Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotes vasodilatation mediated by both bradykinin B2and M1 muscarinic acetylcholine receptors.

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (

Bj-PRO-5a, a natural angiotensin-converting enzyme inhibitor, promotesvasodilatation mediated by both bradykinin B2 and M1 muscarinicacetylcholine receptors

Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venomglands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme(ACE) described. Bj-PRO-5a (

Analysis of the ontogenetic variation in the venom proteome/peptidome of bothrops jararaca reveals different strategies to deal with prey

Previous studies have demonstrated that the pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Variation in the venom proteome is a well-documented phenomenon; however, variation in the venom peptidome is poorly understood. We report a comparative proteomic and peptidomic analysis of venoms from newborn and adult specimens of B. jararaca and correlate it with the evaluation of important venom features. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. The coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of SDS-PAGE, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and concanavalin A-binding proteins. Furthermore, we report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles and were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species.

Bothrops protease A, a unique highly glycosylated serine proteinase, is a potent, specific fibrinogenolytic agent.

BACKGROUND: The hemostatic system is the major target of snake venom serine proteinases (SVSPs) that act on substrates of the coagulation, fibrinolytic and kallikrein-kinin systems. Bothrops protease A (BPA), the most glycosylated SVSP, is a non-coagulant, thermostable enzyme. A cDNA encoding BPA showed that the protein has a calculated molecular mass of 25 409 Da, implying that approximately 62% of its molecular mass as assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis (67 kDa) is due to carbohydrate moieties. RESULTS: Here we show that BPA is a potent fibrinogenolytic agent in vitro, as it readily degraded human and rat fibrinogen at a very low enzyme concentration. Partially N-deglycosylated BPA (p-N-d-BPA) generated similar fibrinogen products, but with enhanced fibrinogenolytic activity. In vivo, injection of 0.75 nmoles of BPA in rats completely avoided thrombus formation induced by stasis in the vena cava, or by endothelium injury in the jugular vein. Moreover, it decreased the fibrinogen plasma level and prolonged the recalcification time. Cleavage of fibrinogen in human and rat plasma was observed with native BPA and p-N-d-BPA by electrophoresis followed by western blot using an anti-fibrinogen antibody. BPA did not cause unspecific degradation of plasma proteins and did not cleave isolated albumin, vitronectin and fibronectin at the same concentration used with fibrinogen. Serine proteinase inhibitors failed to inhibit BPA, probably due to steric hindrance caused by its huge carbohydrate moieties. CONCLUSIONS: To the best of our knowledge, this investigation underscores a new, thermostable, specific defibrinogenating agent that may have an application in the prevention of thrombus formation.

A new fluorometric assay for neutral endopeptidase (EC 3.4.24.11)

An intramolecularly quenched fluorogenic peptide structurally related to Leu-enkephalin, Abz-GGdFLRRV-EDDnp, was selectively hydrolyzed at the R-V bond by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) with kinetic parameters (Km = 3 µM,Kcat = 127 / min and Kcat / Km = 42 / min µM) similar to those of Leu-enkephalin. The specificity of the assay for NEP was demostrated by incubating Abz-GGdFLRRV-EDDnp with a kidney homogenate and with crude membrane preparations of brain and lung. For all three homogenates the complementary fragments Abz-GGdFLRRnp accounted for more than 95 percent of the products wich were totally inhibited by 1 µM thiorphan, a highly specific NEP inhibitor. A continuous fluorometric assay for only 5 min was sufficient to quantify the NEP activity with a minimum sensitivity of 5 ng of purified NEP or the equivalent enzymatic activity in crude tissue preparations.