Lauro Morhy

Ontogenetic variation of metalloproteinases and plasma coagulant activity in venoms of wild Bothrops atrox specimens from Amazonian rain forest.

A comparative study of venoms from juvenile, sub-adult and adult wild Bothrops atrox specimens captured in Manaus region (Brazil) was performed. All venoms tested had acidic pH (5.5) and the human plasma coagulant activity was higher in venoms from juvenile and sub-adult specimens than in adults. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the most intense bands in adult venoms corresponded to polypeptides of 23 and 50kDa. The 23kDa protein was not detected in juvenile venoms. The 23 and 50kDa proteins were purified by two steps of reversed phase-HPLC followed by size exclusion HPLC. Partial amino acid sequence of the 23kDa protein showed homology to metalloproteinases from other snake venoms. Electrospray ionization mass spectrometric analysis (ESI-MS) showed that the 23kDa band contained at least three isoforms of 23030, 23300 and 23645Da. The 50kDa polypeptide was N-terminally blocked for Edman degradation and presented molecular masses ranging from 46.8 to 49.4kDa by ESI-MS. Both proteins were detected by anti-mutalysin II antibodies in immunoblotting assay indicating that they belong to the metalloproteinase family. Immunoblotting analysis also showed that the 23kDa band increased in intensity from juvenile to adult specimens.SDS-PAGE analysis of juvenile and adult venoms following autoproteolysis in pH 7.4 suggested that endogenous venom metalloproteinases can digest the 50kDa metalloproteinase, originating a new protein band of 27kDa. It was also demonstrated in juvenile venoms that the 23kDa band was not the result of proteolytic processing of the 50kDa metalloproteinase.

Compared chemical properties of dermonecrotic and lethal toxins from spiders of the genus Loxosceles (Araneae)

Loxosceles spider venom usually causes a typical dermonecrotic lesion in bitten patients, but it may also cause systemic effects that may be lethal. Gel filtration on Sephadex G-100 ofLoxosceles gaucho, L. laeta, orL. intermedia spider venoms resulted in three fractions (A, containing higher molecular mass components, B containing intermediate molecular mass components, and C with lower molecular mass components). The dermonecrotic and lethal activities were detected exclusively in fraction A of all three species. Analysis by SDS-PAGE showed that the major protein contained in fraction A has molecular weight approximately 35 kDa inL. gaucho andL. intermedia, but 32 kDa inL. laeta venom. These toxins were isolated from venoms ofL. gaucho, L. laeta, andL. intermedia by SDS-PAGE followed by blotting to PVDF membrane and sequencing. A database search showed a high level of identity between each toxin and a fragment of theL. reclusa (North American spider) toxin. A multiple sequence alignment of theLoxosceles toxins showed many common identical residues in their N-terminal sequences. Identities ranged from 50.0% (L. gaucho andL. reclusa) to 61.1% (L. intermedia andL. reclusa). The purified toxins were also submitted to capillary electrophoresis peptide mapping afterin situ partial hydrolysis of the blotted samples. The results obtained suggest thatL. intermedia protein is more similar toL. laeta toxin thanL. gaucho toxin and revealed a smaller homology betweenL. intermedia andL gaucho. Altogether these findings suggest that the toxins responsible for most important activities of venoms ofLoxosceles species have a molecular mass of 32–35 kDa and are probably homologous proteins.

HOMOLOGY BETWEEN THE SEED CYTOLYSIN ENTEROLOBIN AND BACTERIAL AEROLYSINS

Enterolobin, a 55-kDa cytolytic, inflammatory, and insecticidal protein isolated from seeds of the Brazilian treeEnterolobium contortisiliquum (Leguminosae-Mimosoideae) has been further purified and partially sequenced by using both manual and automated methods. A computational search of enterolobin partial amino acid sequence against the PIR database revealed possible sequence similarities with aerolysins, cytolytic proteins fromAeromonas species. An alignment of enterolobin partial sequence to the amino acid sequences ofA. hydrophila andA. sobria aerolysins showed several similar regions with many residue identites. The seed protein enterolobin and the bacterial aerolysins may be homologous proteins despite the distant phylogenetic relationship.

TEMPERATURE-DEPENDENT GERMINATION AND ENDO-β- MANNANASE ACTIVITY IN SESAME SEEDS†

O efeito da temperatura sobre a germinacao e a atividade de endo-b-mananase em sementes de Sesamum indicum foi investigada. A temperatura minima de germinacao (Tmin) esta localizada entre 12,8°C e 13,2°C enquanto a temperatura maxima (Tmax) encontra-se entre 45,5°C e 46°C. As germinabilidades (G%) nao foram significativamente diferentes da viabilidade estimada (88%) entre 18,8°C e 43,2°C. O teste de Mann-Whitney apontou o intervalo de 31,9°C a 35,1°C como sendo a faixa de temperatura otima (Tot). Quando sementes incubadas a temperaturas proximas ou abaixo de Tmin e proximas ou acima de Tmax foram transferidas para 30°C, aquelas submetidas a baixas temperaturas atingiram germinabilidade elevada. Por outro lado, quanto maior a temperatura de pre-incubacao acima de Tmax, menor a germinabilidade alcancada. O principal monossacarideo encontrado na parede celular do endosperma das sementes foi manose. Somente em temperatura supra-otima foi observada elevacao na atividade de endo-b-mananase na regiao micropilar do endosperma anterior a germinacao.

