RESUMEN
The funneled energy landscape theory suggests that the folding pathway of homologous proteins should converge at the late stages of folding. In this respect, proteins displaying a broad energy landscape for folding are particularly instructive, allowing inferring both the early, intermediate and late stages of folding. In this paper we explore the folding mechanisms of human frataxin, an essential mitochondrial protein linked to the neurodegenerative disorder Friedreich's ataxia. Building upon previous studies on the yeast homologue, the folding pathway of human frataxin is thoroughly examined, revealing a mechanism implying the presence of a broad energy barrier, reminiscent of the yeast counterpart. Through an extensive site-directed mutagenesis, we employed a Φ -value analysis to map native-like contacts in the folding transition state. The presence of a broad energy barrier facilitated the exploration of such contacts in both early and late folding events. We compared results from yeast and human frataxin providing insights into the impact of native topology on the folding mechanism and elucidating the properties of the underlying free energy landscape. The findings are discussed in the context of the funneled energy landscape theory of protein folding.
Asunto(s)
Frataxina , Pliegue de Proteína , Humanos , Frataxina/química , Frataxina/genética , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , TermodinámicaRESUMEN
The adaptor protein Grb2, or growth factor receptor-bound protein 2, possesses a pivotal role in the transmission of fundamental molecular signals in the cell. Despite lacking enzymatic activity, Grb2 functions as a dynamic assembly platform, orchestrating intracellular signals through its modular structure. This study delves into the energetic communication of Grb2 domains, focusing on the folding and binding properties of the C-SH3 domain linked to its neighboring SH2 domain. Surprisingly, while the folding and stability of C-SH3 remain robust and unaffected by SH2 presence, significant differences emerge in the binding properties when considered within the tandem context compared with isolated C-SH3. Through a double mutant cycle analysis, we highlighted a subset of residues, located at the interface with the SH2 domain and far from the binding site, finely regulating the binding of a peptide mimicking a physiological ligand of the C-SH3 domain. Our results have mechanistic implications about the mechanisms of specificity of the C-SH3 domain, indicating that the presence of the SH2 domain optimizes binding to its physiological target, and emphasizing the general importance of considering supramodular multidomain protein structures to understand the functional intricacies of protein-protein interaction domains.
Asunto(s)
Proteína Adaptadora GRB2 , Unión Proteica , Pliegue de Proteína , Dominios Homologos src , Humanos , Sitios de Unión , Proteína Adaptadora GRB2/metabolismo , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/genética , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
Protein-protein interactions play crucial roles in a wide range of biological processes, including metabolic pathways, cell cycle progression, signal transduction, and the proteasomal system. For PPIs to fulfill their biological functions, they require the specific recognition of a multitude of interacting partners. In many cases, however, protein-protein interaction domains are capable of binding different partners in the intracellular environment, but they require precise regulation of the binding events in order to exert their function properly and avoid misregulation of important molecular pathways. In this work, we focused on the MATH domain of the E3 Ligase adaptor protein SPOP in order to decipher the molecular features underlying its interaction with two different peptides that mimic its physiological partners: Puc and MacroH2A. By employing stopped-flow kinetic binding experiments, together with extensive site-directed mutagenesis, we addressed the roles of specific residues, some of which, although far from the binding site, govern these transient interactions. Our findings are compatible with a scenario in which the binding of the MATH domain with its substrate is characterized by a fine energetic network that regulates its interactions with different ligands. Results are briefly discussed in the context of previously existing work regarding the MATH domain.
