RESUMO
We report on the first studies on the characterization of venom from Phoneutria boliviensis (Aranae:Ctenidae) (F. O. Pickard-Cambridge, 1897), done with Colombian species. After the electrostimulation extraction process, the venom showed physicochemical properties corresponding to a colorless and water-soluble liquid with a density of 0.86 mg/mL and 87% aqueous content. P. boliviensis venom and RP-HPLC fractions showed hemolytic activity and hydrolyzed the synthetic substrate 4-nitro-3-octanoyloxy-benzoic acid, indicating the presence of phospholipases A2 enzymes. The electrophoretic profile showed an important protein content with molecular masses below 14 kDa, and differences between male and female protein content were also revealed. The RP-HPLC venom profile exposes differences between males and female content consistent with the electrophoretic profile. Five fractions collected from the RP-HPLC displayed significant larvicidal activity. Mass analysis indicates the presence of peptides ranging from 1047.71 to 3278.07 Da. Two peptides, Ctenitoxin-Pb48 and Ctenitoxin-Pb53, were partially identified using HPLC-nESI-MS/MS, which showed a high homology with other Ctenitoxins (family Tx3) from Phoneutria nigriventer, Phoneutria keyserlingi and Phoneutria reidyi affecting voltage-gated calcium receptors (Cav 1, 2.1, 2.2 and 2.3) and NMDA-glutamate receptors.
Assuntos
Venenos de Aranha/química , Venenos de Aranha/farmacologia , Aedes/efeitos dos fármacos , Animais , Benzoilarginina Nitroanilida/metabolismo , Cromatografia Líquida de Alta Pressão , Feminino , Hemólise , Larva/efeitos dos fármacos , Masculino , Peptídeos/análise , Fosfolipases A2/metabolismo , Proteólise , Espectrometria de Massas por Ionização por Electrospray , Aranhas , Espectrometria de Massas em TandemRESUMO
Serine proteinases from three strains of Sitophilus zeamais (Coleoptera: Curculionidae), one susceptible and two resistant to insecticides--one exhibiting fitness cost (resistant cost strain) and the other lacking it (resistant no-cost strain), were partially purified using an aprotinin-agarose affinity column providing purification factors ranging from 36.5 to 51.2%, with yields between 10 and 15% and activity between 529 and 875 microM/min/mg protein with the substrate N-alpha-benzoyl-L-Arg-p-nitroanilide (L-BApNA). SDS-PAGE of the purified fraction revealed a 56,000 Da molecular mass band in all strains and a 70,000 Da band more visible in the resistant no-cost strain. The purified proteinases from all strains were inhibited by phenylmethyl sulphonyl fluoride (PMSF), N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), aprotinin, benzamidine and soybean trypsin inhibitor (SBTI) characterizing them as trypsin-like serine proteinases. Trypsin-like proteinases from the resistant strains exhibited higher affinity for L-BApNA. The resistant no-cost strain exhibited V(max)-values 1.5- and 1.7-fold higher than the susceptible and resistance cost strains, respectively. A similar trend was also observed when using N-alpha-p-tosyl-L-Arg methyl ester (L-TAME) as substrate. These results provide support to the hypothesis that the enhanced serine proteinase activity may be playing a role in mitigating physiological costs associated with the maintenance of insecticide resistance mechanisms in some maize weevil strains.
