The amino acid sequence and properties of an edema-inducing Lys-49 phospholipase A2 homolog from the venom of Trimeresurus mucrosquamatus

Three phospholipase A2 enzymes or homologs were purified from the venom of Trimeresurus mucrosquamatus (Taiwan habu). The most abundant one was found to be a phospholipase homolog without enzyme activity, and its complete amino acid sequence was determined using oligopeptide fragments derived from digestion by endopeptidases Glu-C, Asp-N, Lys-C and alpha-chymotrypsin, and by means of gas-phase sequencing. The sequence revealed that the protein belonged to the Lys-49 family of snake venom phospholipase A2. This protein's function was characterized as edema-inducing. The Lys-49 protein has the potential to bind membrane phospholipid and Ca2+ (Kd = 1.6 x 10(-4) M) as shown by ultraviolet difference spectra; however, the catalytic site appeared to be inactive and the edematous response was independent of the protein's hydrolytic activity. Mast cells and platelets were shown to be subject to activation by the Lys-49 protein.     

Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom

BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom, was structurally and functionally characterized. Several biological activities were assayed and compared with those of the chemically modified toxin involving specific amino acid residues. The cDNA produced from the total RNA by RT-PCR contained approximately 400 bp which codified its 121 amino acid residues with a calculated pI and molecular weight of 8.9 and 13,727, respectively. Its amino acid sequence showed strong similarities with several Lys49 phospholipase A(2) homologues from other Bothrops sp. venoms. By affinity chromatography and gel diffusion, it was demonstrated that heparin formed a complex with BnSP-7, held at least in part by electrostatic interactions. BnSP-7 displayed bactericidal activity and promoted the blockage of the neuromuscular contraction of the chick biventer cervicis muscle. In addition to its in vivo myotoxic and edema-inducing activity, it disrupted artificial membranes. Both BnSP-7 and the crude venom released creatine kinase from the mouse gastrocnemius muscle and induced the development of a dose-dependent edema. His, Tyr, and Lys residues of the toxin were chemically modified by 4-bromophenacyl bromide (BPB), 2-nitrobenzenesulfonyl fluoride (NBSF), and acetic anhydride (AA), respectively. Cleavage of its N-terminal octapeptide was achieved with cyanogen bromide (CNBr). The bactericidal action of BnSP-7 on Escherichia coli was almost completely abolished by acetylation or cleavage of the N-terminal octapeptide. The neuromuscular effect induced by BnSP-7 was completely inhibited by heparin, BPB, acetylation, and CNBr treatment. The creatine kinase releasing and edema-inducing effects were partially inhibited by heparin or modification by BPB and almost completely abolished by acetylation or cleavage of the N-terminal octapeptide. The rupture of liposomes by BnSP-7 and crude venom was dose and temperature dependent. Incubation of BnSP-7 with EDTA did not change this effect, suggesting a Ca(2+)-independent membrane lytic activity. BnSP-7 cross-reacted with antibodies raised against B. moojeni (MjTX-II), B. jararacussu (BthTX-I), and B. asper (Basp-II) myotoxins as well as against the C-terminal peptide (residues 115-129) from Basp-II.     

Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom

Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.     

Purification and some properties of membrane-associated phospholipase A2 of human spleen

Membrane-associated phospholipase A2 was purified to homogeneity from human spleen. The enzyme was solubilized from the particulate fraction by the addition of KBr, and purified by reverse-phase high-performance liquid chromatography. The estimated molecular weight of the enzyme was 14,000. The enzyme had a pH optimum around 9.5, required the presence of Ca2+ for its activity, and hydrolyzed phosphatidylethanolamine more efficiently than phosphatidylcholine.     

Ligand binding inhibitors of A1 adenosine receptor from Rana rugosa are phospholipase A2s.

