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.     

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.     

Lysosomotropic agents activate the capacity for calcium dependent pinocytosis in starvedAmoeba proteus: evidence for a mechanism involving phospholipase A2

Summary Pinocytosis induced by Na⁺ was assayed by phase contrast microscopy in 8–12 days starvedAmoeba proteus. These cultures were inactive with respect to calcium-dependent Na⁺-induced pinocytosis, but treatment with amino acid methyl and ethyl esters increased their capacity for pinocytosis. Besides promoting pinocytosis these compounds also stimulated calcium-sensitive secretion of lysosomal enzymes from normal, 2–3 days starved, cells. Only uncharged 1-forms of the amino acid esters were effective. Also other lysosomotropic compounds including monodansylcadaverine, glycine-phenylalanine-2-naphthylamide, NH₄Cl, and the ionophores monensin and A23187 activated starved cells. The effect of these agents (except A23187) was inhibited by the drug dantrolene suggesting that activation is a consequence of release of Ca²⁺ from intracellular stores. Several of the lysosomotropic agents also lost their activating effect in the presence of phospholipase A₂ (PLA₂) inhibitors. To investigate whether or not PLA₂ activity in the cell culture could imitate the effect of the lysosomotropic agents, we incubated starved cells with snake venom PLA₂s. These enzymes caused rapid, dantrolene-sensitive activation of the cells. Measurement of endogenous PLA₂in normal cells revealed significant cellular activity but no significant secretion of the enzyme into the culture medium was observed. Together the studies with enzyme inhibitors and dantrolene suggest that the process by which lysosomotropic agents affect pinocytosis involves activation of PLA₂ and release of Ca²⁺ from intracellular stores.Abbreviations AnBOMe amino-n-butyric acid methylester - Et ethylester - GPN glycine-1-phenylalanine-2-naphthylamide - MDC monodansylcadaverine - MDTC monodansylthiacadaverine - Me methylester - pBPB p-bromo phenacylbromide - PLA₂ phospholipase A₂     

Isolation and characterization of bioactive compounds of Clematis gouriana Roxb. ex DC against snake venom phospholipase A2 (PLA2) computational and in vitro insights

Bioactive compounds were isolated from Clematis gouriana Roxb. ex DC. The compounds were separated, characterized, the structures elucidated and submitted to the PubChem Database. The PubChem Ids SID 249494134 and SID 249494135 were tested against phospholipases A₂ (PLA₂) of Naja naja (Indian cobra) venom for PLA₂ activity. Both the compounds showed promising inhibitory activity; computational data also substantiated the results. The two compounds underwent density functional theory calculation to observe the chemical stability and electrostatic potential profile. Molecular interactions between the compounds and PLA₂ were observed at the binding pocket of the PLA₂ protein. Further, this protein–ligand complexes were simulated for a timescale of 100 ns of molecular dynamics simulation. Experimental and computational results showed significant PLA₂ inhibition activity.     

Dynamics of formation of lysoforms on enzymatic hydrolysis of phosphatidylalkanols

Hydrolysis of 1,2-dioleoyl-sn-glycero-3-phosphomethanol (DOPM), 1,2-dioleoyl-sn-glycero-3-phosphoethanol (DOPEt), 1,2-dioleoyl-sn-glycero-3-phosphoethyleneglycol (DOPEG), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) catalyzed by phospholipase A₂ (PLA₂) from porcine pancreas was studied in single-component and binary model bilayer membranes (liposomes). The presence of anionic phosphatidylalkanols increases the rate of hydrolysis of zwitterionic DOPC in liposomes by the action of PLA₂. The rate of formation of lysoforms of anionic (acidic) lipids at the initial reaction stage in single-component liposomes increased in the following sequence: DOPEG < DOPM < DOPEt (compared with that for the zwitterionic DOPC). In binary liposomes formation of lyso-DOPC increased in the presence of acidic lipids in the following sequence: DOPM < DOPEt < DOPEG. This indicates that the size of polar fragment of the second substrate and the presence of free hydroxy groups in the head of DOPEG may possibly activate DOPC hydrolysis by the action of PLA₂ in the presence of anionic phospholipids including cardiolipin; the studied phospholipids model fragments of the latter.     

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.     

