RESUMO
Different peptides have been isolated from a wide range of animal species. It is has become increasingly clear that due to the development of antibiotic-resistant microbes, antibacterial and antifungal peptides have attracted the attention in recent years, in order to find new therapeutic agents. In this work, a novel peptide with high inhibitory activity against fungi growth have been isolated from the venom of the Brazilian snake Bothrops jararaca. A Sephacryl S-100 gel filtration column was employed for further separation of proteins. The FV fraction with high antifungal activity was named Pep5Bj, pooled and submitted to reverse-phase chromatography in HPLC. The fraction containing the isolated peptide inhibited the growth of different phytopathogenic fungi (Fusarium oxysporum and Colletotrichum lindemuthianum) and yeast (Candida albicans and Saccharomyces cerevisiae). The peptide minimal inhibitory concentration is comparable to other known antifungal peptides, like insect defensins and cecropins, found in the last years in a large diversity of animals. We investigate F. oxysporum cells membrane permeabilization using SYTOX Green uptake, an organic compound that fluoresces upon interaction with nucleic acids after penetration in cell with compromised plasma membranes. When viewed under fluorescence optical microscopy, F. oxysporum cells exposed to Pep5Bj display strong SYTOX Green fluorescence in the cytosol, especially in the nuclei. The SYTOX Green data suggested that this effect is related to membrane permeabilization. The molecular masses of this peptide was obtained by MALDI-TOF spectrometry and corresponded to 1370Da.
Assuntos
Antifúngicos/farmacologia , Bothrops , Venenos de Crotalídeos/química , Fungos/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Antifúngicos/química , Antifúngicos/isolamento & purificação , Venenos de Crotalídeos/farmacologia , Corantes Fluorescentes/metabolismo , Fungos/crescimento & desenvolvimento , Glucose/metabolismo , Compostos Orgânicos , Peptídeos/química , Peptídeos/isolamento & purificaçãoRESUMO
Canatoxin is a toxic protein from Canavalia ensiformis seeds, lethal to mice (LD(50)=2 mg/kg) and insects. Further characterization of canatoxin showed that its main native form (184 kDa) is a non-covalently linked dimer of a 95 kDa polypeptide containing zinc and nickel. Partial sequencing of internal peptides indicated homology with urease (EC 3.5.1.5) from the same seed. Canatoxin has approx. 30% of urease's activity for urea, and K(m) of 2-7 mM. The proteins differ in their affinities for metal ions and were separated by affinity chromatography on a Zn(2+) matrix. Similar to canatoxin, urease activates blood platelets and interacts with glycoconjugates. In contrast with canatoxin, no lethality was seen in mice injected with urease (10 mg/kg). Pretreatment with p-hydroxymercuribenzoate irreversibly abolished the ureolytic activity of both proteins. On the other hand, p-hydroxymercuribenzoate-treated canatoxin was still lethal to mice, and both treated proteins were fully active in promoting platelet aggregation and binding to glycoconjugates. Taken together, our data indicate that canatoxin is a variant form of urease. Moreover, we show for the first time that these proteins display several biological effects that are unrelated to their enzymic activity for urea.