Asunto(s)
Tiopronina , Ubiquitina-Proteína Ligasas , Tiopronina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Histonas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Ingeniería de Proteínas , Unión ProteicaRESUMEN
PTB (PhosphoTyrosine Binding) domains are protein domains that exert their function by binding phosphotyrosine residues on other proteins. They are commonly found in a variety of signaling proteins and are important for mediating protein-protein interactions in numerous cellular processes. PTB domains can also exhibit binding to unphosphorylated ligands, suggesting that they have additional binding specificities beyond phosphotyrosine recognition. Structural studies have reported that the PTB domain from FRS2 possesses this peculiar feature, allowing it to interact with both phosphorylated and unphosphorylated ligands, such as TrkB and FGFR1, through different topologies and orientations. In an effort to elucidate the dynamic and functional properties of these protein-protein interactions, we provide a complete characterization of the folding mechanism of the PTB domain of FRS2 and the binding process to peptides mimicking specific regions of TrkB and FGFR1. By analyzing the equilibrium and kinetics of PTB folding, we propose a mechanism implying the presence of an intermediate along the folding pathway. Kinetic binding experiments performed at different ionic strengths highlighted the electrostatic nature of the interaction with both peptides. The specific role of single amino acids in early and late events of binding was pinpointed by site-directed mutagenesis. These results are discussed in light of previous experimental works on these protein systems.
Asunto(s)
Péptidos , Dominios Homologos src , Dominios Proteicos , Fosfotirosina/metabolismo , Ligandos , Sitios de Unión , Péptidos/metabolismo , Unión ProteicaRESUMEN
In an effort to investigate the molecular determinants of ligand recognition of the C-terminal SH2 domain of the SHP2 protein, we conducted extensive site-directed mutagenesis and kinetic binding experiments with a peptide mimicking a specific portion of a physiological ligand (the scaffold protein Gab2). Obtained data provided an in-depth characterization of the binding reaction, allowing us to pinpoint residues topologically far from the binding pocket of the SH2 domain to have a role in the recognition and binding of the peptide. The presence of a sparse energetic network regulating the interaction with Gab2 was identified and characterized through double mutant cycle analysis, performed by challenging all the designed site-directed variants of C-SH2 with a Gab2 peptide mutated at +3 position relative to its phosphorylated tyrosine, a key residue for C-SH2 binding specificity. Results highlighted non-optimized residues involved in the energetic network regulating the binding with Gab2, which may be at the basis of the ability of this SH2 domain to interact with different partners in the intracellular environment. Moreover, a detailed analysis of kinetic and thermodynamic parameters revealed the role of the residue at +3 position on Gab2 in the early and late events of the binding reaction with the C-SH2 domain.
Asunto(s)
Péptidos , Dominios Homologos src , Ligandos , Péptidos/metabolismo , Mutagénesis Sitio-Dirigida , Tirosina/metabolismo , Unión ProteicaRESUMEN
SPOP (Speckle-type POZ protein) is an E3 ubiquitin ligase adaptor protein that mediates the ubiquitination of several substrates. Furthermore, SPOP is responsible for the regulation of both degradable and nondegradable polyubiquitination of a number of substrates with diverse biological functions. The recognition of SPOP and its physiological partners is mediated by two protein-protein interaction domains. Among them, the MATH domain recognizes different substrates, and it is critical for orchestrating diverse cellular pathways, being mutated in several human diseases. Despite its importance, the mechanism by which the MATH domain recognizes its physiological partners has escaped a detailed experimental characterization. In this work, we present a characterization of the binding mechanism of the MATH domain of SPOP with three peptides mimicking the phosphatase Puc, the chromatin component MacroH2A, and the dual-specificity phosphatase PTEN. Furthermore, by taking advantage of site-directed mutagenesis, we address the role of some key residues of MATH in the binding process. Our findings are briefly discussed in the context of previously existing data on the MATH domain.
