RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Scutia buxifolia is a tree native to South America and is used as a cardiotonic agent; however, this property has not been associated with a clear mechanism or a specific compound. AIM OF THE STUDY: Given the importance of Na(+),K(+)-ATPase as a drug target in the treatment of heart failure, this study aimed to investigate the possible inhibitory effect of S. buxifolia crude extract and fractions (dichloromethane, ethyl acetate, and butanolic fractions), and identified compounds with effects on the activity of Na(+),K(+)-ATPase in vitro. MATERIALS AND METHODS: First, we characterized the crude extract and fractions by high-performance liquid chromatography, and then monitored their effects on the activity of Na(+),K(+)-ATPase obtained from heart muscle and brain membranes of adult male Wistar rats. RESULTS: We identified gallic acid, chlorogenic acid, caffeic acid, rutin, quercitrin, quercetin, and ursolic acid in S. buxifolia stem bark and leaves; quercitrin and ursolic acid were the main compounds in the ethyl acetate and dichloromethane fractions from leaves and stem bark. The crude extract (3 and 30mg/ml), and the ethyl acetate and dichloromethane fractions (0.1 and 1mg/ml) of both the stem bark and leaves inhibited Na(+),K(+)-ATPase activity in heart and brain samples. We found that, of the identified compounds, only ursolic acid (0.1mg/ml) was able to diminish Na(+), K(+)-ATPase activity in heart and brain samples. CONCLUSIONS: These data indicated that the cardiotonic effects of S. buxifolia may be due to the inhibition of Na(+),K(+)-ATPase activity in heart muscle, supporting the popular use of this plant as a treatment for heart failure.
Assuntos
Miocárdio/enzimologia , Extratos Vegetais/farmacologia , Rhamnaceae/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Triterpenos/farmacologia , Animais , Encéfalo/metabolismo , Coração/efeitos dos fármacos , Masculino , Membranas/efeitos dos fármacos , Membranas/enzimologia , Casca de Planta/química , Folhas de Planta/química , Caules de Planta/química , Ratos , Ratos Wistar , Solventes , Ácido UrsólicoRESUMO
Dipeptidyl peptidase IV is an ectopeptidase with multiple physiological roles including the degradation of incretins, and a target of therapies for type 2 diabetes mellitus. Divalent cations can inhibit its activity, but there has been little effort to understand how they act. The intact membrane-bound form of porcine kidney dipeptidyl peptidase IV was purified by a simple and fast procedure. The purified enzyme hydrolyzed Gly-Pro-p-nitroanilide with an average V(max) of 1.397±0.003 µmol min(-1) mL(-1), k(cat) of 145.0±1.2 s(-1), K(M) of 0.138±0.005 mM and k(cat)/K(M) of 1050 mM(-1) s(-1). The enzyme was inhibited by bacitracin, tosyl-L-lysine chloromethyl ketone, and by the dipeptidyl peptidase IV family inhibitor L-threo-Ile-thiazolidide (K(i) 70 nM). The enzyme was inhibited by the divalent ions Ca(2+), Co(2+), Cd(2+), Hg(2+) and Zn(2+), following kinetic mechanisms of mixed inhibition, with K(i) values of 2.04×10(-1), 2.28×10(-2), 4.21×10(-4), 8.00×10(-5) and 2.95×10(-5) M, respectively. According to bioinformatic tools, Ca(2+) ions preferentially bound to the ß-propeller domain of the porcine enzyme, while Zn(2+) ions to the α-ß hydrolase domain; the binding sites were strikingly conserved in the human enzyme and other homologues. The functional characterization indicates that porcine and human homologues have very similar functional properties. Knowledge about the mechanisms of action of divalent cations may facilitate the design of new inhibitors.