Amino acid sequence of a myotoxic Lys49-phospholipase A2 homologue from the venom of Cerrophidion (Bothrops) godmani

The complete amino acid sequence of myotoxin II (godMT-II), a myotoxic phospholipase A2 (PLA2) homologue from the venom of the Central American crotaline snake Cerrophidion (Bothrops) godmani, was determined by direct protein sequencing methods. GodMT-II is a class II PLA2 showing a Lys instead of Asp at position 49. An additional substitution in the calcium binding loop region (Asn instead of Tyr at position 28) suggests the lack of enzymatic activity observed in this toxin is due to loss of its ability to bind the co-factor Ca2+, since the residues involved in forming the catalytic network of PLA2s (His-48, Tyr-52 and Asp-99) are conserved in godMT-II. This myotoxin shows highest sequence homology with other Lys-49 PLA2 s from Bothrops, Agkistrodon and Trimeresurus species, suggesting that they constitute a conserved family of proteins, yet in contrast presents lower homology with Bothrops asper myotoxin III, a catalytically-active PLA2. The C-terminal region of godMT-II, which is rich in cationic and hydrophobic residues, shares high sequence homology to the corresponding region in the myotoxin II from B. asper, which has been proposed to play an important role in the Ca(2+)-independent membrane damaging activity.

Effects of the cytolytic seed protein enterolobin on coleopteran and lepidopteran insect larvae

Enterolobin, a novel 55 KDa cytolytic and inflammatory protein from Enterolobium contortisiliquum seeds, was tested for its toxic effects on larvae of the coleopteran Callosobruchus maculatus and the lepidopteran Spodoptera littoralis. Bioassays performed with enterolobin incorporated into artificial seeds showed that the phytocytolysin was toxic to larvae of C. maculatus, causing 70% mortality at a concentration of 0.01% (w/w) and 100% mortality at 0.025%. The protein proved to be innocuous to larvae of S. littoralis. In vitro proteolysis studies using larval gut enzymes, analysed on SDS‐PAGE, showed that only S. littoralis proteases could digest enterolobin, suggesting that the insects digestive proteases were able to inactivate the cytolysin before it could exert any toxic effect; C. maculatus proteases, on the other hand, were unable to hydrolyse enterolobin. The mechanism of toxicity of enterolobin did not appear to involve any damage to the microvilli of the epithelial gut cells of C. maculatus as shown by electron microscopy. Some tentative hypotheses are considered in order to explain the toxic mechanism of action of enterolobin towards C. maculatus.

Pro-inflammatory activity of enterolobin: a haemolytic protein purified from seeds of the Brazilian tree Enterolobium contortisiliquum

The pro-inflammatory activity of enterolobin, a haemolytic protein from Enterolobium contortisiliquum seeds, was investigated. In doses ranging from 1 to 20 micrograms/site, enterolobin induced a dose-dependent paw oedema and pleurisy in rats. The effect was apparent after 15 min, peaked at 6 hr and decreased 24 hr after enterolobin was administered. One hour after the intrathoracic injection of enterolobin, the total leukocyte content of the pleural cavity increased significantly, mainly due to mononuclear and neutrophil accumulation. At 24 hr, although the number of mononuclear and neutrophil cells tended to decrease, a great rise in eosinophil counts was noted. Intraperitoneal treatment with the dual lipoxygenase and cyclooxygenase blockers, BW 755c (25 mg/kg) and NDGA (50 mg/kg) or the corticosteroid dexamethasone (0.1 mg/kg) inhibited enterolobin-induced paw oedema by 35, 38 and 47% respectively, whereas indomethacin (2 mg/kg) was inactive. The H1 antagonist, meclizine (25 mg/kg), was also effective against enterolobin oedema while the PAF-antagonists WEB 2086 and PCA 4248 (20 mg/kg) did not modify the reaction. It was concluded that enterolobin is a potent inducer of pleural exudation, cellular infiltration and paw oedema. Furthermore, enterolobin-induced oedema is partially dependent on lipoxygenase metabolites and histamine, while PAF and prostaglandins did not seem to be important in this reaction.

Amino acid sequence of the Phaseolus vulgaris var. "fogo na serra" inhibitor and interactive surface modeling for the enzyme-inhibitor complex.

A trypsin and chymotrypsin inhibitor from seeds ofPhaseolus vulgaris var. “Fogo na Serra” (PFSI) was purified and its complete amino acid sequence was determined using Edman degradation methods. The inhibitor was found to belong to the Bowman-Birk family of enzymatic inhibitors; it has 82 amino acid residues and a 8.985-kDa molecular mass. The PFSI/α-chymotrypsin binary complex has been modeled using the Turkey ovomucoid inhibitor third domain (OMTKY3) bound toα-chymotrypsin [Fujinagaet al. (1987),J. Mol. Biol.,195, 397–418. template. The model allowed identification of the binding surface.