Assuntos
Lectinas/química , Lectinas/metabolismo , Phaseolus/enzimologia , Proteínas de Plantas , Toxinas Biológicas , Urease/química , Urease/fisiologia , Sequência de Aminoácidos , Animais , Plaquetas/enzimologia , Cromatografia em Gel , Dimerização , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Hemaglutininas/metabolismo , Hidroximercuribenzoatos/farmacologia , Cinética , Camundongos , Dados de Sequência Molecular , Peso Molecular , Lectinas de Plantas , Ligação Proteica , Coelhos , Homologia de Sequência de Aminoácidos , Ureia/metabolismo , Urease/metabolismo , Zinco/metabolismoRESUMO
This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical Km on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80 percent homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii
Assuntos
Animais , Coagulação Sanguínea/efeitos dos fármacos , Bothrops , Venenos de Crotalídeos/enzimologia , Fibrinogênio/química , Trombina/isolamento & purificação , Sequência de Aminoácidos , Western Blotting , Venenos de Crotalídeos/farmacologia , Eletroforese em Gel de Poliacrilamida , Testes de Precipitina , Trombina/químicaRESUMO
This article describes the presence of two new forms of a thrombin-like enzyme, both with apparent molecular masses of 38 kDa, in Bothrops atrox venom. Both share the ability to cleave fibrinogen into fibrin and to digest casein. Both present identical K(m) on the substrate BApNA. Their N-terminal amino acid sequences are identical for 26 residues, sharing 80% homology with batroxobin and flavoxobin. Two groups of monoclonal antibodies (mAbs) raised against the purified enzyme forms recognized different epitopes of the putative corresponding enzymes present in B. atrox crude venom. On Western blotting analysis of B. atrox crude venom, mAbs 5DB2C8, 5AA10 and 5CF11, but not mAbs 6CC5 and 6AD2-G5, revealed two or more protein bands ranging from 25 to 38 kDa. By immunoprecipitation assays, the 6AD2-G5 mAb was able to precipitate protein bands of 36-38 kDa from B. atrox, B. leucurus, B. pradoi, B. moojeni, B. jararaca and B. neuwiedii crude venoms. Fibrinogen-clotting activity was inhibited when the same venom specimens were pre-incubated with mAb 6AD2-G5, except for B. jararaca and B. neuwiedii.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Bothrops , Venenos de Crotalídeos/enzimologia , Fibrinogênio/química , Trombina/isolamento & purificação , Animais , Western Blotting , Venenos de Crotalídeos/farmacologia , Eletroforese em Gel de Poliacrilamida , Isoenzimas/isolamento & purificação , Isoenzimas/farmacologia , Testes de Precipitina , Trombina/químicaRESUMO
Viperine and crotaline snake venoms contain one or more hemorrhagic metalloproteases called hemorrhagins. The most potent hemorrhagins belong to the P-III class and have, in addition to the protease domain, disintegrin-like and cysteine-rich domains. Although proteolytic degradation of vascular endothelium basement membrane has been established to be the main factor responsible for hemorrhage, several studies reveal other factors that actually do facilitate this process. Recent evidence has shown that the nonprotease domains of the P-III class hemorrhagins are able to inhibit the platelet aggregation by blocking essential procoagulant integrins on platelets. In this study we report the identification of a hemorrhagin from Bothrops atrox venom. This enzyme, a P-III class metalloprotease, undergoes an apparent spontaneous degradation, releasing a proteic fragment containing the disintegrin-like/cysteine-rich domains. This fragment shows the capability to induce an edematogenic process, suggesting the existence of a still unknown nonenzymatic mechanism of vascular permeability increase.
Assuntos
Venenos de Crotalídeos , Desintegrinas/toxicidade , Edema/induzido quimicamente , Endopeptidases/toxicidade , Inibidores da Agregação Plaquetária/toxicidade , Sequência de Aminoácidos , Animais , Bothrops , Cisteína , Endopeptidases/química , Endopeptidases/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
A protein purification procedure using sonication extraction from polyacrylamide gels (PAGE), which involves identification of a particular protein band and its excision, homogenization, sonication, and subsequent passage through a Sephadex G-25 minicolumn, is reported. Our results show a high degree of recovery regardless of the nature of the protein (soluble or membrane bound) or the characteristics of the gel (SDS-PAGE, native gels, or Tricine-SDS-PAGE). The percentage of recovery was dependent on the protein concentration applied in the gel. This technique is fast, gives high yield, provides good resolution, and can be used without any specialized equipment. The method was tested with a wide variety of membranes and soluble proteins and was found to give good results and to be applicable to different studies. The amino acid sequence of one of the purified proteins (Rf 0.45 stallion ejaculated sperm protein) was determined successfully.
