RESUMO
Alpha-glucosidase inhibitors play an important role in Diabetes Mellitus (DM) treatment since they prevent postprandial hyperglycemia. The Glycoside Hydrolase family 13 (GH13) is the major family of enzymes acting on substrates containing α-glucoside linkages, such as maltose and amylose/amylopectin chains in starch. Previously, our group identified glycoconjugate 1H-1,2,3-triazoles (GCTs) inhibiting two GH13 α-glycosidases: yeast maltase (MAL12) and porcine pancreatic amylase (PPA). Here, we combined kinetic studies and computational methods on nine GCTs to characterize their inhibitory mechanism. They all behaved as reversible inhibitors, and kinetic models encompassed noncompetitive and various mechanisms of mixed-type inhibition for both enzymes. Most potent inhibitors displayed Ki values of 30 µM for MAL12 (GPESB16) and 37 µM for PPA (GPESB15). Molecular dynamics and docking simulations indicated that on MAL12, GPESB15 and GPESB16 bind in a cavity adjacent to the active site, while on the PPA, GPESB15 was predicted to bind at the entrance of the catalytic site. Notably, despite its putative location within the active site, the binding of GPESB15 does not obstruct the substrate's access to the cleavage site. Our study contributes to paving the way for developing novel therapeutic strategies for managing DM-2 through GH13 α-glycosidases inhibition.
Assuntos
Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Cinética , Ligantes , Suínos , Inibidores de Glicosídeo Hidrolases/farmacologia , Inibidores de Glicosídeo Hidrolases/química , Animais , Domínio Catalítico , alfa-Glucosidases/metabolismo , alfa-Glucosidases/química , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Triazóis/química , Triazóis/farmacologia , Modelos MolecularesRESUMO
The advanced glycation end products (AGEs) constitute a wide variety of substances synthesized from interactions between amino groups of proteins and reducing sugars, which excess induces pathogenesis of chronic diseases. Brazil is the major producer of citrus, a low-cost source of hesperidin, which is a polyphenol recognized for its capacity to inhibit AGEs formation. This is the first work to evaluate the effects of a polyphenolic fraction derived from citrus wastes on the antiglycation and on the inhibition properties of digestive enzymes on the possibility to process these wastes in high value-added products. At concentrations of 10, 15 and 20 mg/mL inhibition of AGEs was higher than 60%. The extracts were able to inhibit by 76% the activity of pancreatic lipase and by 98% the activity of α-glucosidase. For the α-amylase the inhibition capacity was lower than 50%. Strong correlation was obtained among anti-glycation with polyphenolic content and antioxidant capacity.
Assuntos
Citrus/química , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/antagonistas & inibidores , Lipase/antagonistas & inibidores , Polifenóis/química , alfa-Amilases/antagonistas & inibidores , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Bovinos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Glicosilação/efeitos dos fármacos , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Polifenóis/isolamento & purificação , Polifenóis/farmacologia , Saccharomyces cerevisiae/enzimologia , SuínosRESUMO
The aqueous and ethanolic extracts of Lippia sidoides Cham. were chemically characterized and tested for their action on enzymes involved in processes such as inflammation, blood coagulation, and digestion. Both extracts potentiated the activity of phospholipases A2 present in the venom of Bothrops atrox in 12 % and completely inhibited the hemolysis induced by B. jararacussu and B. moojeni venoms in the proportions between 1 : 0.5 and 1 : 5 (venom/extracts (w/w)). They inhibited the thrombolysis induced by B. moojeni (10 to 25 %), potentiated the thrombolysis induced by the Lachesis muta muta venom (30 to 80 %), prolonged the coagulation time induced by B. moojeni and L. muta muta venoms, and presented antigenotoxic action. Both extracts reduced the activity of α-glycosidases, the aqueous extract inhibited lipases, and the ethanolic extract inhibited α-amylases. The results demonstrate the modulatory action of the extracts on proteases, phospholipases, and digestive enzymes. In addition, the rich phenolic composition of these extracts highlights their potential for nutraceutical use.