Asunto(s)
Proteínas Nucleares , Proteínas Represoras , Humanos , Proteínas Represoras/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , UbiquitinaciónRESUMEN
Histamine is a biogenic amine implicated in various biological and pathological processes. Convenient cellular models are needed to screen and develop new antihistamine agents. This report aimed to characterize the response of neurons differentiated from mouse P19 embryonal carcinoma cells to histamine treatment, and to investigate the modulation of this response by antihistamine drugs, vegetal diamine oxidase, and catalase. The exposure of P19 neurons to histamine reduced cell viability to 65% maximally. This effect involves specific histamine receptors, since it was prevented by treatment with desloratadine and cimetidine, respectively, H1 and H2 antagonists, but not by the H3 antagonist ciproxifan. RT-PCR analysis showed that P19 neurons express H1 and H2 receptors, and the H3 receptor, although it seemed not involved in the histamine effect on these cells. The H4 receptor was not expressed. H1 and H2 antagonists as well as vegetal diamine oxidase diminished the intracellular Ca2+ mobilization triggered by histamine. The treatment with vegetal diamine oxidase or catalase protected against mortality and a significant reduction of H2O2 level, generated from the cells under the histamine action, was found upon treatments with desloratadine, cimetidine, vegetal diamine oxidase, or catalase. Overall, the results indicate the expression of functional histamine receptors and open the possibility of using P19 neurons as model system to study the roles of histamine and related drugs in neuronal pathogenesis. This model is less expensive to operate and can be easily implemented by current laboratories of analysis and by Contract Research Organizations.
Asunto(s)
Amina Oxidasa (conteniendo Cobre) , Productos Biológicos , Animales , Ratones , Histamina/farmacología , Histamina/metabolismo , Cimetidina/farmacología , Catalasa , Peróxido de Hidrógeno/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Receptores Histamínicos/genética , Antagonistas de los Receptores Histamínicos H1/farmacología , Neuronas/metabolismo , Productos Biológicos/farmacologíaRESUMEN
Although cooperativity is a well-established and general property of folding, our current understanding of this feature in multidomain folding is still relatively limited. In fact, there are contrasting results indicating that the constituent domains of a multidomain protein may either fold independently on each other or exhibit interdependent supradomain phenomena. To address this issue, here we present the comparative analysis of the folding of a tandem repeat protein, comprising two contiguous PDZ domains, in comparison to that of its isolated constituent domains. By analyzing in detail the equilibrium and kinetics of folding at different experimental conditions, we demonstrate that despite each of the PDZ domains in isolation being capable of independent folding, at variance with previously characterized PDZ tandem repeats, the full-length construct folds and unfolds as a single cooperative unit. By exploiting quantitatively, the comparison of the folding of the tandem repeat to those observed for its constituent domains, as well as by characterizing a truncated variant lacking a short autoinhibitory segment, we successfully rationalize the molecular basis of the observed cooperativity and attempt to infer some general conclusions for multidomain systems.
Asunto(s)
Conformación Proteica , Pliegue de Proteína , Proteínas , Cinética , Modelos Moleculares , Proteínas/química , Dominios ProteicosRESUMEN
SH2 (Src Homology 2) domains are among the best characterized and most studied protein-protein interaction (PPIs) modules able to bind and recognize sequences presenting a phosphorylated tyrosine. This post-translational modification is a key regulator of a plethora of physiological and molecular pathways in the eukaryotic cell, so SH2 domains possess a fundamental role in cell signaling. Consequently, several pathologies arise from the dysregulation of such SH2-domains mediated PPIs. In this review, we recapitulate the current knowledge about the structural, folding stability, and binding properties of SH2 domains and their roles in molecular pathways and pathogenesis. Moreover, we focus attention on the different strategies employed to modulate/inhibit SH2 domains binding. Altogether, the information gathered points to evidence that pharmacological interest in SH2 domains is highly strategic to developing new therapeutics. Moreover, a deeper understanding of the molecular determinants of the thermodynamic stability as well as of the binding properties of SH2 domains appears to be fundamental in order to improve the possibility of preventing their dysregulated interactions.