Assuntos
Cátions Bivalentes/farmacologia , Dipeptidil Peptidase 4/metabolismo , Córtex Renal/enzimologia , Animais , Sítios de Ligação , Cálcio/metabolismo , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/isolamento & purificação , Inibidores da Dipeptidil Peptidase IV/farmacologia , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Íons , Cinética , Membranas/efeitos dos fármacos , Membranas/enzimologia , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Sus scrofa , Temperatura , Zinco/metabolismoRESUMO
Detergent-resistant membranes (DRMs) are a class of specialized microdomains that compartmentalize several signal transduction processes. In this work, DRMs were isolated from cerebral cortex synaptic endings (Syn) on the basis of their relative insolubility in cold Triton X-100 (1%). The lipid composition and marker protein content were analyzed in DRMs obtained from adult and aged animals. Both DRM preparations were enriched in Caveolin, Flotillin-1 and c-Src and also presented significantly higher sphingomyelin (SM) and cholesterol content than purified Syn. Total phospholipid-fatty acid composition presented an increase in 16:0 (35%), and a decrease in 20:4n-6 (67%) and 22:6n-3 (68%) content in DRM from adults when compared to entire synaptic endings. A more dramatic decrease was observed in the 20:4n-6 and 22:6n-3 content in DRMs from aged animals (80%) with respect to the results found in adults. The coexistence of phosphatidylcholine-specific-phospholipase C (PC-PLC) and phospholipase D (PLD) in Syn was previously reported. The presence of these signaling pathways was also investigated in DRMs isolated from adult and aged rats. Both PC-PLC and PLD pathways generate the lipid messenger diacylglycerol (DAG) by catalyzing PC hydrolysis. PC-PLC and PLD1 localization were increased in the DRM fraction. The increase in DAG generation (60%) in the presence of ethanol, confirmed that PC-PLC was also activated when compartmentalized in DRMs. Conversely, PLD2 was excluded from the DRM fraction. Our results show an age-related differential fatty acid composition and a selective localization of PC-derived signaling in synaptic DRMs obtained from adult and aged rats.
Assuntos
Detergentes/farmacologia , Fosfatidilcolinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Envelhecimento/metabolismo , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Diglicerídeos/metabolismo , Ácidos Graxos/metabolismo , Membranas/efeitos dos fármacos , Membranas/enzimologia , Ratos , Ratos Wistar , Sinapses/enzimologia , Fosfolipases Tipo C/metabolismoRESUMO
The significance of the coexistence of 2 mannose-6-phosphate receptors (MPRs) in most cell types still remains poorly understood. In this study, expression of the cation-dependent MPR (CD-MPR) and the cation-independent MPR (CI-MPR) was measured by Western blot in rat organs at 3 ages, i.e. in newborn and 10- and 90-day-old rats. It was observed that expression of the CI-MPR tends to diminish from newborns to adults in 5 of the 6 organs studied, whereas the CD-MPR did not show a clear tendency over time. In pancreas, conversely, both MPRs increased progressively from newborns to adults. The activity of 2 acid hydrolases was also measured at the different ages, and a low correlation was found with the expression of the 2 MPRs. With the exception of the pancreas, it is possible that the CI-MPR is mostly occupied with the clearance of insulin-like growth factor-II at early stages of development, and that later both MPRs may participate in the maturation of the lysosomal apparatus. We propose that in the pancreas, both receptors may be involved in increasing the proteolytic activity of this exocrine gland during postnatal development.
Assuntos
Crescimento e Desenvolvimento , Especificidade de Órgãos , Receptor IGF Tipo 2/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Hidrolases/metabolismo , Membranas/enzimologia , Ratos , Ratos Sprague-DawleyRESUMO
Copper is a divalent cation with physiological importance since deficiency of copper homeostasis can cause serious neurological diseases. ATP is an important signalling molecule stored at nerve endings and its inactivation is promoted by ecto-nucleotidases. In this study, we verified the effect of acute and subchronic copper treatments on ecto-nucleotidase activities in zebrafish brain membranes. Treatment with copper sulfate (15 microg/L) during 24h inhibited ATP hydrolysis (16%), whereas ADP and AMP hydrolysis were not altered. Nevertheless, a 96-h exposure with the copper concentration mentioned above inhibited NTPDase (31% and 42% for ATP and ADP hydrolysis, respectively) and ecto-5'-nucleotidase (40%) activities. NTPDase1, NTPDase2_mg and NTPDase2_mv transcripts were decreased after copper exposures during 24 and 96 h. Subchronic copper treatment also reduced the NTPDase2_mq and ecto-5'-nucleotidase expression. In vitro assays demonstrated that NTPDase activities were reduced after copper exposure during 40 min. ATP hydrolysis was inhibited at 0.25, 0.5 and 1mM (13%, 31% and 48%, respectively) and ADP hydrolysis also had a significant decrease at these same copper concentrations (41%, 63% and 68%, respectively). In contrast to the subchronic exposure, no significant changes on ecto-5'-nucleotidase were observed after in vitro assays. Lineweaver-Burk plots suggested that both inhibitory effects on nucleotide hydrolysis may occur in a non-competitive manner. Altogether, these findings indicate that copper is able to promote distinct changes on ecto-nucleotidases after in vivo and in vitro treatments and, consequently, it could control the nucleotide and nucleoside levels, modulating the purinergic signalling.
