Lipid-induced aggregation of phospholipase A2: sucrose density gradient ultracentrifugation and crosslinking studies

The aggregation behavior of cobra venom (Naja naja naja) phospholipase A2 in the presence of lipids and Ca2+ was examined using ultracentrifugation and crosslinking techniques. Velocity sedimentation experiments were performed in sucrose gradients. The sedimentation coefficients of the cobra phospholipase A2 and various controls, including bovine serum albumin (BSA), malate dehydrogenase, carbonic anhydrase and pancreatic phospholipase A2, were calculated both in the presence and absence of ligands. The monomeric phospholipid, diheptanoylphosphatidylcholine, and the phospholipid analogue, dodecylphosphocholine (DPC), increased the sedimentation coefficient of the cobra phospholipase A2 from 2.2 S to 2.9 S, a value that is consistent with the formation of an enzyme dimer. The control proteins were unaffected by the presence of phospholipid, except for BSA, which apparently binds large amounts of DPC. Crosslinking experiments with glutaraldehyde showed that in the presence of diheptanoylphosphatidylcholine or DPC, the amount of crosslinked enzyme increased. Ca2+ had no effect on the aggregation state of the enzyme as measured by either technique. Both the ultracentrifugation data and crosslinking data are consistent with the hypothesis that the cobra venom phospholipase A2 exists as a dimer or higher-order aggregate in the presence of lipid substrate, although it is yet to be determined whether the functional subunit is a monomer, dimer or higher-order oligomer.     

Activation, aggregation, and product inhibition of cobra venom phospholipase A2 and comparison with other phospholipases

The kinetics of phospholipid hydrolysis by cobra venom phospholipase A2 were examined and compared to those of phospholipase A2 from porcine pancreas, Crotalus adamanteus (rattlesnake) venom, and bee venom. Only the enzyme from Naja naja naja (cobra) venom was found to be activated significantly by phosphorylcholine-containing compounds when hydrolyzing phosphatidylethanolamine. The cobra venom enzyme was also the only one in which these activators induced protein aggregation. The parallel specificity for activators and aggregators suggests that these two phenomena are linked. Product effects were also shown to vary between these four phospholipases. These effects manifest themselves in nonlinear time courses, in changes in steady state velocity, and in the differential effects of serum albumin on reaction rates. Different effects were even seen for the same enzyme when acting on different substrates. A model is presented to account for these observations; its main features are enzyme activation by an activator molecule, whose specificity depends on the enzyme, and an activator-induced aggregation of the enzyme.     

1-Stearyl,2-stearoylaminodeoxy phosphatidylcholine, a potent reversible inhibitor of phospholipase A2

1-stearyl, 2-stearoylaminodeoxy phosphatidylcholine, a structurally modified phospholipid substrate analog exhibits potent and reversible inhibition of phospholipase A2 from cobra venom (N. naja naja). The apparent KI values determined in two different assay systems employing phosphatidylcholine-surfactant mixed micelles are in reasonable agreement (40 microM and 16 microM) and indicate that the inhibitor binds to the enzyme as much as two orders of magnitude more tightly than does dipalmitoyl phosphatidylcholine. With phosphatidylethanolamine as substrate, the kinetics are more complicated as the analog also exhibits activation, presumably at a second binding site on the enzyme.     

Purification of a Class B1 platelet aggregation inhibitor phospholipase A2 from Indian cobra (Naja Naja) venom

A platelet aggregation inhibitor phospholipase A(2) (NND-IV-PLA(2)) was isolated from Naja naja (Eastern India) venom by a combination of cation and anion exchange chromatography. NND-IV-PLA(2) is the most catalytically active enzyme isolated from the Indian cobra venom. The acidic PLA(2) profile of Eastern regional Indian cobra venom is distinctly different from that of the western regional venom. However the acidic PLA(2)s from both the regions follow the pattern of increasing catalytic activity with increase in acidic nature of the PLA(2) isoform. NND-IV-PLA(2) is a Class B1 platelet aggregation inhibitor and inhibits platelet aggregation induced by ADP, collagen and epinephrine. Modification of active site histidine abolishes both catalytic activity and platelet aggregation inhibition activities while aristolochic acid, a phospholipase A(2) inhibitor has only partial effect on the two activities.     