Assuntos
Eletroforese em Gel de Poliacrilamida/métodos , Proteínas/isolamento & purificação , Sonicação , Animais , Cavalos , Masculino , Proteínas de Membrana/isolamento & purificação , Espermatozoides/químicaRESUMO
The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement (C) dependent haemolysis. The aim of this study was to characterise the toxins in the venom responsible for the different biological effects. We have previously shown that a 35 kDa protein, named F35, purified from Loxosceles intermedia venom, incorporates into the membranes of human erythrocytes and renders them susceptible to the alternative pathway of autologous C. Here we have further purified the F35 protein which was resolved by reversed phase chromatography into three tightly contiguous peaks termed P1, P2, and P3. P1 and P2 were shown to be homogeneous by SDS-PAGE and N-terminal aminoacid analysis, while P3 consisted of two highly homologous proteins. N-terminal sequencing of all four proteins showed a high degree of homology, which was confirmed by cross-reactivity of antisera raised against the individual purified proteins. Functional characterisation of P1 and P2 indicated the presence of sphingomyelinase activity and either protein in isolation was capable of inducing all the in vivo effects seen with whole spider venom, including C-dependent haemolysis and dermonecrosis. In all assays, P2 was more active than P1, while P3 was completely inactive. These data show that different biological effects of L. intermedia venom can be assigned to the sphingomyelinase activity of two highly homologous proteins, P1 and P2. Identification of these proteins as inducers of the principal pathological effects induced by whole venom will aid studies of the mechanism of action of the venom and the development of a effective therapy.
Assuntos
Dermotoxinas/farmacologia , Hemólise/efeitos dos fármacos , Esfingomielina Fosfodiesterase/farmacologia , Venenos de Aranha/farmacologia , Sequência de Aminoácidos , Animais , Ensaio de Atividade Hemolítica de Complemento , Reações Cruzadas , Dermotoxinas/sangue , Ensaio de Imunoadsorção Enzimática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Necrose , Fragmentos de Peptídeos/isolamento & purificação , Coelhos , Alinhamento de Sequência , Esfingomielina Fosfodiesterase/sangue , Esfingomielina Fosfodiesterase/química , Venenos de Aranha/sangue , Venenos de Aranha/enzimologiaRESUMO
An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS-PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S]methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3.5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.
Assuntos
Ácido Aspártico Endopeptidases/isolamento & purificação , Precursores Enzimáticos/isolamento & purificação , Carrapatos/enzimologia , Tecido Adiposo/enzimologia , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/química , Western Blotting , Cromatografia DEAE-Celulose , Ovos , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/química , Feminino , Hemoglobinas/metabolismo , Hemolinfa/enzimologia , Intestinos/enzimologia , Túbulos de Malpighi/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Coelhos , Carrapatos/crescimento & desenvolvimentoRESUMO
Two peptides with kinin-like biological properties were isolated by chromatography on a Sephadex G-10 column followed by high-performance liquid chromatography, from the venom of the spider Scaptocosa raptoria. The isolated peptides (peptide-S and peptide-R) were shown to cause contraction on the isolated guinea-pig ileum at amounts equivalent to those shown by bradykinin. Both peptides relaxed the isolated rat duodenum, increased the capillary permeability, caused decreasing and biphasic effect of the arterial blood pressure in conscious rats and induced oedema in the rat paw. The peptides had activity and structural similarities to other peptides (kinin-like) isolated from venoms. The complete amino acid analysis gave peptide-S a structure with 36 amino acid residues and peptide-R 22 amino acid residues. The mol. wts were estimated to be in the range of 4000 and 2870, respectively.
Assuntos
Músculo Liso/efeitos dos fármacos , Venenos de Aranha/isolamento & purificação , Sequência de Aminoácidos , Animais , Permeabilidade Capilar/efeitos dos fármacos , Feminino , Cobaias , Masculino , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Venenos de Aranha/farmacologia , Relação Estrutura-AtividadeRESUMO
Crude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources. LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was strongly inhibited by LM-PLA2 as well as ATP-release. Treatment of the protein with p-bromophenacyl bromide or 2-mercaptoethanol, as well as thermal inactivation studies, suggested that the platelet inhibitory effect of LM-PLA2 is dependent on its enzymatic activity. Thus, the platelet inhibitory activity of LM-PLA2 was shown to be dependent on the hydrolysis of plasma phospholipids and/or lipoproteins, most probably those rich in phosphatidylcholine. Surprisingly, lysophosphatidylcholine released by LM-PLA2 from plasma was shown to preferentially inhibited collagen-induced platelet aggregation, in contrast to other PLA2s, whose plasma hydrolytic products indistinctly affect platelet's response to several agonists.