipase A2 and beta-bungarotoxin may have different substrate specificities and prefer different conformational states of the membrane for binding. This may explain, at least in part, why beta-bungarotoxin is neurotoxic, while phospholipase A2 is not.     

Lipophilicity of acceptor substrate as a factor in "late foetal" rat liver microsomal UDP-glucuronosyltransferase activity

The UDP-glucuronosyltransferase activity towards phenolic compounds, as measured by initial velocity, has been directly related to lipophilicity of acceptor substrates, as obtained by measurement of octanol-buffer or octanol-water partition. The acceptor substrates examined include 17 compounds, all probably conjugated by the “late foetal” enzyme activity. Rat liver microsomal enzyme activity towards five acceptor substrates of the “late foetal” enzyme was examined under different activation conditions. A statistically significant, partition-dependent increase in activity was observed when the effects of ageing or Triton X-100 treatment were studied. With n-pentane, phospholipase C or UDP-N-acetylglucosamine, although the enzyme activity depended on the partition coefficient of the acceptor substrate, activity towards each substrate was enhanced by a similar amount. Mild trypsin treatment (which did not itself activate the enzyme) or ageing converted the n-pentane dependent general activation into a partition related form by reducing the activity of the enzyme towards the less lipophilic substrates. Removal of phospholipid from the membrane by n-pentane or hydrolysis by phospholipase C resulted in the partition independent activation. Protein release, which by itself did not activate the enzyme, was also required for a partition-dependent effect. As enzyme activity towards the five substrates was induced by 3-methylcholanthrene, but not by phenobarbital, the “late foetal” enzyme was being studied. The induced enzyme activity appeared similar to the non-induced activity.     

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

Purified phospholipase A2 from Naja naja (common Indian cobra) venom, a basic non-toxic 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.     

Catalytic properties of two phospholipases A from cobra (Naja nigricollis) venom.

The catalytic properties of two proteins with phospholipase A activity, isolated from Naja nigricollis venom, have been studied. They are of type A₂ (E.C. 3.1.1.4) toward phosphatidyl choline and phosphatidyl ethanolamine. These enzymes are active in the presence of sodium deoxycholate (DOC) or Triton X-100. Ca²⁺ does not appear to be necessary for enzyme activity. Their optimum pH is 8·50.     

Agelotoxin: a phospholipase A(2) from the venom of the neotropical social wasp cassununga (Agelaia pallipes pallipes) (Hymenoptera-Vespidae).

The neotropical wasp Agelaia pallipes pallipes is aggressive and endemic in southeast of Brazil, where very often it causes stinging accidents in rural areas. By using gel filtration on Sephadex G-100, followed by high performance reversed phase chromatography in a C-18 column under acetonitrile/water gradient, the agelotoxin was purified: a toxin presenting phospholipase A(2) (PLA(2)) activity, which occurs under equilibrium of three different aggregation states: monomer (mol. wt 14 kDa), trimer (mol. wt 42 kDa) and pentamer (mol. wt 74 kDa).The enzyme presents high sugar contents attached to the protein chain (22% [w/w]) and a transition of the values of pH optimum for the substrate hydrolysis from 7.5 to 9.0, under aggregation from monomer to pentamer. All the aggregation states present Michaelian steady-state kinetic behavior and the monomer polymerization caused a decreasing of phospholipasic activity due a non-competitive inhibition promoted by the formation of a quaternary structure. The PLA(2) catalytic activity of agelotoxin changes according to its state of aggregation (from 833 to 12533 micromol mg(-1) min(-1)) and both the monomeric and oligomeric forms present lowest activities than the PLA(2) from Apis mellifera venom and hornetin from Vespa basalis. Agelotoxin is also a very potent direct hemolysin; the monomer of agelotoxin presented hemolytic actions until 200 times higher than the PbTx from P. paulista, 740 times higher than the PLA(2) from A. mellifera, 570 times higher than that of neutral PLA(2) from N. nigricolis and about 1250 times than that of cardiotoxin from Naja naja atra venom.     