Assuntos
Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Encéfalo/efeitos dos fármacos , Cobre/toxicidade , Monofosfato de Adenosina/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hidrólise , Membranas/efeitos dos fármacos , Membranas/enzimologia , Membranas/metabolismo , RNA Mensageiro/metabolismo , Peixe-ZebraRESUMO
The development of perimicrovillar membranes (PMM) from midgut cells of starved and fed Dysdercus peruvianus was studied by using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and assays for specific enzymatic markers of the perimicrovillar membranes (alpha-glucosidase), perimicrovillar space (aminopeptidase) and microvillar membranes (beta-glucosidase). High activities of these enzymes were observed 6h post-feeding and significant production of membranes was observed at 30 h post-feeding. In the gut cells of starved insects, the rough endoplasmic reticulum was organized in concentric bundles, with a greater number of mitochondria in the cellular apex. The presence of electron dense double-membrane vesicles and the production of PMM were not observed in this condition. Thirty hours post-feeding, a disorganization of the rough endoplasmic reticulum was observed, and it was possible to see double-membrane vesicles close to the cell apex. The membrane system formation was evident with a significant development of PMM in the midgut lumen. The luminal surface of the midgut during starvation and up to 48 h post-feeding was monitored using SEM. It was demonstrated that in the starved condition, the PMM was virtually absent from gut cells, except at the base of the microvilli. At 6h post-feeding, the microvilli were already completely covered with PMM, but with a maximum of PMM formation seen at 30 h post-feeding. Signals of PMM degradation were observed 48 h after pulse feeding.
Assuntos
Sistema Digestório/metabolismo , Hemípteros/fisiologia , Aminopeptidases/metabolismo , Animais , Celulases/metabolismo , Sistema Digestório/enzimologia , Feminino , Hemípteros/enzimologia , Hemípteros/ultraestrutura , Membranas/enzimologia , Membranas/fisiologia , Membranas/ultraestrutura , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Inanição , alfa-Glucosidases/metabolismoRESUMO
NADH dehydrogenase-2 (NDH-2) from Escherichia coli respiratory chain is a membrane-bound cupric-reductase encoded by ndh gene. Here, we report that the respiratory system of a ndh deficient strain suffered a faster inactivation than that of the parental strain in the presence of tert-butyl hydroperoxide due to endogenous copper. The inactivation was similar for both strains when copper concentration increased in the culture media. Furthermore, several ndh deficient mutants grew less well than the corresponding parental strains in media containing either high or low copper concentrations. A mutant strain complemented with ndh gene almost recovered the parental phenotype for growing in copper limitation or excess. Then, NDH-2 gives the bacteria advantages to diminish the susceptibility of the respiratory chain to damaging effects produced by copper and hydroperoxides and to survive in extreme copper conditions. These results suggest that NDH-2 contributes in the bacterial oxidative protection and in the copper homeostasis.