Cobra venom phospholipase A2 inhibition by manoalide. A novel type of phospholipase inhibitor

Manoalide, an unusual nonsteroidal sesterterpenoid recently isolated from sponge, antagonizes phorbol-induced inflammation but not that induced by arachidonic acid, suggesting that manoalide acts prior to the cyclooxygenase step in prostaglandin synthesis, possibly by inhibiting phospholipase A2. We have now studied the inhibitory effect of manoalide on a homogeneous preparation of phospholipase A2 from cobra venom. For a given concentration of manoalide, the inhibition of phospholipase A2 activity toward dipalmitoylphosphatidylcholine/Triton X-100 mixed micelles is time-dependent and plateaus at about 85% inhibition of the initial velocity even after extensive preincubation. Metal ions (Ca2+, Ba2+, Mn2+) increase the inhibition, while lysophosphatidylcholine and substrate micelles protect. Increasing manoalide concentration shows increasing inhibition of the initial velocity until a plateau is reached, giving a typical saturation curve with a linear double-reciprocal plot. Under typical conditions (20-min preincubation, 40 degrees C, pH 7.1), 50% inhibition is achieved at a manoalide concentration of about 2 X 10(-6) M. The data indicate that manoalide is a potent inhibitor of the cobra venom phospholipase A2. Manoalide is now shown to react irreversibly with lysine residues in the enzyme. Surprisingly, the cobra venom phospholipase normally acts poorly on phosphatidylethanolamine as substrate, but after reaction with manoalide, the enzyme is somewhat more active toward this substrate rather than being inhibited. This suggests that a lysine residue may be important in understanding the substrate specificity of phospholipase A2.     

A simple method to obtain a covalent immobilized phospholipase A(2).

In the present work, we obtained an immobilized phospholipase A(2) system through covalent coupling by using an acrylic polymer Eupergit C as support. The immobilized enzyme from cobra venom (Naja naja naja) showed good retention activity and excellent stability. Both properties are of great importance for biomedical applications such as hypercholesterolemia treatments.     

Cobra venom phospholipase A2: A review of its action toward lipid/water interfaces

Summary This review focuses on the mechanism of action of phospholipase A₂ from cobra venom (Naja naja naja) toward the lipid/water interface. Particular points of interest include dramatic changes in the enzyme activity if the physical state of its substrate is altered and the activation of the enzyme by phosphorylcholine containing lipids. The experimental findings include the following: Micellar substrates are hydrolyzed faster by the enzyme than various bilayer forms of substrate aggregation. The activity of the enzyme toward short chain phospholipids increases suddenly above their critical micelle concentrations. An abrupt change in susceptibility to the enzyme is observed at the thermotropic phase transition of phospholipid vesicles. The enzyme shows the kinetic phenomena of surface dilution and activation by certain lipids, which suggest a two-step mechanism of action. A model is discussed which accommodates the present data both for the action of this enzyme at various lipid/water interfaces as well as its interaction with synthetic monomeric ligands and substrates.     

Cobra venom acetylcholinesterase. Purification and molecular properties.

Acetylcholinesterase from cobra (Naja naja oxiana) venom has been purified by affinity chromatography to an homogeneous state, as ascertained by sodium dodecylsulfate/polyacrylamide gel electrophoresis and sedimentation analysis. The specific activity of the preparation was 5000 IU/mg with acetylcholine as substrate. Unlike acetylcholinesterases from insoluble cell structures, the native molecule of the cobra venom enzyme consists of a single polypeptide chain of molecular weight 67,000 +/- 2000. At high enzyme concentrations (greater than 0.2 mg/ml, greater than 1 microM) and ionic strength 0.1 M, it reversibly tends to form higher-molecular-weight 7.1-S aggregates. Despite the apparent structural simplicity of the venom acetylcholinesterase, the disc electrophoresis and isoelectric focusing experiments revealed that the enzyme exists in a number of forms with a common molecular weight but with different isoelectric points. Neuraminidase treatment did not reduce the number of the forms.     

