Snake inhibitors of phospholipase A2 enzymes

Phospholipase A₂ (PLA₂) enzymes consist of a large family of proteins which share the same enzymatic function and display considerable sequence homology. These enzymes have been identified and characterised in mammalian tissue and snake venoms. Numerous physiological functions have been attributed to mammalian PLA₂s and they are nontoxic. In comparison, venom PLA₂s are toxic and induce a variety of pharmacological effects that are probably mediated via membrane receptors. Snake PLA₂ inhibitors (PLIα), with a similar structure to the M-type receptor, have been identified as soluble complexes in the serum of viperinae and crotalinae snakes. These inhibitors showed selective binding to crotalid group II PLA₂s and appeared to be restricted to the serum of this snake family. Analysis of PLA₂ binding to recombinant fragments of PLIα indicated that the CRD region was most likely responsible for enzyme inhibition. A second type of inhibitor, PLIβ, has been identified in serum from one viperid snake and consists of a leucine-rich structure. The third type of inhibitor, PLIγ, was found in the serum of five snake families and contains a pattern of cysteine residues that define a three-finger structure. PLIγ inhibitors isolated from the serum of Elapidae, Hydrophidae, Boidae and Colubridae families were able to inhibit a broad range of enzymes including the nontoxic mammalian group IB and IIA PLA₂s, and bee venom group III PLA₂. However, differences in the binding affinities indicated specificity for particular PLA₂s. A different representation has emerged for crotalid and viperid snakes. Their PLIγs did not inhibit bee venom group III, mammalian group IB and IIA enzymes. Furthermore, inhibition data for the γ-type inhibitor from Crotalus durissus terrificus (CICS) showed that this inhibitor was specific for viperid β-neurotoxins and did not inhibit β-neurotoxins from elapids [1]. Further studies are required to determine if this phenomenon is true for all γ-type inhibitors from Crotalidae snakes. The relative distribution of these inhibitors, their specificities and the structural features involved in binding are discussed in this review.     

An acidic phospholipase A₂ with antibacterial activity from Porthidium nasutum snake venom

Snake venoms are complex mixtures of proteins among which both basic and acidic phospholipases A₂ (PLA₂s) can be found. Basic PLA₂s are usually responsible for major toxic effects induced by snake venoms, while acidic PLA₂s tend to have a lower toxicity. A novel PLA₂, here named PnPLA₂, was purified from the venom of Porthidium nasutum by means of RP-HPLC on a C18 column. PnPLA₂ is an acidic protein with a pI of 4.6, which migrates as a single band under both non-reducing and reducing conditions in SDS-PAGE. PnPLA₂ had a molecular mass of 15,802.6 Da, determined by ESI-MS. Three tryptic peptides of this protein were characterized by HPLC-nESI-MS/MS, and N-terminal sequencing by direct Edman degradation showing homology to other acidic PLA₂s from viperid venoms. PnPLA₂ displayed indirect hemolytic activity in agarose erythrocyte-egg yolk gels and bactericidal activity against Staphylococcus aureus in a dose-dependent manner, with a MIC and MBC of 32 μg/mL. In addition, PnPLA₂ showed a potent inhibitory effect on platelet aggregation with doses up to 40 μg/mL. This acidic PLA₂, in contrast to basic enzymes isolated from other viperid snake venoms, was not cytotoxic to murine skeletal muscle myoblasts C₂C₁₂. This is the first report on a bactericidal protein of Porthidium nasutum venom.     

The molecular cloning of a phospholipase A2 from Bothrops jararacussu snake venom: Evolution of venom group II phospholipase A2's may imply gene duplications

The sequence coding for a snake venom phospholipase A₂ (PLA₂), BJUPLA₂, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA₂'s. A striking feature of the cDNA is the high sequence conservation of the 5 and 3 untranslated regions in cDNAs coding for PLA₂'s from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA₂ with group II PLA₂'s in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA₂'s.The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X76289.Correspondence to: A.M. Moura-da-Silva     

The Amino Acid Sequence of Bothropstoxin-II, an Asp-49 Myotoxin from Bothrops jararacussu (Jararacucu) Venom with Low Phospholipase A2 Activity

The complete amino acid sequence of bothropstoxin-II (BthTX-II), a myotoxin isolated from Bothrops jararacussu snake venom, is reported. The results show that BthTX-II is an Asp-49 phospholipase A₂ (PLA₂)-like protein composed of a single polypeptide chain of 120 amino acid residues (M _r = 13,976), containing one methionine and 14 half-cystines. Despite a high degree of homology with other PLA₂'s and the presence of the strategic residues known to compose the Ca²⁺-binding loop, namely Tyr-28, Gly-30, Gly-32, and especially Asp-49, besides His-48, Tyr-52, and Asp-99, all of them directly or indirectly involved in catalysis, BthTX-II revealed a very low PLA₂ activity when assayed on egg yolk phosphatidylcholine. We attribute this low catalytic activity to the existence of extra mutations, e.g., Trp-5 for Phe-5, which points to the need of considering other strategic positions, since only Lys-49 PLA₂'s have been considered to be devoid of this enzymatic activity.     