Studies on the arachidonic acid cascade—I: Inhibition of phospholipase A2in vitro and in vivo by several novel series of inhibitor compounds

A series of compounds have been discovered which are potent inhibitors in vitro of hog pancreas, cobra venom, and bee venom phospholipase A₂. Collagen-induced aggregation of human platelets was prevented by representatives of this series. The inhibition was reversed by aggregatory concentrations of arachidonate, indicating the hydrolysis of esterified arachidonate had been prevented. In the isolated perfused guinea pig lung sensitized to ovalbumin, the release of prostanoids, resulting from a challenge dose of the antigen, was prevented by exemplars of these compounds. Subsequent administration of arachidonate in the presence of the inhibitor resulted in full prostanoid synthesis and secretion, again indicating that the block was at the phospholipase level. Administration of some of these compounds to guinea pigs by subcutaneous or intraperitoneal routes delayed the onset, and decreased the severity, of erythema induced in depilitated skin by the controlled application of ultraviolet light. This result was, also, consistent with phospholipase A₂ inhibition. Kinetic experiments with hog pancreas phospholipase A₂ demonstrated that, with representatives of this series, the inhibition induced was noncompetitive, and appropriate dissociation constants have been calculated.     

Accessibility of the active site of crotoxin B in the crotoxin complex

G. Canziani, C. Seki and J. C. Vidal. Accessibility of the active site of crotoxin B in the crotoxin complex. Toxicon20, 809–822, 1982.—Basic phospholipases A and the crotoxin complex isolated from Crotalus durissus terrificus venom exhibited similar initial reaction rates, time course and degree of hydrolysis of synthetic short chain lecithins in the monomeric state. Although monomeric lecithins seem to promote dissociation of crotoxin up to a certain extent, this cannot explain the high activity observed with the complex. The crotoxin complex is able to bind the non-hydrolyzable analog d-diheptanoyllecithin, as demonstrated by equilibrium gel-filtration, with a dissociation constant of 0.12mM. This value is similar to the dissociation constant of the crotoxin B-d-diheptanoyllecithin complex (about 0.13mM), estimated from the protection against enzyme inactivation by p-bromophenacyl bromide, which further supports the free accessibility of the substrate to the enzyme active site in the crotoxin complex. The lack of enzyme inactivation when crotoxin is treated with p-bromophenacyl bromide may be interpreted in terms of the specific requirements of the reagent to react with the enzyme rather than protection of the active site. Crotoxin B inhibition by complex formation with crotoxin A, which is not apparent on monomeric substrates, seems not to involve the active site of the enzyme.     

Effects of detergents and organic solvents on rat liver microsomal UDP-glucuronosyltransferase activity towards phenolic substrates

1. The effects of several detergents (Brij 58, deoxycholate and Lubrol 12A9) and ether on the initial rate of UDP-glucuronosyltransferase activity towards fixed concentrations of five phenolic acceptor substrates of widely different octanol-buffer (pH 7.4) partition coefficient have been compared with those observed in non-activated and Triton X-100-and n-pentane-activated rat liver microsomes.

2. Enzymic activity was dependent on the lipid-solubility of acceptor substrate. Each activator, except Triton X-100, enhanced enzymic activity towards all substrates by a similar factor, which was independent of the octanol-buffer partition coefficient. For Triton X-100 microsomes, the activation was also partition-dependent.

3. The highest activation factor was seen with ether. Pre-incubation of ether-activated microsomes for 30?min at 37°C before assay resulted in inactivation of the enzyme towards more water-soluble substrates. Tryptic digestion (30?min at 37°C) of the ether-activated microsomes resulted in marked reduction of enzyme activity towards all substrates.

4. Ether, and the two detergents, Brij 58 and Lubrol 12A9, released small amounts of protein (5-12% total present); both detergents also released some (8-12%) phospholipid.

5. The K^app_m towards acceptor substrate also depended on the octanol-buffer partition coefficient, and was largely unchanged on activation by n-pentane. V_mak was not dependent on partition coefficient and was significantly increased on activation.     

Comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A2 from Hemachatus haemachatus snake venom—I: Enzymatic activity on free and membrane bound substrates