Assuntos
Inflamação/tratamento farmacológico , Lippia/química , Fenóis/farmacologia , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia , Folhas de Planta/química , Coagulação Sanguínea/efeitos dos fármacos , Etanol/química , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Hemostasia/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Lipase/antagonistas & inibidores , Lipase/metabolismo , Lippia/metabolismo , Fenóis/química , Fenóis/metabolismo , Fosfolipases A2/metabolismo , Compostos Fitoquímicos/química , Compostos Fitoquímicos/metabolismo , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Folhas de Planta/metabolismo , Água/química , alfa-Amilases/metabolismoRESUMO
Glucosinolates are secondary metabolites occurring in Brassicaceae plants whose hydrolysis may yield isothiocyanates, widely recognized as health-promoting compounds. Myrosinase catalyzes this conversion. The chemical mechanism involves an unstable intermediary (thiohydroxamate-O-sulfonate) that spontaneously decomposes into isothiocyanates or other non-bioactive compounds depending on pH and cofactors. At acidic pH, non-bioactive compounds such as nitriles and thiocyanates are formed, while at neutral pH isothiocyanates are obtained. Broccoli myrosinase has been poorly studied so far. Recently, its amino acidic sequence was elucidated, and a structural model was built. The aim of this work was to study the molecular interaction of broccoli myrosinase with different ligands at acidic pH to propose possible inhibitors that prevent formation of undesirable compounds at acidic pH, and that at neutral pH dissociate from the enzyme, allowing formation of isothiocyanates. The interaction between broccoli myrosinase and 40 ligands was studied by molecular docking simulations. Both the enzyme and each inhibitor were set at pH 3.0. Amygdaline and arbutin showed the highest affinity to broccoli myrosinase in this condition. The residues that stabilize the complexes agree with those that stabilize the substrate (Gln207, Glu429, Tyr352, and Ser433). Accordingly, amygdaline and arbutin would perform as competitive inhibitors of myrosinase at pH 3.0.
Assuntos
Brassica/química , Inibidores Enzimáticos/química , Glicosídeo Hidrolases/química , Simulação de Acoplamento Molecular , Inibidores Enzimáticos/farmacologia , Glucosinolatos/química , Glicosídeo Hidrolases/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Hidrólise , Simulação de Dinâmica Molecular , Especificidade por SubstratoRESUMO
Enantiomeric 2,3,4-tris(hydroxyalkyl)-5-phenylpyrrolidines have been synthesized from the major cycloadducts obtained by the 1,3-dipolar cycloaddition of sugar enones with azomethine ylides derived from natural amino acids. Reduction of the ketone carbonyl group of the cycloadducts, which possess a basic structure of bicyclic 6-(menthyloxy)hexahydropyrano[4,3-c]pyrrol-7(6H)one, afforded a number of pyrrolidine-based bicyclic systems. A sequence of reactions, which involved hydrolysis of the menthyloxy substituent, reduction, N-protection, and degradative oxidation, afforded varied pyrrolidine structures having diverse configurations and patterns of substitution; in particular, polyhydroxylated derivatives have been obtained. The unprotected products were isolated as pyrrolidinium trifluoroacetates. Because of the furanose-like nature of the target trihydroxyalkyl pyrrolidines, these molecules have been evaluated as inhibitors of the ß-galactofuranosidase from Penicillium fellutanum. The compounds showed practically no inhibitory activity for concentration of pyrrolidines in the range of 0.1-1.6 mM.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Pirrolidinas/síntese química , Pirrolidinas/farmacologia , Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/farmacologia , Reação de Cicloadição , Hidrólise , Modelos Moleculares , Conformação Molecular , Oxirredução , Penicillium/efeitos dos fármacos , Penicillium/enzimologia , EstereoisomerismoRESUMO