Structural and Functional Properties of Cr 5, a New Lys49 Phospholipase A2 Homologue Isolated from the Venom of the Snake Calloselasma rhodostoma

Cr 5 PLA₂ homologous (K49) was isolated from Calloselasma rhodostoma venom in only one chromatographic step in reverse phase HPLC (RP-HPLC) (on μ-Bondapack C-18). A molecular mass of 13.965 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that Cr 5 had a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical residues of a basic PLA₂. The complete amino acid sequence of Cr 5 PLA₂ contains 120 residues, resulting in a calculated pI value of 5.55. This sequence shows high identity values when compared to other K49 PLA₂s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA₂s. The sequence found was SLVELGKMIL QETGKNPAKS YGAYGCNCGV LGRHKPKDAT DRCCFVHKCC YKKLTGCDPK KDRYSYSWKD KTIVCGENNP CLKEMCECDK AVAICLRENL DTYNKKYRYL KPFCKKADDC. In mice, Cr 5 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD₅₀ of Cr 5 was 0.070 mg/kg of the animal weight, by intracerebroventricular (i.c.v.) route. In vitro, the toxin caused rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. The isolation of this PLA₂ and the combined structural and functional information obtained classify Cr 5 as a new member of the K49 PLA₂ family, since it presents typical features from such proteins.     

Plant low-molecular-weight phospholipase A2s (PLA2s) are structurally related to the animal secretory PLA2s and are present as a family of isoforms in rice (Oryza sativa)

Recently, we purified to homogeneity and characterized a low-molecular-weight calcium-dependent phospholipase A₂ (PLA₂) from developing elm seed endosperm. This represented the first purified and characterized PLA₂ from a plant tissue. The full sequences of two distinct but homologous rice (Oryza sativa) cDNAs are given here. These encode mature proteins of 119 amino acids (PLA₂-I, preceded by a 19 amino acid signal peptide) and 128 amino acids (PLA₂-II, preceded by a 25 amino acid signal peptide), and were derived from four expressed sequence tag (EST) clones. Both proteins were homologous to the N-terminal amino acid sequence of the elm PLA₂. They contained twelve conserved cysteine residues and sequences that are likely to represent the Ca²⁺-binding loop and active-site motif, which are characteristic of animal secretory PLA₂s. A soluble PLA₂ activity was purified 145 000-fold from green rice shoots. This had the same biochemical characteristics as the elm and animal secretory PLA₂s. The purified rice PLA₂ consisted of two proteins, with a molecular weight of 12 440 and 12 920, that had identical N-terminal amino acid sequences. This sequence was different from but homologous to the PLA₂-I and PLA₂-II sequences. Taken together, the results suggest that at least three different low-molecular-weight PLA₂s are expressed in green rice shoots. Southern blot analysis suggested that multiple copies of such genes are likely to occur in the rice and in other plant genomes.     

Interisland Mutation of a Novel Phospholipase A<Subscript>2</Subscript> from <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom and Evolution of Crotalinae Group II Phospholipases A<Subscript>2</Subscript>

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A₂ (PLA₂). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA₂, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD₅₀ for mice was 1.34 µg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA₂s based on the amino acid sequences revealed that neurotoxic PLA₂s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA₂ groups such as PLA₂ type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA₂s showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA₂s, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA₂s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation. The nucleotide sequences reported in this paper are available from the GenBank/EMBL/DDBJ databases under accession numbers AB102728 and AB102729.     

Biological and Structural Characterization of a New PLA<Subscript>2</Subscript> from the Crotalus durissus collilineatus Venom

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A₂ (PLA₂) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80 with crotalic PLA₂s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50 if compared to other sources of PLA₂s such as from the Bothrops snake venom. Also, this PLA₂ induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and q-BPB showed a similar reduction. The q-BPB did not reduce significantly the myotoxic activity induced by the PLA₂, but the anhydrous acetic acid treatment and the pre-incu-bation of PLA₂ with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiflorae (Gram-negative), which was drastically reduced by incubation of this PLA₂ with q-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or b-wing region may be involved in the binding of this PLA₂ to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.     