Assuntos
Cobre/toxicidade , Escherichia coli/efeitos dos fármacos , Homeostase/efeitos dos fármacos , NADH Desidrogenase/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases/fisiologia , terc-Butil Hidroperóxido/toxicidade , Cobre/metabolismo , Meios de Cultura , Relação Dose-Resposta a Droga , Transporte de Elétrons/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Homeostase/fisiologia , Membranas/enzimologia , Mutação , NADH Desidrogenase/metabolismo , Estresse Oxidativo/fisiologia , Oxirredutases/metabolismo , Fenótipo , Fatores de Tempo , terc-Butil Hidroperóxido/metabolismoRESUMO
Carbofuran and malathion are broad spectrum pesticides widely used in agricultural practice throughout the world. Toxicity of these pesticides has been correlated with their inhibitory effects on acetylcholinesterase activity. Nucleotides are extracellular signaling molecules, which trigger multiple biological effects. Studies have demonstrated the co-transmission of acetylcholine and ATP at the nerve endings. The control of neurotransmitter ATP levels is promoted by enzymes named ectonucleotidases, which include nucleoside triphosphate diphosphohydrolase (NTPDase) family and ecto-5'-nucleotidase. Since acetylcholine and ATP are co-released at the synapse and the acetylcholinesterase inhibition is an important target for pesticide action, here we verified the effect of exposure in vitro and in vivo to carbofuran and malathion on ectonucleotidase activities from brain membranes of zebrafish. To verify if carbofuran and malathion have a direct inhibitory effect on NTPDase and 5'-nucleotidase activities in brain membranes of zebrafish, we have tested in vitro concentrations of pesticides varying from 0.25 to 5 mM. Carbofuran, in vitro, inhibited ATP and ADP hydrolysis in an uncompetitive manner, but no effect was observed on AMP hydrolysis. Malathion decreased ATP and ADP hydrolysis in competitive and an uncompetitive manner, respectively, but not altered AMP hydrolysis. After exposure to carbofuran (50 and 500 microg/L) during 7 days, ADP hydrolysis was significantly decreased in both concentrations tested (by 19 and 24.5%, respectively). Malathion, at 500 microg/L, was able to inhibit ADP and AMP hydrolysis (by 28 and 58.5%, respectively). This study has shown that ectonucleotidases from brain membranes of zebrafish can be a potential target for pesticide neurotoxicity.
Assuntos
5'-Nucleotidase/metabolismo , Encéfalo/efeitos dos fármacos , Carbofurano/toxicidade , Inibidores da Colinesterase/toxicidade , Inseticidas/toxicidade , Malation/toxicidade , Nucleosídeo-Trifosfatase/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/enzimologia , Hidrólise , Técnicas In Vitro , Membranas/efeitos dos fármacos , Membranas/enzimologia , Peixe-ZebraRESUMO
High continuous GH levels in vivo produce desensitization of the Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway of GH signaling in the liver. To evaluate the mechanisms involved in this desensitization, transgenic mice overexpressing bovine GH were used. In these animals, GH receptor and membrane-associated JAK2 kinase are increased 4.5- and 6-fold, respectively. However, JAK2. STAT5a and -5b do not become tyrosine phosphorylated in response to GH stimulus, nor are these STAT proteins recruited to membranes, suggesting that they cannot bind to the receptor. The content of the suppressor cytokine-inducible src homology 2 (SH2)-containing protein (CIS), both total and membrane-associated, is markedly increased in the liver of GH transgenic mice. This could account for the inhibition of STAT5 activation, because CIS competes with STAT5 for GH receptor docking sites. Existence of an alternative mechanism of negative regulation of this signaling pathway by chronically elevated GH levels is suggested by the low level of JAK2 phosphorylation that transgenic mice exhibit. Whereas total SH2-containing phosphatase 2 (SHP-2) content is the same in both kinds of mice, membrane-associated SHP-2 protein levels increase 4.5-fold in GH transgenic animals. This could explain the dramatic inhibition of JAK2 phosphotyrosine level, thus contributing to the suppression of GH signaling observed in these transgenic mice.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Hormônio do Crescimento/metabolismo , Proteínas do Leite , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas , Transdução de Sinais/fisiologia , Transativadores/antagonistas & inibidores , Animais , Bovinos , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Janus Quinase 2 , Fígado/metabolismo , Membranas/enzimologia , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos/metabolismo , Proteína Fosfatase 2 , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/metabolismo , Fator de Transcrição STAT5 , Proteínas Supressoras da Sinalização de CitocinaRESUMO
An ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) activity present in alkaline phosphatase-depleted rat osseous plate membranes, obtained 14 days after implantation of demineralized bone particles in the subcutaneous tissue of Wistar rats, was characterized. At pH 7.5, NTPDase1 hydrolyzed nucleotide triphosphates at rates 2.4-fold higher than those of nucleotide diphosphates, while the hydrolysis of nucleotide monophosphates and non-nucleotide phosphates was negligible. NTPDase 1 hydrolyzed ATP and ADP following Michaelis-Menten kinetics with V=1278.7+/-38.4 nmol Pi/min/mg and K(M)=83.3+/-2.5 microM and V=473.9+/-18.9 nmol Pi/min/mg and K(M)=150.6+/-6.0 microM, respectively, but in the absence of magnesium and calcium ions, ATP or ADP hydrolysis was negligible. The stimulation of the NTPDase1 by calcium (V=1084.7+/-32.5 nmol Pi/min/mg; and K(M)=377.8+/-11.3 microM) and magnesium (V=1367.2+/-41.0 nmol Pi/min/mg and K(M)=595.3+/-17.8 microM) ions suggested that each ion could replace the other during the catalytic cycle of the enzyme. Oligomycin, ouabain, bafilomycin A(1), theophylline, thapsigargin, ethacrynic acid, P(1),P(5)-(adenosine-5')-pentaphosphate and omeprazole had negligible effects on the hydrolysis of ATP and ADP by NTPDase1. However, suramin and sodium azide were effective inhibitors of ATP and ADP hydrolysis. To our knowledge this is the first report suggesting the presence of NTPDase1 in rat osseous plate membranes. Considering that the ectonucleoside triphosphate diphosphohydrolase family of enzymes participates in many regulatory functions, such as response to hormones, growth control, and cell differentiation, the present observations raise interesting questions about the participation of this activity in the calcification process.