Inhibitors of the A1 adenosine receptor were isolated from the skin extract of Korean frog, Rana rugosa. The frog-skin extract was prepared by an electrical shock and fractionated with C4 followed by C18 reverse-phase HPLC. Two A1 receptor inhibitors were isolated using a filter binding assay and the molecular masses of the proteins were estimated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to be 15 347 and 15 404 Da, respectively. The inhibitory activity was also measured against other membrane receptors, such as the A2 adenosine receptor, muscarinic acetylcholine receptor and capsaicin receptor. Ligand binding to the A2 and muscarinic receptors was also severely inhibited by these proteins. However, they did not inhibit the functional activation of the capsaicin receptor by its ligand, capsaicin, suggesting that inhibition of ligand-receptor binding occurs specifically. Their N-terminal sequences were determined by Edman degradation. Surprisingly, they showed sequence similarity to the secretory protein, phospholipase A2 from various organisms. The phospholipase A2 activity of both proteins was tested using Dole's assay technique. Both proteins showed phospholipase A2 activity, and therefore, they were designated as PLA2-R1 and PLA2-R2, respectively. In addition, their ligand-binding inhibitory activity depended on their phospholipase A2 activity. This is the first finding that the frog secretes a phospholipase A2 similar to that of snake venoms, which posess inhibitory activity against the adenosine A1, adenosine A2 and muscarinic receptors.     

Cloning and expression of an acidic platelet aggregation inhibitor phospholipase A2 cDNA from Bothrops jararacussu venom gland

The phospholipase A2 (PLA2, E.C. 3.1.1.4) superfamily is defined by enzymes that catalyze the hydrolysis of the sn-2 bond of phosphoglycerides. Most PLA2s from the venom of Bothrops species are basic proteins, which have been well characterized both structurally and functionally, however, little is known about acidic PLA2s from this venom. Nevertheless, it has been demonstrated that they are non-toxic, with high catalytic and hypotensive activities and show the ability to inhibit platelet aggregation. To further understand the function of these proteins, we have isolated a cDNA that encodes an acidic PLA2 from a cDNA library prepared from the poly(A)+ RNA of venom gland of Bothrops jararacussu. The full-length nucleotide sequence of 366 base pairs encodes a predicted gene product with 122 amino acid with theoretical isoelectric point and size of 5.28 and 13,685 kDa, respectively. This acidic PLA2 sequence was cloned into expression vector pET11a (+) and expressed as inclusion bodies in Escherichia coli BL21(DE3)pLysS. The N-terminal amino acid sequence of the 14 kDa recombinant protein was determined. The recombinant acidic PLA2 protein was submitted to refolding and to be purified by RP-HPLC chromatography. The structure and function of the recombinant protein was compared to that of the native protein by circular dichroism (CD), enzymatic activity, edema-inducing, and platelet aggregation inhibition activities.     

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.     

The purification and characterization of a phospholipase A in hamster heart cytosol for the hydrolysis of phosphatidylcholine

Phospholipases A1 and A2 catalyze the hydrolysis of acyl groups of phospholipids at C-1 and C-2, respectively. These phospholipases are important in phospholipid catabolism and the remodeling of the acyl groups of phospholipids. Phospholipase A from hamster heart cytosol was purified by a combination of ion-exchange and gel filtration chromatography. The purity of the enzyme was assessed by nondenaturing polyacrylamide gel electrophoresis, two-dimension polyacrylamide gel electrophoresis, and immunological studies. The purified enzyme exhibited both phospholipase A1 and A2 activities toward phosphatidylcholine and had the ability to hydrolyze the acyl groups of phosphatidylethanolamine. However, the enzyme was not active toward lysophosphatidylcholine, diacylglycerol, or triacylglycerol. By Sepharose 6B chromatography, the molecular weight of the purified enzyme was estimated to be 140,000. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was composed of identical Mr 14,000 subunits. At least six subunits in the native enzyme could be cross-linked by dimethyl suberimidate. Both phospholipase A1 and A2 activities showed similar pH profiles, exhibited no absolute requirements for divalent metallic cations, but displayed a high degree of specificity for the acyl groups of phosphatidylcholine at both C-1 and C-2. The Km of phospholipases A1 and A2 for 1-palmitoyl-2-arachidon-ylglycerophosphocholine was found to be identical (0.5 mM).     