Vaporization of Inorganic Mercury by Cell-free Extracts of Drug Resistant Escherichia coli

The cDNAs encoding venom phospholipase A₂ (PLA₂) inhibitors (PLIs), named Protobothrops elegans (Pe)γPLI-A, PeγPLI-B, PeαPLI-A, and PeαPLI-B, were cloned from the P. elegans liver cDNA library. They were further divided into several constituents due to nucleotide substitutions in their open reading frames. For PeαPLI-A, two constituents, PeαPLI-A_a and PeαPLI-A_b, were identified due to three nonsynonymous substitutions in exon 3. Far-western blot and mass-spectrometry analysis of the P. elegans serum proteins showed the presence of γPLIs, and αPLIs, which can bind venom PLA₂s. In αPLIs from Protobothrops sera, A or B subtype-specific amino acid substitutions are concentrated only in exon 3. A comparison of γPLIs showed that γPLI-As are conserved and γPLI-Bs diversified. Mathematical analysis of the nucleotide sequences of Protobothrops γPLI-B cDNAs revealed that the particular loops in the three-finger motifs diversified by accelerated evolution. Such evolutionary features should have made serum PLIs acquire their respective inhibitory activities to adapt to venom PLA₂ isozymes.     

Interisland Evolution of <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom Phospholipase A<Subscript>2</Subscript> Isozymes

Abstract Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A₂ (PLA₂), named PL-Y, was isolated from Okinawa T. flavoviridis venom and its amino acid sequence was determined from both protein and cDNA. PL-Y was unable to induce edema. In contrast, PLA-B, a PLA₂ from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA₂, named PLA-B′, which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp⁴⁹]PLA₂ isozymes, PL-Y (Okinawa), PLA-B (Tokunoshima), and PLA-B′ (Amami-Oshima), are identical in the N-terminal half but have one to four amino acid substitutions in the β1-sheet and its vicinity. Such interisland sequence diversities among them are due to isolation in the different environments over 1 to 2 million years and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, a major PLA₂, named PLA2, ubiquitously exists in the venoms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that the PLA2 gene is a prototype among T. flavoviridis venom PLA₂ isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component. Phylogenetic analysis based on the amino acid sequences revealed that T. flavoviridis PLA₂ isozymes are clearly separated into three groups, PLA2 type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Basic [Asp⁴⁹]PLA₂-type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA2 type and [Lys⁴⁹]PLA₂ type.     

Purification and Amino Acid Sequence of MP-III 4R D49 Phospholipase A2 from Bothrops pirajai Snake Venom, a Toxin with Moderate PLA2 and Anticoagulant Activities and High Myotoxic Activity

MP-III 4R PLA₂ was purified from the venom of Bothrops pirajai venom (Bahia's jararacussu) after three chromatographic steps which started with RP-HPLC. The complete amino acid sequence of MP-III 4R PLA₂ from Bothrops pirajai was determined by amino acid sequencing of reduced and carboxymethylated MP-III 4R and the isolated peptides from clostripain and protease V8 digestion. MP-III 4R is a D49 PLA₂ with 121 amino acid residues and has a molecular weight estimated at 13,800 Da, with 14 half-cysteines. This protein showed moderate PLA₂ and anticoagulant activity. This PLA₂ does not have a high degree of homology with other bothropic PLA₂-like myotoxins (~75%) and nonbothropic myotoxins (~60%). MP-III 4R is a new PLA₂, which was isolated using exclusively analytical and preparative HPLC methods. Based on the N-terminal sequence and biological activities, MP-III 4R was identified as similar to piratoxin-III (PrTX-III), which was isolated by conventional chromatography based on molecular exclusion ion exchange chromatography. Clinical manifestations indicate that at the site of toxin injection, there may be pain of variable intensity, because animals continue to lick the limb. No clinical sign indicating general toxicity was noticed. Myotoxicity was observed in gastrocnemius muscle cells after exposure to MP-III 4R, with a high frequency (70%) of affected muscle fibers.     

Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus

Phospholipase A₂ (PLA₂) is one of the main components of bee venom. Here, we identify a venom PLA₂ from the bumblebee, Bombus ignitus. Bumblebee venom PLA₂ (Bi-PLA₂) cDNA, which was identified by searching B. ignitus venom gland expressed sequence tags, encodes a 180 amino acid protein. Comparison of the genomic sequence with the cDNA sequence revealed the presence of four exons and three introns in the Bi-PLA₂ gene. Bi-PLA₂ is an 18-kDa glycoprotein. It is expressed in the venom gland, cleaved between the residues Arg44 and Ile45, and then stored in the venom sac. Comparative analysis revealed that the mature Bi-PLA₂ (136 amino acids) possesses features consistent with other bee PLA₂s, including ten conserved cysteine residues, as well as a highly conserved Ca²⁺-binding site and active site. Phylogenetic analysis of bee PLA₂s separated the bumblebee and honeybee PLA₂ proteins into two groups. The mature Bi-PLA₂ purified from the venom of B. ignitus worker bees hydrolyzed DBPC, a known substrate of PLA₂. Immunofluorescence staining of Bi-PLA₂-treated insect Sf9 cells revealed that Bi-PLA₂ binds at the cell membrane and induces apoptotic cell death.     