Assuntos
Apirase/metabolismo , Lâmina de Crescimento/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Alameticina/farmacologia , Fosfatase Alcalina/deficiência , Animais , Antígenos CD , Apirase/antagonistas & inibidores , Apirase/química , Sítios de Ligação , Calcificação Fisiológica , Cálcio , Catálise/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Magnésio , Membranas/enzimologia , Ratos , Especificidade por SubstratoRESUMO
Calcium-dependent protein kinases (CDPKs), the most abundant serine/threonine kinases in plants, are found in various subcellular localizations, which suggests that this family of kinases may be involved in multiple signal transduction pathways. A complete analysis to try to understand the molecular basis of the presence of CDPKs in various localizations in the cell has not been accomplished yet. It has been suggested that myristoylation may be responsible for membrane association of CDPKs. In this study, we used a rice CDPK, OSCPK2, which has a consensus sequence for myristoylation at the N-terminus, to address this question. We expressed wild-type OSCPK2 and various mutants in different heterologous systems to investigate the factors that affect its membrane association. The results show that OSCPK2 is myristoylated and palmitoylated and targeted to the membrane fraction. Both modifications are required, myristoylation being essential for membrane localization and palmitoylation for its full association. The fact that palmitoylation is a reversible modification may provide a mechanism for regulation of the subcellular localization. OSCPK2 is the first CDPK shown to be targeted to membranes by an src homology domain 4 (SH4) located at the N-terminus of the molecule.
Assuntos
Membranas/enzimologia , Ácido Mirístico/metabolismo , Oryza/enzimologia , Ácidos Palmíticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Biológico , Cálcio/farmacologia , DNA Recombinante/genética , DNA Recombinante/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Mutação , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Protoplastos/metabolismo , Zea mays/genéticaRESUMO
Previous results from our laboratory have shown that NADH-supported electron flow through the Escherichia coli respiratory chain promotes the reduction of cupric ions to Cu(I), which mediates damage of the respiratory system by hydroperoxides. The aim of this work was to characterize the NADH-linked cupric reductase activity from the E. coli respiratory chain. We have used E. coli strains that either overexpress or are deficient in the NADH dehydrogenase-2 (NDH-2) to demonstrate that this membrane-bound protein catalyzes the electron transfer from NADH to Cu(II), but not to Fe(III). We also show that purified NDH-2 exhibits NADH-supported Cu(II) reductase activity in the presence of either FAD or quinone, but is unable to reduce Fe(III). The K(m) values for free Cu(II) were 32 +/- 5 pM in the presence of saturating duroquinone and 22 +/- 2 pM in the presence of saturating FAD. The K(m) values for NADH were 6.9 +/- 1.5 microM and 6.1 +/- 0.7 microM in the presence of duroquinone and FAD, respectively. The quinone-dependent Cu(II) reduction occurred through both O(*-)(2)-mediated and O(*-)(2)-independent pathways, as evidenced by the partial inhibitory effect (30-50%) of superoxide dismutase, by the reaction stoichiometry, and by the enzyme turnover numbers for NADH and Cu(II). The cupric reductase activity of NDH-2 was dependent on thiol groups which were accessible to p-chloromercuribenzoate at low, but not at high, ionic strength of the medium, a fact apparently connected to a conformational change of the protein. To our knowledge, this is the first protein with cupric reductase activity to be isolated and characterized in its biochemical properties.