Phospholipase A(2) with platelet aggregation inhibitor activity from Austrelaps superbus venom: protein purification and cDNA cloning

Four phospholipase A(2) (PLA(2)) enzymes (Superbins a, b, c, and d) with varying platelet aggregation inhibitor activities have been purified from Austrelaps superbus by a combination of gel filtration, ion-exchange, and reversed-phase high-pressure liquid chromatography. Purity and homogeneity of the superbins have been confirmed by high-performance capillary zone electrophoresis and mass spectrometry. The electron spray ionization mass spectrometry data showed that their molecular masses range from 13,140 to 13,236 Da. Each of the proteins has been found to be basic and exhibit varying degrees of PLA(2) activity. They also displayed different platelet aggregation inhibitory activities. Superbin a was found to possess the most potent inhibitory activity with an IC(50) of 9.0 nM, whereas Superbin d was found to be least effective with an IC(50) of 3.0 microM. Superbins b and c were moderately effective with IC(50) values of 0.05 and 0.5 microM, respectively. The amino-terminal sequencing confirmed the identity of these superbins. cDNA cloning resulted in the identification of 17 more PLA(2) isoforms in A. superbus venom. It has also provided complete information on the precursor PLA(2). The precursor PLA(2) contained a 27-amino-acid signal peptide and 117- to 125-amino-acid PLA(2) (molecular mass ranging from 13,000 to 14,000 Da). Two of these PLA(2) enzymes resembled more closely (87%) Superbin a in structure. Two unique PLA(2) enzymes containing an extra pancreatic loop also have been identified among the isoforms.     

Effect of forskolin and quinacrine on leukocyte functions under the effect of low-dose radiation

The adenylate cyclase and phospholipase A2 incorporation in the functional responses as well as lipid peroxidation processes and glutathione system homeostasis of animal leukocytes to small doses of ionizing radiation (1-100 mGy) have been estimated. The cells were irradiated by introduction of radioactive isotope 14C-leucine into the incubation medium. It is established that the ionizing radiation has different effects on the modification of cellular functions by the agents, which change adenylate cyclase and phospholipase A2 activity. Neutralization of stimulative irradiation effect on chemokinesis of polymorphonuclear leukocytes by quinacrine (the inhibitor of phospholipase A2) indicates for certain, that metabolism of eicosanoids takes immediate part in the cell response to ionizing radiation. Apparently, adenylate cyclase has no influence on this process, where at indicates the lack of influence of forskolin (the stimulator of adenylate cyclase) on the spontaneous motility, and on the radiation action on this leukocyte function. Rosette forming ability of lymphocytes is regulated by both enzymes because it is modified both by the inhibitor of phospholipase A2, and by the adenylate cyclase stimulant. In this case it is impossible to exclude the action of ionizing radiation both through the adenylate cyclase cascade, and through the eicosanoid metabolism. In all the concentration range the radionuclides do not affect the studied biochemical indexes of the cell, but change the action of the modifiers.     

Inhibitors of liver lysosomal acid phospholipase A1

Lysosomal acid phospholipase A1, as well as other lysosomal enzymes, may be released under pathophysiological conditions into extralysosomal compartments. As shown here, several unspecific mechanisms exist which inhibit the hydrolysis of membrane diacylphospholipids by lysosomal acid phospholipase A1 and hence prevent an uncontrolled membrane destruction. These findings were obtained by employing partially purified rat liver lysosomal acid phospholipase A1 and sonicated radioactively labeled phosphatidylethanolamine or phosphatidylcholine as substrate. The inhibitory principles found include (1) pH, (2) inorganic cations, and (3) various proteins. Inorganic cations and proteins, however, inhibited lysosomal acid phospholipase A1 activity only below pH 6.0, and inhibition never exceeded 96%. Of the inorganic cations studied, the divalent species, as compared to the monovalent one, impaired lysosomal acid phospholipase A1 activity at significantly lower concentrations. Virtually all of the intracellular and extracellular proteins studied inhibited the enzyme activity, but the inhibitory potencies of the different proteins varied considerably. In general, basic and hydrophobic proteins were the most potent inhibitors, whereas glycoproteins appeared to be less inhibitory. The degree of inhibition of the enzyme activity in both proteins and inorganic cations depended on the substrate concentration and not on that of the enzyme. Binding studies provided evidence for inhibitor-substrate and against inhibitor-enzyme interactions.     