Asunto(s)
Tirosina , Dominios Homologos src , Fosfotirosina/metabolismo , Tirosina/metabolismo , Transducción de Señal , Unión Proteica , Sitios de UniónRESUMEN
Two thirds of eukaryotic proteins have evolved as multidomain constructs, and in vivo, domains fold within a polypeptide chain, with inter-domain interactions possibly crucial for correct folding. However, to date, most of the experimental folding studies are based on domains in isolation. In an effort to better understand multidomain folding, in this work we analyzed, through equilibrium and kinetic folding experiments, the folding properties of the Growth factor receptor-bound protein 2 (Grb2), composed by one SRC homology 2 domain flanked by two SRC homology 3 domains. In particular we compared the kinetic features of the multidomain construct with the domains expressed in isolation. By performing single and double mixing folding experiments, we demonstrated that the folding of the SH2 domain is kinetically trapped in a misfolded intermediate when tethered to the C-SH3. Importantly, within the multidomain construct, misfolding occurred independently if refolding is started with C-SH3 in its unfolded or native state. Interestingly, our data reported a peculiar scenario, in which SH2 and C-SH3 domain reciprocally and transiently interact during folding. Altogether, the analysis of kinetic folding data provided a quantitative description of the multidomain folding of Grb2 protein, discussed under the light of previous works on multidomain folding.
Asunto(s)
Péptidos , Dominios Homologos src , Cinética , Péptidos/química , Pliegue de ProteínaRESUMEN
SH2 domains are structural modules specialized in the recognition and binding of target sequences containing a phosphorylated tyrosine residue. They are mostly incorporated in the 3D structure of scaffolding proteins that represent fundamental regulators of several signaling pathways. Among those, Crkl plays key roles in cell physiology by mediating signals from a wide range of stimuli, and its overexpression is associated with several types of cancers. In myeloid cells expressing the oncogene BCR/ABL, one interactor of Crkl-SH2 is the focal adhesion protein Paxillin, and this interaction is crucial in leukemic transformation. In this work, we analyze both the folding pathway of Crkl-SH2 and its binding reaction with a peptide mimicking Paxillin, under different ionic strength and pH conditions, by using means of fluorescence spectroscopy. From a folding perspective, we demonstrate the presence of an intermediate along the reaction. Moreover, we underline the importance of the electrostatic interactions in the early event of recognition, occurring between the phosphorylated tyrosine of the Paxillin peptide and the charge residues of Crkl-SH2. Finally, we highlight a pivotal role of a highly conserved histidine residue in the stabilization of the binding complex. The experimental results are discussed in light of previous works on other SH2 domains.
Asunto(s)
Proteínas Nucleares , Dominios Homologos src , Proteínas Nucleares/metabolismo , Oncogenes , Paxillin , Fosforilación , Unión Proteica , Tirosina/metabolismo , Dominios Homologos src/fisiologíaRESUMEN
The existing zymography method for the detection of diamine oxidase (DAO) activity has been improved by a new staining procedure with the aim to ameliorate its sensitivity. Both procedures used SDS-PAGE gels containing uniformly distributed entrapped peroxidase (that wouldn't migrate during electrophoresis). The new approach with 3,5-dichloro-2-hydroxybenzenesulfonate (DCHBS) as peroxidase substrate and with 4-amino-antipyrine as color stabilizer allows a more sensitive detection of DAO when compared to the previously reported o-phenylenediamine (o-PDA) as peroxidase substrate. The newly improved method appears faster, simple and environmentally friendly. It can be used for most of oxidases releasing hydrogen peroxide as reaction product.