Assuntos
Escherichia coli/enzimologia , FMN Redutase , Oxirredutases/metabolismo , Sítios de Ligação , Transporte de Elétrons , Cinética , Membranas/enzimologia , NAD/metabolismo , NADH Desidrogenase/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oxirredutases/química , Oxirredutases/isolamento & purificação , Conformação Proteica , Reagentes de Sulfidrila/farmacologia , Superóxidos/metabolismo , Ácido p-Cloromercurobenzoico/farmacologiaRESUMO
1. The in vitro effect of valproic acid (VA) (10(-6) to 10(-3) M) on glutamic acid decarboxylase (GAD) activity in whole brain and cerebral cortex (CC) of neonates and of adult rats was examined. 2. VA did not induce changes on GAD activity either in CC or in the rest of the brain (RB) of adult animals. 3. But at 10(-3) M, VA induced an increase in GAD activity in homogenates of noncortical brain areas of neonates; no increments were found in CC of these animals. This latter increase was detected in the membrane-bound fraction of the enzyme and was not due to physicochemical nonspecific changes related to the potential solvent activity of VA at this high concentration. 4. We may conclude that VA induces changes on GAD activity in neonatal stages of development but not in adult brain. Therefore, although a direct enhancement of GAD activity may play a role in the mechanism of action of VA in pediatric patients, this cannot be verified in the adult population.
Assuntos
Animais Recém-Nascidos/metabolismo , Encéfalo/enzimologia , Inibidores Enzimáticos/farmacologia , Glutamato Descarboxilase/antagonistas & inibidores , Ácido Valproico/farmacologia , Envelhecimento , Animais , Encéfalo/crescimento & desenvolvimento , Técnicas In Vitro , Masculino , Membranas/efeitos dos fármacos , Membranas/enzimologia , Ratos , Ratos WistarRESUMO
Ecto-nucleotidases may have a role in the regulation of purinoceptor-mediated responses. ATP-diphosphohydrolase or apyrase has been described as an ecto-nucleotidase, which is characterized by a low specificity for its substrates and bivalent cations. The aim of this work was to demonstrate the presence of apyrase as an ecto-enzyme in the rat kidney. ATPase-ADPase activities of the renal microvillar membrane preparation, which correspond to "right side out' membranes, were characterized. The detection of ATP-diphosphohydrolase in the renal vasculature was done through perfusion of isolated rat kidney. ATPase-ADPase activities of the microvillar membrane preparation and apyrase share similar kinetic properties. These include: low substrate and bivalent metal specificities and insensitivity towards inhibitors like: oligomycin, ouabain, verapamil, levamisole and Ap5A. The M(r) or native ATPase and ADPase activities was determined by the 60Co irradiation-inactivation technique being around 65 kDa for both hydrolytic activities. Immunowestern blot analysis also supports the presence of apyrase in microvilli. Perfusion of isolated rat kidney with ATP and ADP, in the presence or absence of different inhibitors or apyrase antibodies indicated the existence of this enzyme in the vascular endothelium. The identification of ATP-diphosphohydrolase as an ecto-enzyme both in microvilli and vasculature support the proposal that the enzyme may have an important role in the extracellular metabolism of nucleotides.