DESCRIÇÃO DA TECNOLOGIA O eliglustate é uma Terapia de Redução do Substrato e age inibindo parcialmente a enzima ácido ß-glicosidase, evitando assim o acúmulo de glicosilceramida (molécula de gordura) no organismo. REGISTRO DO MEDICAMENTO NO MUNDO: O tartarato de eliglustate (Cerdelga®) não possui registro no Brasil para esta ou outra indicação. Nos Estados Unidos o medicamento foi registrado em 2014, para pacientes com doença de Gaucher tipo 1. PESQUISA CLÍNICA: O tartarato de eliglustate (Cerdelga®) não possui registro no Brasil para esta ou outra indicação. Nos Estados Unidos o medicamento foi registrado em 2014, para pacientes com doença de Gaucher tipo 1. A Comissão Européia concedeu a autorização de comercialização do medicamento na Europa em janeiro de 2015, para o tratamento da doença de Gaucher tipo 1. PESQUISA CLÍNICA: Para coletar informações sobre eficácia e segurança do medicamento, foram selecionadas pesquisas com o medicamento (ensaios clínicos de fase 1d , 2e e 3f ) concluídas e em andamento. Estudos concluídos: Foram localizados três estudos de fase 1 concluídos que testaram o uso de eliglustate em voluntários sadios. Esses estudos analisaram a segurança e a tolerabilidade do eliglustate em dose única e em múltiplas doses, além do efeito dos alimentos sobre o medicamento. Um estudo de fase 2 e sua extensão foram localizados com resultados publicados. O primeiro estudo é multicêntrico, aberto, braço único e avaliou a eficácia, segurança e farmacocinética do eliglustate administrado duas vezes ao dia, numa dosagem de 50 ou 100mg, por 52 semanas, em 26 pacientes com doença de Gaucher tipo 1 não tratados anteriormente com miglustate ou imiglucerase durante os últimos 12 meses ou bifosforados durante os últimos três meses. Vinte pacientes permaneceram no estudo de extensão que completou dois anos de tratamento. Um estudo de fase 3 (EDGE) está concluído, no entanto, não foram encontrados resultados publicados até a data de publicação do presente alerta. Este estudo é randomizado, multicêntrico, duplo-cego e envolveu a participação de 171 pacientes adultos com a doença. O estudo comparou o uso de eliglustate em diferentes dosagens e em uma ou duas vezes ao dia em adultos maiores de 18 anos. Dois estudos de fase 3 (ENGAGE e ENCORE) estão concluídos e possuem resultados publicados. Estes estudos testaram a eficácia do uso do eliglustate em pacientes adultos com a doença de Gaucher tipo 1. No estudo ENGAGE os pacientes não poderiam ter sido tratados previamente com outra TRS ou TRE, num período de, respectivamente, seis e nove meses antes da randomização. Já no estudo ENCORE os pacientes deveriam ter sido tratados previamente com TRE por três anos ou mais. ANÁLISE DOS ESTUDOS DE FASE 3 CONCLUÍDOS: Para atingir o objetivo deste alerta, foram analisados os estudos de fase 3 concluídos (ENGAGE e ENCORE ) com resultados publicados. Importa destacar que, nos estudos analisados, foram utilizadas como parâmetro de eficácia: 1. alterações na porcentagem de parâmetros sanguíneos, como a concentração de hemoglobina e a contagem de plaquetas; 2. alterações no tamanho dos órgãos (fígado e baço). Esses parâmetros foram utilizados considerando a relação entre tais manifestações clínicas e o acometimento de órgãos nessa doença. Além disso, não foram observadas, em nenhum dos estudos, mortes decorrentes dos tratamentos utilizados. A maioria dos eventos adversos foram descritos como não graves, leves ou moderados, como dor nas articulações, dor de cabeça, resfriado comum, diarréia e sinusite. No entanto, cabe destacar que no estudo ENCORE dois pacientes que utilizavam eliglustate e um que utilizava imiglucerase descontinuaram o tratamento devido a eventos adversos: palpitações e infarto do miocárdio (grupo eliglustate) e desordem psicótica (grupo imiglucerase).
Assuntos
Humanos , Projetos de Desenvolvimento Tecnológico e Inovação , Doença de Gaucher/tratamento farmacológico , Glicosídeo Hidrolases/antagonistas & inibidores , Brasil , Eficácia , Análise Custo-BenefícioRESUMO
A novel multienzyme complex, E1C, and a free endoglucanase, E2 (GH5), from Fusarium verticillioides were purified. The E1C contained two endoglucanases (GH6 and GH10), one cellobiohydrolase (GH7) and one xylanase (GH10). Maximum activity was observed at 80 °C for both enzymes and they were thermostable at 50 and 60 °C. The activation energies for E1C and E2 were 21.3 and 27.5 kJ/mol, respectively. The KM for E1C was 10.25 g/L while for E2 was 6.58 g/L. Both E1C and E2 were activated by Mn(2+) and CoCl2 while they were inhibited by SDS, CuSO4, FeCl3, AgNO4, ZnSO4 and HgCl2. E1C and E2 presented endo-ß-1,3-1,4-glucanase activity. E1C presented crescent activity towards cellopentaose, cellotetraose and cellotriose. E2 hydrolyzed the substrates cellopentaose, cellotetraose and cellotriose with the same efficiency. E1C showed a higher stability and a better hydrolysis performance than E2, suggesting advantages resulting from the physical interaction between proteins.
