RESUMO
In this study, fibrinolytic protease was isolated and purified from Perinereis aibuhitensis Grub, and the extraction process was optimized. The properties of the enzyme, such as the amino acid composition, thermal stability, optimal temperature, and pH, were investigated. After detoxification, proteins collected from fresh Clamworm (Perinereis aibuhitensis Grub) were concentrated via ammonium sulfate precipitation. The crude protease was purified using gel filtration resin (Sephadex G-100), anion exchange resin (DEAE-Sepharose FF), and hydrophobic resin (Phenyl Sepharose 6FF). The molecular weight of the protease was determined by polyacrylamide gel electrophoresis (SDS-PAGE). The optimum temperature and optimum pH of the protease were determined. The activity of crude protease in the 40-60% salt-out section was the highest, reaching 467.53 U/mg. The optimal process for purifying crude protein involved the application of DEAE-Sepharose FF and Phenyl Sepharose 6FF, which resulted in the isolation of a single protease known as Asp60-D1-P1 with the highest fibrinolytic activity; additionally, the enzyme activity was measured at 3367.76 U/mg. Analysis by Native-PAGE and SDS-PAGE revealed that the molecular weight of Asp60-D1-P1 was 44.5 kDa, which consisted of two subunits with molecular weights of 6.5 and 37.8 kDa, respectively. The optimum temperature for Asp60-D1-P1 was 40°C, and the optimal pH was 8.0.
Assuntos
Fibrinolisina , Animais , Concentração de Íons de Hidrogênio , Fibrinolisina/metabolismo , Fibrinolisina/isolamento & purificação , Poliquetos/enzimologia , Temperatura , Peso Molecular , Estabilidade Enzimática , Metais/farmacologia , Eletroforese em Gel de Poliacrilamida , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Fibrinolíticos/metabolismoRESUMO
Acetylsalicylic acid (ASA) intoxication is potentially lethal. After ingestion, AAS is rapidly transformed into salicylic acid that dissociates into an hydrogen ion plus salicylate. Salicylate is the main form of AAS in the body and produces multiple alterations. Initially, the stimulation of the ventilatory center promotes a respiratory alkalosis. Then, the mitochondrial dysfunction induced by salicylate, will generate a progressive metabolic acidosis due to the accumulation of ketoacids, lactic acid and dicarboxylic acids among others. Another alterations include hydro electrolytic disorders, gastrointestinal lesions, neurological involvement, ototoxicity and coagulopathy. The correct handling of acetylsalicylic acid intoxication requires an thorough knowledge of its pharmacokinetics and pharmacodynamics. Treatment consists in life support measures, gastric lavage, activated charcoal and urinary alkalization to promote the excretion of salicylates. In some occasions, it will be necessary to start renal replacement therapy as soon as possible.
Assuntos
Humanos , Aspirina/intoxicação , Aspirina/metabolismo , Fibrinolíticos/intoxicação , Fibrinolíticos/metabolismo , Overdose de Drogas/fisiopatologia , Overdose de Drogas/terapia , Acidose/induzido quimicamente , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Aspirina/administração & dosagem , Overdose de Drogas/metabolismo , Hipoglicemia/induzido quimicamente , Hipotensão/induzido quimicamente , Mitocôndrias/efeitos dos fármacosRESUMO
Background Streptomyces sp. DPUA 1576 from Amazon lichens was studied to protease and fibrinolytic production. A 2² factorial experimental design was applied to optimize its protease enzyme production using two independent variables, namely soybean flour and glucose concentrations. Results The optimal conditions to obtain high protease production (83.42 U/mL) were 1.26% soybean flour and 1.23% glucose concentration. A polynomial model was fitted to correlate the relationship between the two variables and protease activity. In relation to fibrinolytic activity, the highest activity of 706.5 mm² was obtained at 1.7% soybean flour and 1.0% glucose concentration, which was 33% higher than plasmin. Fibrinolytic production was not optimized in the studied conditions. Conclusions These results show that the optimization of the culture medium can enhance protease production, thus becoming a good process for further research. In addition, Streptomyces sp. DPUA 1576, isolated from Amazon lichens, might be a potential strain for fibrinolytic protease production.