The purified phospholipase A₂ of Naja nigricollis venom is a basic, relatively toxic protein, while the purified phospholipase A₂ of Hemachatus haemachatus is neutral and relatively non-toxic. In order to establish whether the difference in toxicity correlates with hydrolytic ability, we compared the two enzymes using substrates in various physical states such as mixed micelles, native soluble lipoprotein or organized in membranes. The purified phospholipids were used as mixed micelles with Triton X-100. When compared on purified egg l-α-phosphatidylcholine (PC), the two enzymes showed similar pH-and temperature-dependence and were equally affected by activators and inhibitors. N. nigricollis phospholipase A₂ had a V_max of 250 μ-equiv. per min per mg and a K_m of 4.2 mM, while H. haemachatus phospholipase A₂ had a V_max of 1052 μ-equiv. per min per mg and a K_m of 2.2 mM. Both enzymes favored the substrates in the liquid-crystalline state. With a buffered egg yolk dilution as substrate, a V_max of 356 μ-equiv. per min per mg and a K_m of 29 mM were found for N. nigricollis, while H. haemachatus had a V_max of 616 μ-equiv. per min per mg and a K_m of 25 mM. The hydrolysis of purified PC, l-α-phosphatidylethanolamine (PE), l-α-phosphatidylserine (PS) and l-α-phosphatidylinositol (PI) was followed with the substrates taken either singly or in various combinations. Significant differences in preference of the two enzymes were apparent on single substrates, such as the comparatively high hydrolysis of PC by H. haemachatus phospholipase A₂ and of PE by N. nigricollis phospholipase A₂. On mixtures of the four substrates, taken either in equal amounts or in proportions resembling the phospholipid distribution of human red cells, rat brain or electric eel Sachs organ, the sequence of substrate preference exhibited by the two enzymes was again strikingly different. A main feature of the N. nigricollis phospholipase A₂ was its high ability to hydrolyze PS. There was no essential difference between the actions of the two enzymes on fresh human red cells. However, erythrocytes from stored, outdated blood were hemolyzed and phospholipids were fully hydrolyzed by N. nigricollis phospholipase A₂, while the H. haemachatus enzyme was nonhemolytic and induced only limited hydrolysis. The same disparity in behavior could be demonstrated on fresh guinea pig erythrocytes. A comparison of hydrolysis, in permeable red cell ghosts and in Triton-solubilized membranes, by the phospholipases, revealed that the high preference of N. nigricollis enzyme for PS was masked by sequestration of this phospholipid within the ghosts.     

Two-step inactivation of bee venom phospholipase A2 by scalaradial

Scalaradial (SLD), a marine natural product isolated from the sponge (Cacospongia sp., possesses anti-inflammatory properties in vivo and in vitro (Pharmacologist 32: 168, 1990). In this study we characterize its effects against bee venom phospholipase A2 (PLA2; EC 3.1.1.4). SLD is a potent inactivator of bee venom PLA2 with an IC50 value of 0.07 microM. Inactivation of bee venom PLA2 occurred in a time-dependent, irreversible manner. The rate of inactivation followed first-order reaction kinetics and was dependent on the concentration of SLD. Kinetic analysis suggested a two-step mechanism of inactivation: an initial apparent noncovalent binding (Ki = 4.5 x 10(-5) M) followed by covalent modification. The rate of inactivation was reduced markedly in the presence of excess phosphatidylcholine, suggesting that modification of the enzyme occurs at or near the substrate binding site.     

Effects of phospholipase A2 from cobra and bee venom on the presynaptic membrane

L. G. Magazanik, I. M. Gotgilf, T. I. Slavnova, A. I. Miroshnikov and U. R. Apsalon. Effects of phospholipase A₂ from cobra and bee venom on the presynaptic membrane. Toxicon17, 477–488, 1979.—Phospholipases A₂ from bee venom and cobra venom have been isolated and studied. A parallelism was found between enzymatic activity and the ability to block spontaneous miniature end-plate potentials (m.e.p.p.'s) or end-plate potentials (e.p.p.'s) induced by nerve stimulation in the frog sartorius muscle. Different experimental procedures affected both enzymatic activity and blocking ability in qualitatively the same way. Thus, modification of the histidine residue in cobra venom phospholipase by bromophenacyl bromide or the removal of Ca-ions from the medium abolished both activities. Replacement of Ca²⁺ by Sr²⁺ inhibited both the enzymatic and presynaptic effects of cobra venom phospholipase, but did not inhibit the presynaptic action of bee venom phospholipase and decreased its enzymatic activity only 6-fold. Irreversible binding of cobra and bee venom phospholipase to the presynaptic membrane was found in Ca-free solution but Ca-ions were essential for the presynaptic blocking effect induced by these phospholipases. A reduction in the effect of high K⁺ on m.e.p.p. frequency was observed after cobra venom phospholipase treatment. The similar effects of hypertonic sucrose solution and the mitochondrial poison TTFB (4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole) were changed only slightly by bee and cobra venom phospholipase pretreatment. It is concluded that the mechanism of presynaptic blockade induced by bee venom and cobra venom phospholipase consists mainly of damage to sites of release at the presynaptic membrane. There are also some signs of disturbances of depolarization-secretion coupling and of the process of formation of new quanta. The possible functional role of enzymatic activity in the presynaptic effect is discussed.     