Correlation between the phospholipids domains of the target cell membrane and the extent of Naja kaouthia PLA(2)-induced membrane damage: evidence of distinct catalytic and cytotoxic sites in PLA(2) molecules

Two phospholipase A(2) (PLA(2)) enzymes (NK-PLA(2)-A and NK-PLA(2)-B) were purified from the venom of the monocled cobra Naja kaouthia. The molecular weights of NK-PLA(2)-A and NK-PLA(2)-B, as estimated by mass spectrometry, were 13,619 and 13,303 Da respectively. Both phospholipases were highly thermostable, had maximum catalytic activity at basic pH, and showed preferential hydrolysis of phosphatidylcholine. Intravenous injection of either PLA(2) up to a dose of 10 mg/kg body weight was non-toxic to mice and did not show neurotoxic symptoms. The N. kaouthia PLA(2)s displayed anticoagulant and cytotoxic activity, but poor hemolytic activity. Both the PLA(2)s were more toxic to Sf9 and Tn cells compared to VERO cells. NK-PLA(2) exhibited selective lysis of wild-type baculovirus-infected Sf9 cells compared to normal cells. Amino acid modification studies and heating experiments suggest that separate sites in the NK-PLA(2) molecules are responsible for their catalytic, anticoagulant and cytotoxic activities.     

Studies on phospholipase A2 in human seminal plasma.

1. Human seminal plasma and posterior lobe of prostate was found to have phospholipase A2 (PLA2) activity hydrolysing phosphatidylethanolamine with 14C-labelled linoleic and arachidonic acid. 2. A negative relationship was between sperm count and PLA2 activity in human seminal plasma. 3. The purified PLA2 from human seminal plasma showed high affinity to heparin, sensitivity toward p-bromophenacyl bromide, Pb2+, dithioerythritol and EDTA and it was activated by Ca2+ and Mn2+. 4. The purified PLA2 had alkaline pH optimum (7.5-10.0) and pI-value of 5.3. In SDS-PAGE enzyme preparation resulted in two bands with mol. wt of 14,000 and 16,000.     

The effect of calmodulin and chlorpromazine on the activity of phospholipases A2 from various sources

The effects of calmodulin and chlorpromazine on purified phospholipase A2 preparations from snake venoms: cobra (Naja naja oxiana), echis (Ehis multisquamatus) and Agkistrodon halys halys, as well as on phospholipases A2 from rat liver mitochondria and human platelets were studied. It was shown that within the concentration range of 1-5 microM calmodulin stimulates the phospholipase activity. Chlorpromazine inhibits the activity of these enzymes, the degree of inhibition being different for various phospholipases. Calmodulin was shown to interact with the phospholipases in the absence of exogenous Ca2+. The results obtained indicate that all phospholipases tested are calmodulin-dependent enzymes.     

Contamination of commercial preparations of calmodulin by phospholipase A2

In the course of studies of the possible regulation of cellular phospholipase A2 activities by calcium and calmodulin, it was observed that some of the commercial preparations of calmodulin contained significant phospholipase A2 activity. Six commercially available calmodulin sources were compared for the presence of contaminating phospholipase A2 activity, relative purity by SDS-gel electrophoresis, and relative biological activity in stimulating calmodulin-deficient phosphodiesterase. One of the commercial calmodulin sources contained a relatively high specific phospholipase A2 activity (1.30 +/- 0.11 nmol [1-14C]arachidonic acid released/mg protein per h) and yielded two major bands in SDS-gel electrophoresis. Two of the calmodulin sources tested were relatively free of phospholipase A2 activity, were quite pure (one band on SDS-gel) and had high biological activity in stimulating calmodulin-deficient phosphodiesterase. Thus, investigators using commercially available preparations of calmodulin should be aware of the contamination of some of these sources by phospholipase A2 activity. These findings may be of importance to investigators considering the role of calmodulin in activating a variety of calcium-dependent enzymes, including phospholipase A2.     