Purification and characterization of a potent hemolytic toxin with phospholipase A2 activity from the venom of Indian Russell's viper

A potent toxin with phospholipase A₂ (PLA₂) and hemolytic activity in vitro was purified from the Russell's viper venom of eastern India (RVV-EI). The purified protein (RVV-PFIIc) of 15.3 kDa molecular weight, and a lethal toxicity dose (LD₅₀ i.p.) of 0.1 mg/kg body weight, was the most toxic PLA₂ so far reported from the Indian subcontinent. The material also possessed anticoagulant activity as it enhanced the prothrombin induced plasma clotting time in vitro. The PLA₂ toxin (RVV-PFIIc) was shown to be different from other PLA₂s of RVV in respect to one or more of these parameters e.g. molecular weight, isoelectric pH, in vivo toxicity, specific activity of the enzyme and certain other biological activities. The first 19 amino terminal sequence (NLFQFAEMIVKMTGKEAVH) of RVV-PFIIc showed variable degree of homology (42.1–94.7%) with those of other RVV-PLA₂s described in the literature. Antisera raised against RVV-EI or RVV-PFIIc, though completely neutralized the in vivo lethal toxicity of RVV-EI or RVV-PFIIc, failed to inhibit their PLA₂ activity in vitro thereby suggesting that in vivo toxicity and in vitro activity of the enzyme may not be directly related. Apart from RVV-PFIIc, at least two other PLA₂ isozymes were found to be present in RVV-EI that were distinct from RVV-PFIIc in respect to their molecular, biological as well as serological properties. The significance of these and related data in antivenom therapy is discussed.     

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     

Growth,N2 fixation and photosynthesis in a cyanobacterium,Trichodesmium sp., under Fe stress

Trichodesmium sp., isolated from the Great Barrier Reef lagoon, was cultured in artificial seawater media containing a range of Fe concentration. Fe additions stimulated growth, N₂ fixation, cellular chlorophyll a content, light-saturated chlorophyll a-specific gross photosynthetic capacity (P_m ^chla) and the dark respiration rate (R_d ^chla). Cell yields only doubled for 9 nM Fe relative to zero added Fe, whereas N₂ fixation increased 11-fold considerably for 450 nM Fe. The results suggest that N₂ fixation of Trichodesmium is more sensitive to Fe limitation than are the cell yields.     

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.     

Ca2+ signaling in the transformation of promastigotes to axenic amastigotes of Leishmania donovani

Three acidic phospholipases A₂ from Indian cobra (Naja naja naja) venom inhibited platelet aggregation in platelet rich plasma induced separately by ADP, collagen and epinephrine with different potencies. The order of inhibition was epinephrine > collagen > ADP. They did not inhibit platelet aggregation induced by arachidonic acid (10 M). The inhibition was dependent on concentration of the protein and the time of incubation of the phospholipases A₂ with platelet rich plasma. Parabromophenacyl bromide modified PLA₂ enzymes lost their enzymatic activity as well as platelet aggregation inhibition activity suggesting the involvement of catalytic function in platelet aggregation inhibitory activity.     

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.     

Suppression of Settlement of Larvae of Fouling Organism by Water-Soluble Substances from Epibiotic Bacteria

Twenty-nine bacterial strains were isolated from the surface of the green alga Ulva reticulata, the soft coral Dendronephthya sp., and the sponge Haliclona sp. The bacterial species Vibrio alginolyticus, Vibrio sp. 4, an unidentified α-Proteobacterium, Vibrio sp. 7, Pseudoalteromonas sp. 2, and Pseudoalteromonas sp. 4 were found to suppress the larval settlement of the polychaete Hydroides elegans (Haswell, 1883) and the bryozoan Bugula neritina (Linnaeus, 1758). Aqueous extracts of five bacteria (all those named above except Pseudoalteromonas sp. 2) prevented larval settlement. Bacteria V. alginolyticus, Vibrio sp. 4, and an unidentified α-Proteobacterium were first discovered to produce high-molecular substances (>100 kDa) preventing larval settlement. Their activity was inhibited by amylase treatment, while trypsin and papain did not influence their activity. The data obtained proved that bacteria from the surface of the number of marine organisms excrete water-soluble sacchariferous compounds preventing larval settlement.     

Molecular evolution of group II phospholipases A2

The nucleotide sequences of 13 cDNAs encoding group II phospholipases A₂ (PLA₂ ^S), which are from viperidae snake venoms and from mammalian sources, were aligned and analyzed by phylogenetic trees constructed using various components of the sequences. The evolutionary trees derived from the combined sequences of the untranslated (5 and 3) region and the signal peptide region of cDNAs were in accord with the consequences from taxonomy. In contrast, the evolutionary trees from the mature protein-coding region sequences of cDNAs and from the amino acid sequences showed random patterns. These observations indicated that the mature protein-coding region has evolved through a process differently from the untranslated and signal peptide regions. The trees built from the nucleotide differences at each of three positions of codons in the mature protein-coding region suggested that snakevenom-gland PLA₂ genes have evolved via a process different from mammalian PLA₂ genes. The occurrence of accelerated evolution has been recently discovered in Trimeresurus flavoviridis venom-gland group II PLA₂ isozyme genes (Nakashima et al. 1993, Proc Natl Acad Sci USA 90:5964–5968), so the present phylogenetic analysis together with the estimation of nucleotide divergence of cDNAs provides further evidence that snakevenom-group II PLA₂ isozyme genes have evolved by accelerated evolution to gain diverse physiological activities. Correspondence to: M. Ohno     

Stimulation of metamorphosis in Hydractinia echinata involves generation of lysophosphatidylcholine