Identification and characterization of a phospholipase A2 from the venom of the Saw-scaled viper: Novel bactericidal and membrane damaging activities

Phospholipase A₂ (PLA₂), a common toxic component of snake venom, has been implicated in various pharmacological effects. In this study, a basic myotoxic PLA₂, named EcTx-I was isolated from Echis carinatus snake venom by using gel filtration on Superdex G-75, and reverse phase HPLC on C18 and C8 Sepharose columns. PLA₂, EcTx-I was 13,861.72 molecular weight as estimated by MALDI-TOF (15 kD by SDS-PAGE), and consisted of 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with basic myotoxic PLA₂s from other snake venoms. The purified PLA₂ EcTx-I was evaluated (250 μg/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. EcTx-I showed strong antibacterial activity against B. pseudomallei (KHW) and E. aerogenes among the tested bacteria. Other Gram-negative and Gram-positive bacteria showed only a moderate effect. However, the Gram-positive bacterium E. aerogenes failed to show any effect on EcTx-I protein at tested doses. The most significant bacteriostatic and bactericidal effect of EcTx-I was observed at MICs of >15 μg/ml against (B. pseudomallei, KHW) and MICs >30 μg/ml against E. aerogenes. Mechanisms of bactericidal and membrane damaging effects were proved by ultra-structural analysis. EcTx-I was able to induce cytotoxicity on THP-1 cells in vitro as well as lethality in BALB/c mice. EcTx-I also induced mild myotoxic effects on mouse skin, but was devoid of hemolytic effects on human erythrocytes up to 500 μg/ml. It is shown that the toxic effect induced by E. carinatus venom is due to the presence of myotoxic PLA₂ (EcTx-I). The result also corroborates the hypothesis of an association between toxic and enzymatic domains. In conclusion, EcTx-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.     

Purification and inflammatory edema induced by two PLA2 (Anch TX-I and Anch TX-II) from sea anemone Anthothoe chilensis (Actiniaria: Sagartiidae)

The Anch TX-I and II PLA₂ were purified from Anthothoe chilensis (Lesson, 1830) from the extract of the anemone after only two chromatographic step using molecular exclusion chromatography (Sephadex G-75) and reverse phase HPLC on μ-Bondapak C18 column. Both PLA₂ showed a molecular mass of ~ 14 kDa determined by MALDI-TOF mass spectrometry and showed a high catalytic activity (data not showed). Although homologous with mammalian or snake venom group I PLA₂s, Anch TX-I and II is sufficiently structurally different for the question of its placement into the existing PLA₂ classification scheme to arise. In addition, Anch TX-I and II, despite possessing many common structural features, also differ in some important structural properties. The amino acid sequence of both PLA₂ (Anch TX-I and III) showed high amino acid sequence identity with PLA₂Rhopilema nomadica and Bunodosoma caissarum Cnidaria and PLA₂ of group III protein isolated from the Mexican lizard Heloderma horridum horridum and Heloderma suspectum. In addition, Anch TX-I and Anch TX-II showed high amino acid sequence identity with PLA₂ from group III also showed significant overall homology to bee Apis dorsata, Bombus terrestris and Bombus pennsylvanicus and PLA₂. We also investigated the in vivo edematogenic activity of Anch TX-I and Anch TX-II in a model of paw and skin edema in rats and observed that both are able to induce dose-dependent edema.     