Isolation and characterization of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a new Lys49 phospholipase A2 homologue

Myotoxic phospholipases A2 of class II are commonly found in the venoms of crotalid snakes. As an approach to understanding their structure-activity relationship, diverse natural variants have been characterized biochemically and pharmacologically. This study describes a new myotoxic phospholipase A2 homologue, isolated from the venom of Atropoides (Bothrops) nummifer from Costa Rica. A. nummifer myotoxin 1 is a basic protein, with an apparent subunit molecular mass of 16 kDa, which migrates as a dimer in sodium dodecylsulfate-polyacrylamide gel electrophoresis under nonreducing conditions. It is strongly recognized by antibodies generated against Bothrops asper myotoxin II, by enzyme-immunoassay. The toxin induces rapid myonecrosis upon intramuscular injection in mice (evidenced by an early increase in plasma creatine kinase activity), and significant edema in the footpad assay. It also displays cytolytic activity upon cultured murine endothelial cells. The toxin (up to 50 microg) has no detectable phospholipase A2 activity on egg yolk phospholipids, and does not show an anticoagulant effect on sheep platelet-poor plasma in vitro. N-terminal sequence determination (53 amino acid residues) demonstrated that A. nummifer myotoxin II is a new Lys49 variant of the family of myotoxic, class II phospholipases A2. Sequence comparison with other phospholipases A2 revealed Asn53 as a novel substitution. In addition, this myotoxin is the first Lys49 variant presenting Asn in its N-terminus. Consequently, these findings suggest that neither Ser1 or Lys53, usually found in this family of proteins, are essential amino acid residues for their myotoxic, cytolytic, or edema-inducing effects.     

Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.     

Effects of phospholipase A2 on numbers of histamine H1 receptors in guinea pig lungs

Effects of phospholipase A2 on numbers of histamine H1 receptors and muscarinic acetylcholine receptors were estimated in guinea pig lungs. Histamine H1 and muscarinic acetylcholine receptors in lung membranes were studied by the direct binding technique using 3H-pyrilamine and 3H-quinuclidinyl benzilate, respectively. The control group had two orders (high-affinity and low-affinity) of binding sites of histamine H1 receptors. Pretreatment of lung membranes with phospholipase A2 destroyed high-affinity binding sites, and these sites could not be detected after treatment. Low-affinity sites were not affected by the treatment. In contrast, the numbers of muscarinic acetylcholine receptors did not change significantly in spite of the addition of phospholipase A2. These results indicate that resistance of receptors against phospholipase A2 treatment varies among receptors and even between the binding sites in the same receptor. These variations might modulate pathological conditions associated with inflammation in which phospholipase is activated. Easy establishment of histamine tachyphylaxis might be explained by this mechanism.     

Comparative study of three phospholipase A2s from the venom of Vipera aspis

1. Three phospholipase A2s, PLA2-I, PLA2-II and PLA2-III, were isolated from Vipera aspis venom by gel filtration and ion exchange chromatography. 2. Purified PLA2-I, -II and -III have mol. wts of 30,200, 16,000 and 13,500, and isoelectric points of 9.45, 7.65 and less than 4.1, respectively. 3. PLA2-I consists of an acidic subunit (mol. wt 13,700, pI: less than 3.5) and a basic subunit (mol. wt 16,500, pI: 10.6), which can be separated under highly acidic conditions. 4. PLA2-I possessed lethal activity and LD50 for this preparation was estimated to be 0.288 (0.209-0.397) micrograms/g, while lethality was not observed when PLA2-II, -III or each subunit of PLA2-I were administered. 5. Capillary permeability-increasing activity was found in the samples which possessed basic isoelectric points. Additionally, PLA2-I and its basic subunit drastically prolonged activated partial thromboplastin time of platelet rich plasma. 6. Intramuscular injections of PLA2-I, -II and -III increased serum creatine phosphokinase activity in mice, indicating that damage in muscle was caused by these enzymes. 7. NH2-terminal sequences of the three PLA2s were compared with other phospholipase A2s from snake venoms. Furthermore, antigenicities were tested using antiserum prepared against each sample.