Assuntos
Celulase/metabolismo , Fusarium/enzimologia , Glicosídeo Hidrolases/metabolismo , Complexos Multienzimáticos/metabolismo , Sequência de Aminoácidos , Celulase/antagonistas & inibidores , Celulase/química , Celulose/metabolismo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/química , Concentração de Íons de Hidrogênio , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , Especificidade por Substrato , TemperaturaRESUMO
Trypanosoma cruzi, etiological agent of Chagas' disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas' disease.
Assuntos
Doença de Chagas/fisiopatologia , Glicosídeo Hidrolases/metabolismo , Estágios do Ciclo de Vida/fisiologia , Trypanosoma cruzi/crescimento & desenvolvimento , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Animais , Northern Blotting , Southern Blotting , Western Blotting , Catálise , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Doença de Chagas/tratamento farmacológico , Chlorocebus aethiops , Técnica Indireta de Fluorescência para Anticorpo , Glicosídeo Hidrolases/antagonistas & inibidores , Humanos , Hidroxiureia , Microscopia Eletrônica , Pirrolidinas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Células VeroRESUMO
A new (1â6)-linked thiodisaccharide formed by two galactofuranosyl units has been synthesized. Methyl (methyl α,ß-D-galactofuranosid)uronate was employed as the starting compound, which was per-O-silylated with TBSCl and reduced with LiAlH4 to afford methyl 2,3,5-tri-O-tert-butyldimethylsilyl-ß-D-galactofuranoside (2ß) as a key precursor for the preparation of methyl per-O-tert-butyldimethylsilyl-6-thio-ß-D-galactofuranoside (12). The free thiol group of 12 was glycosylated and the product O-deprotected to afford the target ß-D-Galf-S-(1â6)-ß-d-Galf-OMe (14). The conformations of this thiodisaccharide were preliminarily studied using combined theoretical calculations and NMR data. Furthermore, the glycomimetic 14 showed to be a competitive inhibitor of the ß-galactofuranosidase from Penicillum fellutanum (K(i)=3.62 mM).
Assuntos
Antifúngicos/síntese química , Dissacarídeos/síntese química , Proteínas Fúngicas/antagonistas & inibidores , Galactose/química , Glicosídeo Hidrolases/antagonistas & inibidores , Penicillium/química , Tiogalactosídeos/síntese química , Antifúngicos/química , Configuração de Carboidratos , Dissacarídeos/química , Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Glicosilação , Cinética , Espectroscopia de Ressonância Magnética , Penicillium/enzimologia , Tiogalactosídeos/químicaRESUMO
Derivatives of 5-deoxy-beta-D-galactofuranose (5-deoxy-alpha-L-arabino-hexofuranose) have been synthesized starting from D-galacturonic acid. The synthesis of methyl 5-deoxy-alpha-L-arabino-hexofuranoside (14alpha) was achieved by an efficient strategy previously optimized, involving a photoinduced electron transfer (PET) deoxygenation. Compound 14alpha was converted into per-O-acetyl-5-deoxy-alpha,beta-L-arabino-hexofuranoside (16), an activated precursor for glycosylation reactions. The SnCl4-promoted glycosylation of 16 led to 4-nitrophenyl (19alpha), and 4-methylthiophenyl 5-deoxy-alpha-L-arabino-hexofuranosides (20alpha). The oxygenated analog 4-methylphenyl 1-thio-beta-D-galactofuranoside (23beta) was also prepared. The 5-deoxy galactofuranosides were evaluated as inhibitors or substrates of the exo-beta-D-galactofuranosidase from Penicillium fellutanum, showing that the absence of HO-5 drastically diminishes the affinity for the protein.
Assuntos
Galactosídeos/química , Galactosídeos/metabolismo , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Penicillium/enzimologia , Galactosídeos/síntese química , Especificidade por SubstratoRESUMO
The chemical composition and the biomolecular properties of a series of crude plant extracts were altered without previous knowledge of their detailed chemical composition.