Asunto(s)
Amina Oxidasa (conteniendo Cobre) , Colorantes , Electroforesis en Gel de Poliacrilamida , Peróxido de Hidrógeno , Oxidorreductasas , Peroxidasa , PeroxidasasRESUMEN
Clostridium difficile toxin A (TcdA) impairs the intestinal epithelial barrier, increasing the mucosa permeability and triggering a robust inflammatory response. Lathyrus sativus diamino oxidase (LSAO) is a nutraceutical compound successfully used in various gastrointestinal dysfunctions. Here, we evaluated the LSAO (0.004-0.4 µM) ability to counter TcdA-induced (30 ng/mL) toxicity and damage in Caco-2 cells, investigating its possible mechanism of action. LSAO has improved the transepithelial electrical resistance (TEER) score and increased cell viability in TcdA-treated cells, significantly rescuing the protein expression of Ras homolog family members, A-GTPase (RhoA-GTPase), occludin, and zonula occludens-1 (ZO-1). LSAO has also exhibited an anti-apoptotic effect by inhibiting the TcdA-induced expression of Bcl-2-associated X protein (Bax), p50 nuclear factor-kappa-B (p50), p65nuclear factor-kappa-B (p65), and hypoxia-inducible transcription factor-1 alpha (HIF-1α), and the release of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and vascular endothelial growth factor (VEGF) in the cell milieu. Our data showed that LSAO exerts a protective effect on TcdA-induced toxicity in Caco-2 cells, placing itself as an interesting nutraceutical to supplement the current treatment of the Clostridium difficile infections.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/farmacología , Toxinas Bacterianas/toxicidad , Enterotoxinas/toxicidad , GTP Fosfohidrolasas/metabolismo , Lathyrus/enzimología , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP rhoA/metabolismo , Células CACO-2 , Suplementos Dietéticos , Humanos , Interleucina-6/metabolismo , FN-kappa B/metabolismo , Permeabilidad/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Excess of histamine in gut lumen generates a pronounced gastrointestinal discomfort, which may include diarrhea and peristalsis dysfunctions. Deleterious effects of histamine can be alleviated with antihistamine drugs targeting histamine receptors. However, many antihistamine agents come with various undesirable side effects. Vegetal diamine oxidase (vDAO) might be a relevant alternative owing to its histaminase activity. Mammalian intestinal mucosa contains an endogenous DAO, yet possessing lower activity compared to that of vDAO preparation. Moreover, in several pathological conditions such as inflammatory bowel disease and irritable bowel syndrome, this endogenous DAO enzyme can be lost or inactivated. Here, we tested the therapeutic potential of vDAO by focusing on the well-known effect of histamine on gut motility. Using ex vivo and in vitro assays, we found that vDAO is more potent than commercial anti-histamine drugs at inhibiting histamine-induced contraction of murine distal colon muscles. We also identified pyridoxal 5'-phosphate (the biologically active form of vitamin B6) as an effective enhancer of vDAO antispasmodic activity. Furthermore, we discovered that rectally administered vDAO can be retained on gut mucosa and remain active. These observations make administration of vDAO in the gut lumen a valid alternative treatment for histamine-induced intestinal dysfunctions.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/farmacología , Histamina/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Amina Oxidasa (conteniendo Cobre)/metabolismo , Animales , Colon/metabolismo , Femenino , Peróxido de Hidrógeno/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ratones , Músculo Liso/metabolismoRESUMEN
Over the last few decades, coppercontaining amine oxidase (CuAO) from vegetal sources, and belonging to the class of diamine oxidase, has been documented to exhibit beneficial effects in both in vivo and ex vivo animal models of inflammatory or allergic conditions, including asthmalike reaction and myocardial or intestinal ischemiareperfusion injuries. The aim of the present study was to assess the potential of vegetal CuAO as an antiinflammatory and an antiallergic agent and to clarify its antioxidant properties. In cellfree systems, the reactive oxygen species and reactive nitrogen species scavenging properties of CuAO that is purified from Lathyrus sativus were investigated. Its effect on the formylmethionylleucylphenylalanine peptide (fMLP)activated cellular functions of human neutrophils were subsequently analyzed. The obtained results demonstrated that CuAO is not a scavenger of superoxide or nitric oxide, and does not decompose hydrogen peroxide. However, it inhibits the fMLPdependent superoxide generation, elastase release and cell migration, and interferes with the process of calcium flux, supporting the idea that plant CuAO can interact with human neutrophils to modulate their inflammatory function. Therefore, the importance of these properties on the possible use of vegetal CuAO to control inflammatory conditions, particularly intestinal inflammation, is discussed in the current study.