Assuntos
Besouros/enzimologia , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Inseticidas/toxicidade , Serina Proteases/metabolismo , Zea mays/parasitologia , Animais , Benzoilarginina Nitroanilida/metabolismo , Catálise/efeitos dos fármacos , Besouros/classificação , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Proteínas de Insetos/isolamento & purificação , Cinética , Fluoreto de Fenilmetilsulfonil/farmacologia , Inibidores de Proteases/farmacologia , Serina Proteases/isolamento & purificação , Especificidade da Espécie , Especificidade por Substrato , Temperatura , Tripsina/isolamento & purificação , Tripsina/metabolismoRESUMO
Proteolytic activities isolated from the marine demosponges Geodia cydonium and Suberites domuncula were analyzed by 2-D zymography, a technique that combines IEF and zymography. After purification, a 200 kDa proteolytically active protein band was obtained from G. cydonium when analyzed in gelatin copolymerized 1-D zymograms. The enzymatic activity was quantified using alpha-N-benzoyl-D-arginine p-nitroanilide (BAPNA) as a substrate and corresponded to a serine protease. The protease activity was resistant to urea and SDS. DTT and 2-mercaptoethanol (2-ME) did not significantly change the protease activity, but induced a shift in molecular mass of the proteolytic band to lower M(r) values as detected by zymography. Under mild denaturing conditions, lower M(r) bands (<200 kDa) were identified in 1-D zymograms, suggesting that the protease is composed of subunits which retain the catalytic activity. After 2-D zymography, the protease from G. cydonium revealed a pI of 8.0 and an M(r) shift from 200 to 66 kDa. To contrast these results, a cytosolic sample from S. domuncula was analyzed. The proteolytic activity of this sponge after 2-D zymography corresponded to an M(r) of 40 kDa and a pI of 4.0. The biological function of both sponge proteases is not yet known. This study demonstrates that mild denaturing conditions required for IEF may alter the interpretation of the 2-D zymography, and care must be taken during sample preparation.
Assuntos
Poríferos/enzimologia , Serina Endopeptidases/metabolismo , Animais , Benzoilarginina Nitroanilida/metabolismo , Ditiotreitol/química , Eletroforese em Gel Bidimensional/métodos , Ativação Enzimática , Mercaptoetanol/química , Peso Molecular , Serina Endopeptidases/análise , Dodecilsulfato de Sódio/química , Especificidade por Substrato , Ureia/químicaRESUMO
Podisus nigrispinus (Dallas) is a common predator in agricultural and natural systems in Neotropical America. Its feeding strategy involves extra-oral digestion and to better understand this process its salivary glands were extracted and subjected to morphological and preliminary enzyme characterization. The salivary glands of P. nigrispinus are formed by a pair of main and accessory gland complexes. The main salivary glands are further divided into an anterior and a posterior lobe. The compartmentalization of the salivary gland complex is likely to be important for the production, activation and release of the digestive enzymes used in the extra-oral digestion of prey items. Proteases and lipase, important digestive enzymes involved in zoophagy, were detected in the salivary glands of P. nigrispinus. The prevailing trypsin-like protease activity was characterized by using the serine-protease substrate N-alpha-benzoyl-L-Arg-p-nitroanilidine (L-BApNA) and the trypsin inhibitors tosyl-L-lysine chloromethyl ketone (TLCK) and benzamidine. The KM value obtained for trypsin-like activity was 1.57 mm and the different peaks of optimum pH and temperature activity suggest the presence of multiple forms of this enzyme in P. nigrispinus. Detection of amylase activity in the salivary glands of this predator suggests its ability to digest starch and obtain nutrients from plants, which may have adaptative value under prey scarcity.