Assuntos
Peptídeo Hidrolases/biossíntese , Streptomyces/enzimologia , Fibrinolíticos/metabolismo , Glycine max , Modelos Estatísticos , Actinobacteria , Farinha , Glucose/análise , LíquensRESUMO
The anticlotting and antithrombotic activities of heparin, heparan sulfate, low molecular weight heparins, heparin and heparin-like compounds from various sources used in clinical practice or under development are briefly reviewed. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate from Artemia franciscana and a dermatan sulfate from tuna fish show a potent heparin cofactor II activity. A heparan sulfate derived from bovine pancreas has a potent antithrombotic activity in an arterial and venous thrombosis model with a negligible activity upon the serine proteases of the coagulation cascade. It is suggested that the antithrombotic activity of heparin and other antithrombotic agents is due at least in part to their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate.
Assuntos
Humanos , Animais , Bovinos , Anticoagulantes/farmacologia , Endotélio Vascular/citologia , Fibrinolíticos/farmacologia , Heparina/farmacologia , Heparitina Sulfato/farmacologia , Anticoagulantes/química , Anticoagulantes/metabolismo , Crustáceos , Fibrinolíticos/química , Fibrinolíticos/metabolismo , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/farmacologia , Heparina de Baixo Peso Molecular/química , Heparina de Baixo Peso Molecular/metabolismo , Heparina de Baixo Peso Molecular/farmacologia , Heparina/metabolismo , Heparitina Sulfato/biossíntese , AtumRESUMO
The interaction of plasminogen, tissue plasminogen activator (t-PA) and urokinase with a clinical strain of Helicobacter pylori was studied. Plasminogen bound to the surface of H. pylori cells in a concentration-dependent manner and could be activated to the enzymatic form, plasmin, by t-PA. Affinity chromatography assays revealed a plasminogen-binding protein of 58.9 kDa in water extracts of surface proteins. Surface-associated plasmin activity, detected with the chromogenic substrate CBS 00.65, was observed only when plasminogen and an exogenous activator were added to the cell suspension. The two physiologic plasminogen activators, t-PA and urokinase, were also shown to bind to and remain active on the surface of bacterial cells. epsilon-Aminocaproic acid caused partial inhibition of t-PA binding, suggesting that the kringle 2 structure of this activator is involved in the interaction with surface receptors. The activation of plasminogen by t-PA, but not urokinase, strongly depended on the presence of cells and a 25-fold enhancer effect on the initial velocity of activation by t-PA compared to urokinase was established. Furthermore, a relationship between cell concentration and the initial velocity of activation was demonstrated. These findings support the concept that plasminogen activation by t-PA on the bacterial surface is a surface-dependent reaction which offers catalytic advantages
Assuntos
Humanos , Fibrinolíticos/metabolismo , Helicobacter pylori/metabolismo , Ativadores de Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Aminocaproatos/metabolismo , Cromatografia , Eletroforese em Gel de Poliacrilamida , Helicobacter pylori/isolamento & purificação , Indicadores e Reagentes , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The effect of several ions (Cl-, Na+, K+, Ca2+) on the rate of plasminogen (Pg) activation by recombinant staphylokinase (rSTA) is reported. Both monovalent and divalent ions affect the rate at which Pg is activated by rSTA, in a concentration-dependent manner (range 0-100 mM). In almost all cases, a decrease of the initial velocity of activation was observed. Cl- showed the most striking inhibitory effect at low concentrations (64 percent at 10 mM). However, in the presence of a fibrin surface, this inhibition was attenuated to 38 percent. Surprisingly, 10 mM Ca2+ enhanced the Pg activation rate 21 percent when a polymerized fibrin matrix was present. These data support the idea that ions can modulate the rate of Pg activation through a mechanism that may be associated with changes in the molecular conformation of the zymogen. This effect is strongly dependent on the presence of a fibrin clot