Platelet aggregation inhibitors from Agkistrodon acutus snake venom

Among all the purified components from A. acutus venom, including ADPase, 5'-nucleotidase, phospholipase A2 and fibrinogenases, only the venom ADPase (50-100 micrograms/ml) shows marked inhibitory action on ADP (10 microM)-, collagen (10 micrograms/ml)- and sodium arachidonate (100 microM)-induced platelet aggregations of rabbit platelet-rich plasma. The venom 5'-nucleotidase (100 micrograms/ml) inhibited ADP-induced platelet aggregation by 31 +/- 4% (n = 4, P less than 0.05). Fibrinogenolytic enzymes (fractions I and IX, 100 micrograms/ml) did not significantly inhibit platelet aggregation induced by ADP (10 microM), collagen (10 micrograms/ml) or sodium arachidonate (100 microM). However, when the fibrinogenase (fraction IX, 100 micrograms/ml) was preincubated with platelet-rich plasma for 30 min it inhibited collagen (20 micrograms/ml)- and ADP (10 microM)-induced platelet aggregations by 34 +/- 9% (n = 4, P less than 0.05) and 35 +/- 6% (n = 4, P less than 0.05), respectively. The phospholipase A2 (100 micrograms/ml) did not affect platelet aggregation. The venom ADPase is a single chain polypeptide with a molecular weight of 94,000. The specific ADPase activity is estimated to be 4.3 mu moles Pi/min/mg of protein. It also possesses phosphodiesterase and weak 5'-nucleotidase activities.     

The glycoprotein nature of phospholipase A2, hyaluronidase and acid phosphatase from honey-bee venom

Experiments with immobilized concanavalin A strongly suggest a glycoprotein nature of three honey-bee venom enzymes, phospholipase A₂, hyaluronidase and acid phosphatase. The electrophoretically and chromatographically detectable heterogeneity of phospholipase A₂ results from absence of carbohydrate in a subfraction. Mannose, fucose and N-acetylglucosamine, but not galactose nor f-tylgalactosamineare present in the con A-binding fraction of bee venom. It is therefore concluded that only N-glycosidically linked carbohydrate occurs in bee venom glycoproteins.     

Phospholipase A2 in venom extracts from honey bees (Apis mellifera L.) of different ages

We measured phospholipase A2 activity in the venom of worker honey bees (Apis mellifera L.) of known ages using chemical (titrimetric) and radioallergosorbent methods. The two techniques give similar results. Low levels of phospholipase A2 are present in the venom system at the time of eclosion. Phospholipase A2 activity in the venom increases steadily through the 10 days after eclosion. Maximal phospholipase A2 levels (about 40 micrograms phospholipase A2/venom sac) are maintained through the rest of the life of a worker bee in summer.     

蜂毒中磷酯酶A_2和透明质酸酶的活性分析

运用酶学方法分析了蜂毒中两个主要大分子成分磷酯酶A_2和透明质酸酶的活性,并与部分纯化后的峰毒中上述两个酶蛋白的活性作了比较。结果发现,部分纯化后的蜂毒几乎不含透明质酸酶、磷酯酶A_2,活性由原来的180u/mg下降至16u/mg。     

Effects of the mycotoxin sporidesmin on swelling and respiration of liver mitochondria

In the presence of both oxidizable substrate and phosphate, low concentrations of sporidesmin produced rapid swelling of mitochondria isolated from guinea-pig liver. There is no requirement for alkali metal ions. Uncoupling agents or respiratory inhibitors inhibited the swelling. Mitochondria that had been swollen in the presence of sporidesmin could be contracted by addition of ATP, magnesium ions and bovine serum albumin. The rate of swelling was dependent on the concentration of both sporidesmin and mitochondrial protein and half-maximum effect was observed at 140 nmoles of sporidesmin per mg of protein. A lag period occurred before the maximal effect of sporidesmin and this period was also dependent on the concentration of both sporidesmin and mitochondrial protein. Sporidesmin decreased the respiratory control index of mitochondria by increasing the rate of state 4 respiration and decreasing that of state 3. As sporidesmin had no effect on the respiration of mitochondrial preparations that had either been treated with Triton X-100 or been subject to sonication, the toxin does not directly inhibit the respiratory chain. It is suggested that sporidesmin produces the effects described above by altering the permeability of the mitochondrial membrane.