Assuntos
Extratos Vegetais/química , Sulfonas/química , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Fitoterapia , Extratos Vegetais/farmacologia , Plantas MedicinaisRESUMO
Thiodisaccharides having beta-D-Galf or alpha-L-Araf units as non-reducing end have been synthesized by the SnCl(4)- or MoO(2)Cl(2)-promoted thioglycosylation of per-O-benzoyl-D-galactofuranose (1), its 1-O-acetyl analogue 4, or per-O-acetyl-alpha-L-arabinofuranose (16) with 6-thioglucose or 6-thiogalactose derivatives. After convenient removal of the protecting groups, the free thiodisaccharides having the basic structure beta-D-Galf(1-->6)-6-thio-alpha-D-Glcp-OMe (5) or beta-D-Galf(1-->6)-6-thio-alpha-D-Galp-OMe (15) were obtained. The respective alpha-L-Araf analogues 18 and 20 were prepared similarly from 16. Alternatively, beta-D-Galf(1-->4)-4-thio-3-deoxy-alpha-L-Xylp-OiPr was synthesized by Michael addition to a sugar enone of 1-thio-beta-d-Galf derivative, generated in situ from the glycosyl isothiourea derivative of 1. The free S-linked disaccharides were evaluated as inhibitors of the beta-galactofuranosidase from Penicillium fellutanum, being 15 and 20 the more active inhibitors against this enzyme.
Assuntos
Arabinose/análogos & derivados , Dissacarídeos/síntese química , Inibidores Enzimáticos/síntese química , Glicosídeo Hidrolases/antagonistas & inibidores , Penicillium/enzimologia , Arabinose/química , Catálise , Compostos Clorados/química , Dissacarídeos/química , Dissacarídeos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Galactose/química , Glicosídeo Hidrolases/metabolismo , Compostos de Manganês/química , Óxidos/química , Penicillium/metabolismo , Sulfetos/química , Compostos de Estanho/químicaRESUMO
Intracellular alpha-L-rhamnosidase (EC 3.2.1.40) from the psychrotolerant Pseudoalteromonas sp. 005NJ showed a dose-dependent inhibition for L-rhamnose (IC(50) = 20 mM) and D-ribose (IC(50) = 95 mM), whereas D-glucose and L-fucose presented a lower inhibition, with IC(50) values as high as >0.5 and >0.2 M, respectively. On the other hand, D-fructose enhanced enzyme activity threefold, reaching a plateau of maximum specific activity between 0.2 and 0.4 M of this monosaccharide. Both effects, low inhibition and stimulation, caused by key fruit sugars (glucose and fructose), make this biocatalyst an interesting system in terms of its potential application for debittering fruit juices.
Assuntos
Frutose/metabolismo , Glucose/metabolismo , Glicosídeo Hidrolases/metabolismo , Pseudoalteromonas/enzimologia , Temperatura Baixa , Inibidores Enzimáticos/metabolismo , Indústria Alimentícia , Glicosídeo Hidrolases/antagonistas & inibidores , Concentração Inibidora 50 , Pseudoalteromonas/metabolismo , Ramnose/metabolismo , Ribose/metabolismoRESUMO
In many mammals, sperm associations had been observed, but not in the mouse. In this work, mouse sperm rosettes are morphologically described inside the epididymis and during its dissolution in a culture medium. Also characterized are the saccharides present in the linking material. Sperm association and other epididymal actions are supported by sperm during epididymal transit and are verified at the caudal region, suggesting a relation between epididymal transit and sperm maturation. In drops of epididymal content obtained from distal (cauda), but not from proximal (caput and corpus) regions; dissolved in culture medium, rosettes appear to be 10 to 15 motile sperm joined by their heads. After 3 min, sperm progressively detach, disassembling the rosette. These structures are studied by several techniques, including optic, electronic (scanning electron microscopy and transmission electron microscopy), and video microscopy. At the ultrastructural level, a dense network of electron-dense material was observed between sperm heads, joining them. Based on previous works in rat, several lectins were used to characterize the type of saccharides present in this linking material. To avoid the contact between sperm and epididymal fluid from distal region--that probably exerts an influence on sperm association--a ligature was placed between caput and corpus. This epididymal content isolated from caput did not display any rosettes after 28 days.