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/química , Amina Oxidasa (conteniendo Cobre)/farmacología , Lathyrus/química , Neutrófilos/efectos de los fármacos , Adolescente , Adulto , Anciano , Femenino , Humanos , Peróxido de Hidrógeno/metabolismo , Inflamación/metabolismo , Masculino , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Proteínas de Plantas/metabolismo , Superóxidos/metabolismo , Adulto JovenRESUMEN
Because histamine is a modulator of cancer cell proliferation and invasiveness, this study aimed at investigating the effect of Lathyrus sativus-derived diamine oxidase (LSAO) and its mechanism of action on Caco-2 cell line, considering that LSAO catalizes the oxidative deamination of histamine to the corresponding aldehyde, NH3 and H2 O2 . Histamine (0.01-1 µM) caused a proliferative effect on Caco-2 cells promoting cell migration, invasion and nitric oxide and vascular endothelial growth factor release. Histamine (1 µM) stimulus also down regulated occludin expression, favouring up regulation of pro-proliferative nuclear protein Ki67. Incubation with LSAO (0.004-0.4 µM) resulted in a significant inhibition of histamine-induced effects. LSAO rescued occludin expression and down regulated Ki67, and it inhibited histamine-induced increase of both MMP-2 and 9 expression. Histamine effects were mediated by RhoA-GTP down regulation and inversely related to phospho-p38MAPK/p50/65 up regulation. These effects were counteracted by LSAO incubation. Histamine catabolism by LSAO accounts for a significant down regulation of proliferation and invasiveness of Caco-2 cells. This study highlights the importance to control histamine levels in contrasting pro-angiogenic and metastatization capability of colon cancer cells and expands the knowledge about the diamine oxidase from L. sativus seeding as a phytotherapeutic approach for colon cancer.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/farmacología , Neoplasias del Colon/tratamiento farmacológico , Lathyrus/enzimología , Neovascularización Patológica/tratamiento farmacológico , Células CACO-2 , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/irrigación sanguínea , Histamina , HumanosRESUMEN
Clostridium butyricum, the type species of the genus Clostridium, is considered an obligate anaerobe, yet it has been shown to grow in the presence of oxygen. C. butyricum strains atypically producing the botulinum neurotoxin type E are the leading cause of type E human botulism in Italy. Here, we show that type E botulinum neurotoxin-producing C. butyricum strains growing exponentially were able to keep growing and producing toxin in vitro upon exposure to air, although less efficiently than under ideal oxygen-depleted conditions. Bacterial growth in air was maintained when the initial cell density was higher than 103 cells/ml. No spores were detected in the cultures aerated for 5 h. To understand the biological mechanisms allowing the adaptation of vegetative cells of C. butyricum type E to oxygen, we compared the proteome and metabolome profiles of the clostridial cultures grown for 5 h under either aerated or anaerobic conditions. The results indicated that bacterial cells responded to oxygen stress by slowing growth and modulating the expression of proteins involved in carbohydrate uptake and metabolism, redox homeostasis, DNA damage response, and bacterial motility. Moreover, the ratio of acetate to butyrate was significantly higher under aeration. This study demonstrates for the first time that a botulinum neurotoxin-producing Clostridium can withstand oxygen during vegetative growth. IMPORTANCE Botulinum neurotoxins, the causative agents of the potentially fatal disease of botulism, are produced by certain Clostridium strains during vegetative growth, usually in anaerobic environments. Our findings indicate that, contrary to current understanding, the growth of neurotoxigenic C. butyricum strains and botulinum neurotoxin type E production can continue upon transfer from anaerobic to aerated conditions and that adaptation of strains to oxygenated environments requires global changes in proteomic and metabolic profiles. We hypothesize that aerotolerance might constitute an unappreciated factor conferring physiological advantages on some botulinum toxin-producing clostridial strains, allowing them to adapt to otherwise restrictive environments.