Summary Whilst the significance of the phosphoinositide cycle in the activation of developmental events by extra-cellular signals is well established, the involvement of the phosphatidylcholine (PC) cycle is a matter just emerging. In the present study, the metabolism of phosphatidylcholine in early metamorphosis of Hydractinia echinata (Coelenterata; Hydrozoa) was investigated by incubation of planula larvae with ³H-choline, extraction of the metabolites and isolation of the metabolites by thin-layer chromatography (TLC). Phosphatidylcholine (PC), lysophosphatidylcholine (LPC), acetylcholine and glycerophosphocholine were the labelled metabolites. Induction of metamorphosis did not stimulate an increased incorporation of choline into PC. In larvae preincubated with ³H-choline to a steady state level of incorporation, a significant transient elevation of the radioactive label in LPC was observed 90 min after addition of metamorphosis stimulating agents. LPC probably derived from PC by the action of a phospholipase A₂ (PLA₂). LPCs from bovine and soybean origin as well as isolated larval LPC did not influence metamorphosis. PLA₂ from bee venom promoted Cs⁺-induced metamorphosis but did not influence phorbol ester-induced metamorphosis. The data suggest that a PLA₂ is activated during metamorphosis. This PLA₂ activation does not occur in those putative receptor cells which receive the primary external inducing stimulus but in the many larval cells which resume proliferation or differentiation in response to a second, internally propagated signal. Offprint requests to: T. Leitz     

The endosymbiotic unit ofStentor polymorphus andChlorella sp. morphological and physiological studies

Summary The greenStentor polymorphus harbours unicellular coccoid chlorophycean algae. They are located in food vacuoles, where they show various states of digestion, as well as in individual so-called perialgal vacuoles. According to their characteristic morphological properties the algae belong to the genusChlorella. They can be isolated from the ciliate and cultivated in mass cultures in a sterile defined inorganic medium supplemented with vitamins B₁ and B₁₂. The algae have no secondary carotenoids and excrete maltose by a pH-dependent mechanism. They thus show a conspicuous physiological similarity to the symbiotic chlorellae ofParamecium bursaria andHydra viridis, which also excrete maltose.A comparison of the properties of the chlorellae isolated fromStentor polymorphus and of the intactStentor polymorphus-Chlorella unit with the characteristic features of symbiotic chlorellae and with endosymbiotic systems containingChlorella sp. in general, lead to the conclusion that the greenStentor polymorphus is also a true endosymbiotic system.     

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.     

Kinetics of sulfate uptake by freshwater and marine species ofDesulfovibrio

Apparent half-saturation constants (K _m) and maximum uptake rates (V _max) for sulfate were determined in four species ofDesulfovibrio of freshwater and marine origin using a³⁵S-sulfate tracer technique. The lowerstK _m (5 M) was found in the freshwater speciesDesulfovibrio vulgaris (Marburg) and the highestK _m (77 M) in the marine speciesDesulfovibrio salexigens. Maximum specific rates of sulfate uptake (i.e.,V _max) were proportional to the growth rates observed in batch cultures. The halophilicDesulfovibrio salexigens did not change itsK _m andV _max between 1 and 6,000 M SO ₄ ^2- , and apparently did not induce a low-affinity uptake system at high sulfate concentrations. The low half-saturation constants measured for sulfate uptake explain why high rates of bacterial sulfate reduction occur in surface sediments of freshwater lakes, and why sulfate reduction can be a quantitatively important process in anaerobic carbon mineralization in low-sulfate environments. The results shows that extremely low sulfate concentrations must occur before sulfate reduction is completely outcompeted by methanogenesis.Abbreviations MPB methane producing bacteria - SRB sulfate reducing bacteria     

Determination of the Amino Acid Sequence of a New Phospholipase A<Subscript>2</Subscript> (MIDCA1) Isolated from <Emphasis Type="Italic">Micrurus dumerilii carinicauda</Emphasis> Venom

A new Phospholipase A₂ (PLA₂) from Micrurus dumerilii carinicauda venom was isolated and its primary structure determined. This new PLA₂ showed a low enzymatic activity when compared with other PLA₂s and it is moderately basic with an isoelectric point of 8.0. Its amino acid sequence showed the presence of 120 amino acid residues and its sequence was: NLIQFLNMIQCTTPGREPLVAFANYGCYCGRGGSGTPVDELDRCCQVHDNCYDTAKKVFGCSPYFTMYSYDCSEGKLTCKDNNTKCKAAVCNCDRTAALCFAKAPYNDKNYKIDLTKRCQ. The structural model of MIDCA1, when compared with other strong neurotoxic PLA₂s, such as Naja naja, showed significant differences in the β-wing and neurotoxic sites, despite the high level of amino acid sequence similarity. These observations indicate a dissociation between the biological and catalytic activity of this new PLA₂, supporting the view that other regions of the protein are involved in the biological effects.