Isolation and characterization of an acidic lethal phospholipase Az from Malayan cobra (Naja naja sputatrix) venom

An acidic, lethal phospholipase Az was purified to electrophoretic homogeneity from the venom of the Malayan cobra (Naja naja sputatrix). The enzyme has an isoelectric point of 5.58, a molecular weight of 12000, and a medium lethal dose (LD50) of 0.86 micrograms/g in mice by intravenous injection. The enzyme also exhibited weak anticoagulant and edema-forming activities. The amino acid composition of the enzyme is similar to those of other cobra venom phospholipases Az.     

The interaction of phospholipase A2 with phospholipid analogues and inhibitors

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)     

The anticoagulant activity of Malayan cobra (Naja naja sputatrix) venom and venom phospholipase A2 enzymes

Malayan cobra (Naja naja sputatrix) venom was found to exhibit an in vitro anticoagulant activity that was much stronger than most common cobra (genus Naja) venoms. The most potent anticoagulants of the venom are two lethal phospholipase A2 enzymes with pI's of 6.15 and 6.20, respectively. The anticoagulant activity of the venom is due to the synergistic effect of the venom phospholipase A2 enzymes and polypeptide anticoagulants. Bromophenacylation of the two phospholipase A2 enzymes reduced their enzymatic activity with a concomitant drop in both the lethal and anticoagulant activities.     

Characterization and physical properties of the major form of phospholipase A2 from cobra venom (Naja naja naja) that has a molecular weight of 11,000.

The major form of phospholipase A2 from cobra venom (Naja naja naja) was prepared in 30% yield and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate and on Sephadex G-100 chromatography. The monomer molecular weight is about 11,000 according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ultracentrifugation and molecular sieve techniques were employed to confirm the molecular weight and to demonstrate a concentration-dependent aggregation of the enzyme. It was found that at concentrations below about 0.05 mg ml(-1), the enzyme exists predominantly in the monomeric form; kinetic studies are usually conducted in much more dilute solutions (0.2 mug ml(-1)). The amino acid composition of the enzyme is reported. Of special interest is the presence of five to six disulfide bonds, 1 tryptophan residue, and 1 histidine residue. It is stable at high temperatures and is unusually resistant to denaturing agents. The isoelectric point was found to be 4.95. The findings that the protein is unusually resistant to denaturing agents and that it undergoes a concentration-dependent aggregation help to explain some of the previous reports in the literature on the apparent multiple forms of the cobra enzyme and their separation.     

Kinetic characterization of phospholipase A2 modified by manoalogue

Manoalogue, a synthetic analogue of the sea sponge-derived manoalide, has been previously shown to partially inactivate the phospholipase A2 from cobra venom (Reynolds, L. J., Morgan, B. P., Hite, E. D., Mihelich, E. D., & Dennis, E. A. (1988) J. Am. Chem. Soc. 110, 5172) by reacting with enzyme lysine residues. In the present study, the inactivation of the phospholipases A2 from pig pancreas, bee venom, and cobra (Naja naja naja) venom by manoalogue was studied in detail. Manoalogue-treated enzymes were examined in the scooting mode on vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol. Here the native enzymes bound irreversibly to the vesicles and hydrolyzed all of the phospholipids in the outer monolayer without leaving the surface of the interface. All three manoalogue-treated enzymes showed reduced catalytic turnover for substrate hydrolysis in the scooting mode, and the modified enzymes did not hop from one vesicle to another. Thus, inactivation by manoalogue is not due to the decrease in the fraction of enzyme bound to the substrate interface. This result was also confirmed by fluorescence studies that directly monitored the binding of phospholipase A2 to vesicles. A chemically modified form of the pig pancreatic phospholipase A2 in which all of the lysine epsilon-amino groups have been amidinated was not inactivated by manoalogue, indicating that the modification of lysine residues and not the amino-terminus is required for the inactivation. Several studies indicated that the manoalogue-modified enzymes contain a functional active site. For example, studies that monitored the protection by ligands of the active site from attack by a alkylating agent showed that manoalogue-modified pig phospholipase A2 was capable of binding calcium, a substrate analogue, lipolysis products, and a competitive inhibitor. Furthermore, relative to native enzymes, manoalogue-modified enzymes retained significantly higher catalytic activities when acting on water-soluble substrates than when acting on vesicles in the scooting mode. Intact manoalogue had no affinity for the catalytic site on the enzyme as it did not inhibit the enzyme in the scooting mode and it did not protect the active site from alkylation. Pig pancreatic phospholipase A2 bound to micelles of 2-hexadecyl-sn-glycero-3-phosphocholine was resistant to inactivation by manoalogue, suggesting that the modification of lysine residues on the interfacial recognition surface of the enzyme was required for inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis of a naphthylvinyl-labeled glycerol ether analog of phosphatidylcholine and its use in the assay of phospholipase A2