RESUMEN
Food biogenic amines, in particular, histamine, are often responsible for various enteric and vascular dysfunctions. Several years ago, the oral administration of copper-containing diamine oxidase (DAO), also called histaminase, able to oxidatively deaminate biogenic amines, had been suggested as a food supplement to control food allergy and enteric dysfunctions. This report is aimed to generate a global image on the behavior of orally administrated DAO dosage forms in the intestinal tract. The catalytic stability of DAO from Lathyrus sativus seedlings in various simulated intestinal media with different pH and containing different association of cholic acids, pancreatic proteases, bicarbonate, lipids, or alcohol was investigated. Cholic acids and lipids protected the enzyme in the simulated intestinal fluids. However, they were not able to protect against the inhibitory effect of 24-36% (v/v) ethanol. These observations may be relevant for oral administration of enzymes as food supplements or therapeutic bioactive agents.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/química , Ácidos Cólicos/metabolismo , Mucosa Intestinal/metabolismo , Lathyrus/enzimología , Proteínas de Plantas/química , Amina Oxidasa (conteniendo Cobre)/metabolismo , Aminas Biogénicas/metabolismo , Ácidos Cólicos/química , Estabilidad de Enzimas , Humanos , Concentración de Iones de Hidrógeno , Intestinos/química , Lathyrus/química , Lathyrus/metabolismo , Modelos Biológicos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Zymography on peroxidase-entrapped gels enables the determination of the intrinsic specific activity (ISA) of H2O2-producing oxidases in the presence of interfering enzymes including catalase (decomposing hydrogen peroxide). To reveal the searched oxidase, the zymography gel is incubated first in the developing solution containing the appropriate substrate of the oxidase and an oxidizable chromogen such as o-phenylenediamine (o-PDA) peroxidase substrate. The zymographic gel, after image analysis, can be restained by Coomassie Blue for molecular weights, for the whole electrophoretic pattern and for the protein concentration, thus allowing for the determination of oxidase ISA.
Asunto(s)
Electroforesis en Gel de Poliacrilamida , Oxidorreductasas , Proteínas , Animales , Catalasa/análisis , Catalasa/química , Bovinos , Activación Enzimática , Peróxido de Hidrógeno/química , Oxidorreductasas/análisis , Oxidorreductasas/química , Proteínas/análisis , Proteínas/química , Colorantes de Rosanilina , Espectrofotometría/métodos , Coloración y Etiquetado , Especificidad por SustratoRESUMEN
The purpose of this investigation was to elaborate a fast zymographic assay of oxidase enzymes in the presence of interfering enzymes as catalase (which disturbs current dosages based on H2O2 detection). This method also allows the determination of intrinsic specific activity (ISA) of oxidases, such as diamine oxidase (DAO) or glucose oxidase (GOD). The SDS-PAGE gels with entrapped peroxidase have been obtained by polymerization of acrylamide and bis-acrylamide in the presence of horse-radish peroxidase. The entrapped peroxidase was uniformly distributed in the PolyacrylAmide (PAA) material and did not migrate during electrophoresis. The obtained PAA gels allow the electrophoretic separation of various oxidases from contaminating proteins. As an example, to reveal DAO, the resulting PAA-gel should be incubated after the electrophoretic run in the developing solution containing putrescine (a DAO substrate) and o-phenylenediamine (a HRP substrate) to give coloured bands on the gel in the presence of DAO-generated H2O2. The results showed that is possible to determine the DAO in the presence of interfering catalase because they migrate differently. Thus, the H2O2 released in situ by DAO is no more decomposed by catalase because of its different mobility. It was also found that the same electrophoretic gel, after zymography, can be restained by Coomassie Blue for quantitation of proteins corresponding to the zymographic bands. With the obtained enzyme units and protein concentration it is also possible to calculate the intrinsic specific activity of DAO directly from the intensities of enzyme bands in zymography and from those of protein bands (Coomassie Blue staining), quantified by densitometry.