Assuntos
Epididimo/ultraestrutura , Oligossacarídeos/metabolismo , Maturação do Esperma , Motilidade dos Espermatozoides , Espermatozoides/ultraestrutura , 1-Desoxinojirimicina/farmacologia , Animais , Adesão Celular , Agregação Celular , Inibidores Enzimáticos/farmacologia , Epididimo/metabolismo , Epididimo/cirurgia , Glicosídeo Hidrolases/antagonistas & inibidores , Glicosídeo Hidrolases/metabolismo , Imino Furanoses/farmacologia , Ligadura , Masculino , Manitol/análogos & derivados , Manitol/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Vídeo , Pirrolidinas/farmacologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Swainsonina/farmacologia , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
Broad-spectrum antimicrobial activity of an invertase inhibitory protein (IIP) isolated from Cyphomandra betacea ripe fruits is documented. Minimal inhibitory concentration (MIC) values were determined by agar macrodilution and broth microdilution assays. This IIP inhibited the growth of xylophagous and phytopatogenic fungi (Ganoderma applanatum, Schizophyllum commune, Lenzites elegans, Pycnoporus sanguineous, Penicillium notatum, Aspergillus niger, Phomopsis sojae and Fusarium mango) and phytopathogenic bacteria (Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae pv. syringae and Erwinia carotovora var carotovora). The IIP concentration required to completely inhibit the growth of all studied fungi ranged from 7.8 to 62.5 microg/ml. Phytopatogenic bacteria were the most sensitive, with MIC values between 7.8 and 31.25 microg/ml. Antifungal and antibacterial activities can be associated with their ability to inhibit hydrolytic enzymes. Our results indicate the possible participation of IIP in the plant defense mechanism and its potential application as a biocontrol agent against phytopathogenic fungi and bacteria.
Assuntos
Anti-Infecciosos/farmacologia , Inibidores Enzimáticos/química , Frutas/metabolismo , Glicosídeo Hidrolases/antagonistas & inibidores , Peptídeos/química , Antifúngicos/farmacologia , Inibidores Enzimáticos/metabolismo , Proteínas Fúngicas/química , Testes de Sensibilidade Microbiana , Proteínas de Plantas/químicaRESUMO
An acid invertase from the fern Pteris deflexa Link was purified and the effect of reaction products on enzyme activity was studied. Fructose and glucose were competitive and non-competitive inhibitors of the enzyme, respectively. Since proteins suppressed glucose and fructose inhibition of the enzyme, an invertase modulation by reaction products is unlikely; nevertheless, an invertase proteinaceous inhibitor previously reported could have a role in this respect. The purified enzyme was an heterodimer Mr 90,000 Daltons composed of subunits of 66,000 and 30,000 Daltons. The enzyme had beta-fructofuranosidase activity and hydrolyzed mainly sucrose but also raffinose and stachyose, with Km of 3.22, 10.80 and 38.50 mM, respectively. Invertase activity with an optimum pH at 5.0 was present in almost every leaf fern tissue. Pinnas (sporophyll leaflets) had the higher enzyme levels. Invertase histochemical and immunochemical localization studies showed the enzyme mainly in phloem cells. Epidermis, collenchyma and parenchyma cells also showed invertase protein.
Assuntos
Inibidores Enzimáticos/química , Frutose/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/farmacologia , Pteris/enzimologia , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/isolamento & purificação , Cinética , Especificidade por Substrato , beta-FrutofuranosidaseRESUMO
The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family. Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, alpha-L-arabinofuranosidase and beta-glucosidase are also shown to be inhibited. Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.