The synthesis of a naphthylvinyl-labeled glycerol ether analog of phosphatidylcholine, 1-O-[12-(2-naphthyl)-dodec-11-enyl]-2-O-decanoyl-sn-glycerol-3- phosphocholine (NVPC), is described. This involves a Wittig reaction between 2-naphthaldehyde and a phosphonium salt which gives the trans-naphthylvinyl group as the predominant isomer. Lyso NVPC was prepared from NVPC by phospholipase A2 action. NVPC absorbs strongly at 248 nm (epsilon = 58,300 M-1 cm-1) and gives broad fluorescence emission with maxima at 343 nm and 360 nm and a quantum yield of 0.10 in ethanol. An assay for phospholipase A2 was developed using high performance liquid chromatography with fluorescence detection to separate and quantify NVPC and lyso NVPC. Activities as low as 1-2 pmol/min in an assay volume of 0.1 ml can easily be measured. The assay was used with a pure enzyme from cobra venom and a crude enzyme from synovial fluid. Enzyme specificities for phosphatidylcholine and NVPC with cobra venom and porcine pancreatic phospholipases A2 were compared using a titrametric assay. The use of the assay with NVPC to study the metabolism of platelet activating factor is discussed.     

Exploring the action and specificity of cobra venom phospholipase A2 toward human erythrocytes, ghost membranes, and lipid mixtures

We have investigated the action and substrate specificity of phospholipase A2 (EC 3.1.1.4) purified from cobra venom (Naja naja naja) toward intact and Triton-solubilized human erythrocytes, toward ghost membranes, and toward extracted ghost lipids in mixed micelles with Triton X-100. We have found that: (i) phospholipids in the outer surface of intact erythrocytes are extremely poor substrates for the phospholipase, (ii) phospholipids in ghost erythrocyte membranes and in Triton-solubilized erythrocytes are suitable substrates for the enzyme, (iii) in these latter systems which contain a mixture of lipids, phosphatidylethanolamine is preferentially hydrolyzed, whereas in model studies on individual phospholipid species in mixed micelles with Triton, phosphatidylcholine is the preferred substrate of the enzyme, and (iv) the preferential hydrolysis of phosphatidylethanolamine is also observed for extracted ghost lipid mixtures in mixed micelles. These results demonstrate a dependence of phospholipase A2 activity on the ghosting procedure and a dependence of substrate specificity on the presence of other lipids. The relevance of these findings to the interpretation of membrane lipid asymmetry studies utilizing phospholipases is considered in detail.     

A comparative study of cobra (Naja) venom enzymes

1. The L-amino acid oxidase, hyaluronidase, alkaline phosphomonoesterase, protease, phosphodiesterase, acetylcholinesterase, phospholipase A and 5'-nucleotidase activities of 47 samples of venoms from all the six species of cobra (Naja), including five subspecies of Naja naja, were examined. 2. The results demonstrated interspecific differences in the venom contents of phospholipase A, acetylcholinesterase, hyaluronidase and phosphodiesterase. These differences in venom enzyme contents can be used for the differentiation of species of the genus Naja. 3. Thus, our results revealed a correlation between the enzyme composition of venom and the taxonomic status of the snake at the species level for the genus Naja.     