Assuntos
Heterópteros/enzimologia , Heterópteros/ultraestrutura , Amilases/análise , Amilases/metabolismo , Animais , Benzamidinas/farmacologia , Benzoilarginina Nitroanilida/metabolismo , Digestão/fisiologia , Feminino , Heterópteros/fisiologia , Concentração de Íons de Hidrogênio , Hidrólise , Lipase/análise , Lipase/metabolismo , Masculino , Microscopia Eletrônica de Varredura/veterinária , Glândulas Salivares/enzimologia , Glândulas Salivares/ultraestrutura , Serina Endopeptidases/análise , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Temperatura , Tosilina Clorometil Cetona/farmacologiaRESUMO
At present the physiological role of most oviductal proteins remains unknown. In this work, we present evidence that the oviductal secretion as well as the crude oviductal tissue-extract show proteolytic-like esterase and amidase activity. The proteolytic activity of the oviductal enzymes was higher in the oviducts of superovulated hamster females than in those of normal ones, indicating that gonadotrophic hormones would stimulate the synthesis and secretion of these enzymes. Some of their properties were analyzed in the 15,600-g supernatant of both oviductal tissue extracts (OE) and oviductal fluid (OF). The enzymatic activity toward the synthetic substrates p-tosyl-l-arginine methyl ester-HCl (TAME) and alpha-N-benzoyl-dl-arginine-p-nitroanilide HCl (BAPNA) was activated by calcium ions, reached a maximum at pH 7.5, and was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-l-lysine chloromethyl ketone HCl (TLCK), phenyl methyl sulfonyl fluoride (PMSF), and benzamidine. The OE glycoprotein fraction recognized by WGA-Sepharose affinity columns (37% total proteins) showed proteolytic activity with properties similar to the OE and OF enzymes. The protease activity could be ascribed to a plasminogen activator (PA) detected in the Triton X-100 treated tissue crude membrane fraction (Triton-CMF) and in the oviductal secretion of the superovulated females. In the Triton-CMF fraction, 100% of the proteolytic activity was plasminogen-dependent. The use of amiloride, a selective urokinase-type plasminogen activator (uPA) inhibitor, shows that 90% of this activity was due to a tissue-type plasminogen activator (tPA) and 10% to uPA whereas in the uterus 100% of the activity was tPA. Only a small percentage of the OF proteolytic activity was plasminogen-dependent, probably due to the presence of PA inhibitors in this medium.
Assuntos
Tubas Uterinas/enzimologia , Mesocricetus/metabolismo , Ativadores de Plasminogênio/metabolismo , Amidoidrolases/metabolismo , Animais , Benzoilarginina Nitroanilida/metabolismo , Cálcio/farmacologia , Cloreto de Cálcio/farmacologia , Compostos Cromogênicos/farmacologia , Cricetinae , Relação Dose-Resposta a Droga , Epitélio/efeitos dos fármacos , Epitélio/enzimologia , Esterases/metabolismo , Tubas Uterinas/efeitos dos fármacos , Feminino , Concentração de Íons de Hidrogênio , Inibidores de Serina Proteinase/farmacologia , Tosilarginina Metil Éster/metabolismo , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
We have measured sperm-bound amidase activity in fresh, cooled and frozen/thawed ram spermatozoa, in order to study if freezing and thawing led to some degree of acrosome damage of motile/viable spermatozoa not detected by optical methods. This assay was based on the fact that membrane damage would result in an increased access of the enzyme substrate to the sperm acrosome. Semen was collected from adult Australian Merino rams, and spermatozoa were washed by centrifugation through a Ficoll solution. Sperm-bound amidase activity was measured in whole spermatozoa using the protease substrate benzoyl-arginyl-p-nitroanilide (BAPNA). Acrosomal status was also assessed by light microscopy after Giemsa staining. Most amidase activity was shown to be sperm-bound, as only a minor fraction of the enzyme activity was release into the medium after induced damage. Simultaneous assessment of sperm-bound amidase activity and the percentage of spermatozoa with microscopically evident acrosomal damage, after mild sonication for different times, showed a high correlation between both parameters (r = 0.97, p < 0.001). In separate experiments, fresh, cooled and frozen/thawed semen samples were filtered through Sephadex G-10 to obtain a subpopulation of motile, mostly acrosome-intact spermatozoa. As controls, spermatozoa from the same samples to which extensive acrosome damage was induced were evaluated. Slow cooling to 4 degrees C had no effect on amidase activity or percent acrosomal damage with respect to fresh samples. Freezing and thawing resulted in a sperm population that, after filtration through Sephadex, had a low percentage of acrosome damage (9.4%, vs. 2.1% for fresh filtered controls), which was 11% of that obtained after extensive acrosome damage (83%). However, amidase activity in these samples was markedly increased, showing values of activity that were 56% of those obtained in extensively damaged spermatozoa. This effect was not due to an alteration in the enzyme kinetics. We conclude that sperm-bound amidase activity is useful to detect subtle changes, provoked by a standard freezing/thawing procedure, in the permeability of acrosomes from ram spermatozoa which are not detected by direct observation of the acrosomes after Giemsa staining.