Assuntos
Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/antagonistas & inibidores , Bactérias Gram-Negativas/efeitos dos fármacos , Tubérculos/enzimologia , Inibidores Enzimáticos/isolamento & purificação , Bactérias Gram-Negativas/crescimento & desenvolvimento , Hidrólise/efeitos dos fármacos , Extratos Vegetais/química , Proteínas de Plantas/antagonistas & inibidores , Tubérculos/química , beta-FrutofuranosidaseRESUMO
The kinetics of thermal inactivation of A. terreus alpha-rhamnosidase was studied using the substrate p-nitrophenyl alpha-L-rhamnoside between 50 degrees C and 70 degrees C. Up to 60 degrees C the inactivation of the purified enzyme was completely reversible, but samples of crude or partially purified enzyme showed partial reversibility. The presence of the product rhamnose, the substrate naringin, and other additives reduced the reversible inactivation, maintaining in some cases full enzyme activity at 60 degrees C. A mechanism for the inactivation process, which permitted the reproduction of experimental results, was proposed. The products rhamnose (inhibition constant, 2.1 mM) and prunin (2.6 mM) competitively inhibited the enzyme reaction. The maximum hydrolysis of supersaturated naringin solution, without enzyme inactivation, was observed at 60 degrees C. Hydrolysis of naringin reached 99% with 1% naringin solution, although the hydrolysis degree of naringin was only 40% due to products inhibition when the initial concentration of flavonoid was 10%. The experimental results fitted an equation based on the integrated Michaelis-Menten's, including competitive inhibition by products satisfactorily.
Assuntos
Aspergillus/efeitos dos fármacos , Flavanonas , Flavonoides/metabolismo , Glicosídeo Hidrolases/antagonistas & inibidores , Proteínas de Plantas , Algoritmos , Aspergillus/enzimologia , Reagentes de Ligações Cruzadas , Meios de Cultura , Globulinas/farmacologia , Temperatura Alta , Hidrólise , Cinética , TemperaturaRESUMO
Invertase and urease are enzyme entities highly associated with the cells of the astaxanthin-producer yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) during any stage of its cell growth cycle. In this study cellobiose was a more efficient carbon source than sucrose or its hexose counterparts for invertase expression. Extensive ultrasonication or abrasion with glass pearls were required in order to promote enzyme release. In contrast to the yeast whose growth declines above 27 degrees C, the released enzymes displayed a higher optimum temperature range when assayed in vitro. Isoforms from both enzymes could be resolved either by FPLC on DEAE-Sepharose or by an affinity approach on immobilized Concanavalin. The zymogram for invertase showed a pI somewhat less acidic than that of the similar enzyme from S. cerevisiae.
Assuntos
Celobiose/metabolismo , Inibidores Enzimáticos/farmacologia , Glicosídeo Hidrolases/metabolismo , Sacarose/metabolismo , Urease/metabolismo , Leveduras/enzimologia , Divisão Celular/fisiologia , Cromatografia de Afinidade , Eletroforese , Indução Enzimática , Glicosídeo Hidrolases/antagonistas & inibidores , Sonicação , Especificidade por Substrato , Temperatura , Ureia/metabolismo , Urease/antagonistas & inibidores , Leveduras/metabolismo , beta-FrutofuranosidaseRESUMO
Plant invertases play important roles in sucrose metabolism. Cell wall invertase was reported to participate in phloem loading and unloading. Soluble invertases would be involved in hexose level regulation in mature tissues and in stored sucrose utilization within vacuoles. Invertase inhibitory proteins were described as one of the possible mechanisms for invertase activity regulation in some plant species; nevertheless, these proteins were found only in sink tissues, suggesting that this mechanism would not be relevant in the sucrose turnover of leaves. This report describes the purification of invertase from Pteris deflexa fronds and the occurrence of an invertase inhibitory protein in this fern organ, as well as its purification and invertase-inhibitor interactions. The Mr of the invertase and of its inhibitory protein were 90,000 and 18,000, respectively. SDS-PAGE in the presence of 2-mercaptoetanol gave two subunits for the enzyme (Mr=66,000 and 30,000) and only one for the inhibitor. The inhibitor protein is a glycoprotein (12% w/w of neutral sugars) that did not show agglutinating activity like some others, and also showed a high heat stability at pH 5.0. The optimum pH of invertase activity is 5.0, while invertase inhibitory protein caused maximal inhibition at the same pH value. Invertase-inhibitor complex formation occurs in an immediate manner and a protease activity was discarded. The inhibition is non-competitive (Ki=1.5 x 10(-6) M) without interactions among the binding sites. The complex is slightly dissociable and sucrose was able to partially reduce the inhibitory effect. Up to the present, invertase inhibitory proteins have been found solely in heterotrophic tissues. In this work we demonstrate that this protein is also present in an autotrophic tissue of a lower vascular plant.