Effect of phospholipases A2 from bee and cobra venom on the phospholipid composition and Na+-,K+-ATPase activity of synaptosomes

The bee and cobra venom phospholipases A2 as well as partially acetylated cobra venom phospholipase A2 are studied for their effect on phospholipid composition of synaptosomes and their Mg2+- and Na+,K+-ATPase activity. It is established that these phospholipases induce the splitting of phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine, inhibition of the Na+,K+-ATPase activity and activation of Mg2+-ATPase. Bee venom phospholipase A2 is more effective than cobra venom phospholipase A2, the both phospholipases splitting phosphatidylethanolamine most intensively. The ATPase activity may be partially or completely restored by exogenic phosphatidylcholine and phosphatidylserine; exogenic phosphatidylethanolamine is not efficient in this respect.     

Effect of ethanol on structural and functional characteristics of rat brain cortical membranes in vitro

When finding the mechanism of the in vitro ethanol membrane action it was established that alcohol in moderate concentrations enhanced the inactivation of the rat brain cortex Na+, K(+)-ATPase after modification of the microsomal fraction by phospholipase A2 from cobra venom. It is supposed that ethanol sensitivity is determined by the structural state of the functional protein-lipid enzyme complexes in membrane. The decrease of the intrinsic tryptophan fluorescence of the membrane proteins and fluorescence of the probe 1-anilinonaphthalene-8-sulfonate (both without and under membrane surface charge shielding) in the presence of ethanol was revealed. It is concluded, that conformational changes of the membrane polar surface area coincide with the enzyme inactivation by ethanol.     

Kinetic analysis of phospholipase A2 activity toward mixed micelles and its implications for the study of lipolytic enzymes.

A detailed kinetic scheme is proposed for the action of phospholipase A2 on mixed micelles of phospholipid and surfactant: see article. where E is the enzyme, A is the mixed micelle, and B is the phospholipid substrate in the mixed micelle. This scheme takes into account quantitatively the involvement of the lipid-water interface in the action of this enzyme toward substrate in macromolecular lipid complexes. The kinetic equation for this scheme is derived and four simplifying assumptions which are necessary for its practical application are described. Kinetic data are reported for the action of cobra venom phospholipase A2 (Naja naja naja) on 1,2-dipalmitosyl-sn-glycero-3-phosphorylcholine in mixed micelles with the nonionic surfactant Triton X-100, and these data are analyzed in terms of the kinetic equation presented. At 40 degrees, pH 8.0, and in the presence of 10 mM Ca2+, V was found to be about 4 X 10(3) mumol min(-1) mg of protein(-1). KsA, which is the dissociation constant for the enzyme-mixed micelle complex, is about 5 X 10(-4) M. KmB, the Michaelis constant for the catalytic step, which is (k-2 + k3)/k2, is 1 to 2 X 10(-10) mol cm-2. This kinetic treatment, together with the fact that the mixed micelle system allows the concentration of the substrate in the lipid-water interface to be varied, has made possible the quantitative separation of the association of a lipolytic enzyme with the lipid-water interface (expressed as KsA) and the binding to the substrate in the interface (reflected in the KmB term). The implications of this kinetic scheme for the analysis of phospholipase A2 from other sources acting on other aggregated forms of phospholipid and for the study of other phospholipases and lipases is considered.     

Isolation and characterization of a lethal phospholipase A2 (NN-IVb1-PLA2) from the Indian cobra (Naja naja naja) venom.

Indian cobra (Naja naja naja) venom is reported to contain multiple forms of phospholipase A2. Only a couple of them have been isolated and characterized. A lethal phospholipase A2 (NN-IVb1-PLA2) from Naja naja naja venom has been purified in three steps involving CM-Sephadex C-25, Sephadex G-50 and rechromatography on CM-Sephadex C-25 columns. It is a basic protein with pl value between 7-7.5 and has molecular weight between 11,000-11,500. The LD50 of NN-IVb1-PLA2 is 1.2 mg/K g body weight. It induces neurotoxic symptoms in the experimental mice and is devoid of myotoxic, anticoagulant, edema inducing and direct hemolytic activities.