Accelerated Evolution and Molecular Surface of Venom Phospholipase A2 Enzymes

Multiple phospholipase A₂ (PLA₂) isoenzymes found in a single snake venom induce a variety of pharmacological effects. These multiple forms are formed by gene duplication and accelerated evolution of exons. We examined the amino acid sequences of 127 snake venom PLA₂ enzymes and their homologues to study in which location most natural substitutions occur. Our data show that hot spots of amino acid substitutions in this group of proteins occur mostly on the surface. A logistic model correlating the substitution rates of each amino acid residue with their surface accessibility indicates that the probability of natural substitutions occurring in the fully exposed residue is 2.6–3.5 times greater than that of substitutions occurring in buried residues. These surface substitutions play a significant role in the evolution of new PLA₂ isoenzymes by altering the specificity of targeting to various tissues or cells, resulting in distinct pharmacological effects. Thus natural substitutions in PLA₂ enzymes, in contrast to popular belief, are not random substitutions but appear to be directed toward modifying the molecular surface. Received: 11 May 1998 / Accepted: 29 June 1998     

Structural and functional properties of BaTX,a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Bothrops alternatus

BaTX PLA₂, a K49 phospholipase A₂ homologue was purified from Bothrops alternatus venom after two chromatographic steps, molecular exclusion on Superdex 75 and reverse phase HPLC on μ-Bondapack C-18. A molecular mass of 13898.71 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that BaTX has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA₂. The complete amino acid sequence of BaTX PLA₂ contains 121 residues, resulting in a calculated pI value of 8.63. This sequence shows high identity values when compared to other K49 PLA₂s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA₂s. The sequence was SLFELGKMIL QETGKNPAKS YGAYYCYCGW GGQGQPKDAT DRCCYVHKCC YKKLTGCNPK KDRYSYSWKD KTIVCGENNS CLKELCECDK AVAICLRENL NTYNKKYRYY LKPLCKKADA C. In mice, BaTX induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. The LD₅₀ of BaTX was 7 μg/g body weight, by intravenous route. In vitro, the toxin caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparations. The blockage 50% was achieved at a concentration of 0.03 μM: 40 ± 0.4 min and 0.07 μM: 35 ± 0.3 min. Moreover, this protein induced a rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. Thus, the combined structural and functional information obtained identify BaTX as a new member of the K49 PLA₂ family, which presents the typical bioactivities described for such proteins.     

Taxonomic distribution and quantitative analysis of free purine and pyrimidine nucleosides in snake venoms

The nucleoside content of 32 elapid and viperid venoms was examined. Free purines, principally adenosine (ADO), inosine (INO), and guanosine (GUA), comprised as much as 8.7% of the solid components of some venoms. Thus, purines are far more abundant in some venoms than many proteinaceous toxins. Hypoxanthine (HYP) was found in about half of elapid and viperine venoms, in which it is a relatively minor constituent (<60 microg/g). Adenosine monophosphate (AMP) was tentatively identified in only three elapid and two viperid venoms. The pyrimidines, uridine (URI) and cytidine (CYT), were also found in most elapid and viperine venoms. In most of these, the amount of uridine was substantially greater than that of cytidine. Thymidine (THY) was not found in any venom, indicating that DNA from disintegration of glandular cells is not the source of venom nucleosides. In contrast to elapid and viperine venoms, most crotaline venoms are devoid of free nucleosides. Elapid and viperine venoms also contained other minor, low molecular weight constituents that could not be positively identified. Some had spectra identical to those of adenosine, nicotinamide adenine dinucleotide (NAD), inosine, xanthosine (XAN), and guanosine, while others had unique spectra. There is no apparent correlation between quantities of venom nucleosides and literature values for the three dominant venom enzymes that release endogenous nucleosides, 5'-nucleotidase (5NUC), phosphodiesterase (PDE), and alkaline phosphomonoesterase (PME).     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.     

Up-regulation of the expressions of phospholipase A2 inhibitors in the liver of a venomous snake by its own venom phospholipase A2

Venomous snakes such as Gloydius brevicaudus have three distinct types of phospholipase A₂ inhibitors (PLIα, PLIβ, and PLIγ) in their blood so as to protect themselves from their own venom phospholipases A₂ (PLA₂s). Expressions of these PLIs in G. brevicaudus liver were found to be enhanced by the intramuscular injection of its own venom. The enhancement of gene expressions of PLIα and PLIβ in the liver was also found to be induced by acidic PLA₂ contained in this venom. Furthermore, these effects of acidic PLA₂ on gene expression of PLIs were shown to be unrelated to its enzymatic activity. These results suggest that these venomous snakes have developed the self-protective system against their own venom, by which the venom components up-regulate the expression of anti-venom proteins in their liver.     

Isolation and enzymatic characterization of a basic phospholipase A2 from Bothrops jararacussu snake venom

A novel basic phospholipase A₂ (PLA2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY . . .) showed a high degree of homology with basic D49 PLA₂ myotoxins from other Bothrops venoms. Bj IV had high PLA₂ activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45°C. Full PLA₂ activity required Ca²⁺ but was inhibited by Cu²⁺ and Zn²⁺, and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA₂ was similar to that in the basic PLA₂ of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA₂ could partly explain the low PLA₂ activity of Bothrops venoms.