Assuntos
Apirase/metabolismo , Endotélio Vascular/enzimologia , Rim/enzimologia , Adenosina Trifosfatases/metabolismo , Animais , Fenômenos Químicos , Físico-Química , Endotélio Vascular/ultraestrutura , Focalização Isoelétrica , Rim/irrigação sanguínea , Rim/ultraestrutura , Cinética , Membranas/enzimologia , Microvilosidades/enzimologia , Perfusão , Ratos , SolubilidadeRESUMO
Biochemical abnormalities have been implicated in possible mechanisms underlying the epileptic phenomena. Some of these alterations include changes in the activity of several enzymes present in epileptic tissues. Systemic administration of pilocarpine in rats induces electrographic and behavioral limbic seizures and status epilepticus, that is followed by a transient seizure-free period (silent period). Finally a chronic phase ensues, characterized by spontaneous and recurrent seizures (chronic period), that last for the rest of the animal's life. The present work aimed to study the activity of the enzyme Na+K+ ATPase, in rat hippocampus, during the three phases of this epilepsy model. The enzyme activity was determined at different time points from pilocarpine administration (1 and 24 h of status epilepticus, during the silent and chronic period) using a spectrophotometric assay previously described by Mishra and Delivoria-Papadopoulos [Neurochem. Res. (1988) 13, 765-770]. The results showed decreased enzyme activities during the acute and silent periods and increased Na+K+ ATPase activity during the chronic phase. These data show that changes in Na+K+ ATPase activity could be involved in the appearance of spontaneous and recurrent seizures following brain damage induced by pilocarpine injection.
Assuntos
Epilepsia/enzimologia , Hipocampo/enzimologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Modelos Animais de Doenças , Epilepsia/induzido quimicamente , Masculino , Membranas/enzimologia , Pilocarpina , Ratos , Ratos WistarRESUMO
The cell wall-less fz, sg, os-1 ("slime") triple mutant of Neurospora crassa lacks (1,3)-beta-D-glucan synthase activity. fz, sg, os-1 segregants from slime x wild-type crosses initially germinate as a plasmodium (slime-like), but develop hyphae in a few hours and acquire a stable mycelial phenotype (mycelial intermediate). The cell wall-less phenotype (stable slime) can be reisolated from mycelial intermediates by filtration-enrichment selection in medium of high osmolarity. Pairs of mycelial intermediate and stable slime obtained from a single slime-like segregant were comparatively studied. Mycelial intermediate strains synthesize a cell wall with normal amounts of (1,3)-beta-glucan, chitin, and other polysaccharides and possess (1,3)-beta-glucan synthase activity with apparently normal properties (i.e., association with membranes, stability, Km app, Vmax, stimulation by GTP). The enzyme was dissociated by treatment with Tergitol NP-40 and NaCl into a membrane-bound catalytic center and a soluble factor which activates the enzyme in the presence of GTP. Heterologous reconstitution assays demonstrated that stable slime spheroplasts had normal activity of the soluble activating factor, but were severely deficient in membrane-bound activity. The genetic composition of the viable progeny of stable slime or mycelial intermediate x wild-type crosses failed to show differences between the two extreme phenotypes of slime. However, the analysis of heterokaryons demonstrated that the stable slime homokaryotic progeny of stable slime/wild-type heterokaryons were not viable. In contrast, the behavior of mycelial intermediate/wild-type heterokaryons was normal. Apparently, stable slime strains differed from the original mycelial intermediate in a mutation(s) which arose spontaneously during the filtration-enrichment selection applied to mycelial intermediates in order to obtain the cell wall-less phenotype. This new trait impaired conidial germination and might be the actual cause of the loss of (1,3)-beta-glucan synthase activity and cell wall.
Assuntos
Parede Celular/genética , Glucosiltransferases/deficiência , Proteínas de Membrana , Mutação , Neurospora crassa/enzimologia , Proteínas de Schizosaccharomyces pombe , Fracionamento Celular/métodos , Parede Celular/química , Contagem de Colônia Microbiana , Cruzamentos Genéticos , Teste de Complementação Genética , Hexosaminas/análise , Hexoses/análise , Membranas/enzimologia , Fenótipo , Polissacarídeos/químicaRESUMO
Ursodeoxycholic acid and its endogenous metabolite tauroursodeoxycholic acid inhibited in vitro the microsomal bilirubin UDP-glucuronosyltransferase from rat liver. The magnitude of the inhibition correlated well with the loss of integrity of microsomal vesicles, suggesting that bile salts needed to reach the lumen to exert their inhibitory effects. The endogenous bile acids cholic acid, chenodeoxycholic acid and deoxycholic acid also exhibited inhibitory effects on bilirubin glucuronidation in digitonin-disrupted microsomes. Ursodeoxycholic acid inhibitory capacity was similar to that of chenodeoxycholic acid and deoxycholic acid but greater than that of cholic acid, the major endogenous bile salt. Kinetic studies, performed in detergent-activated preparations, showed that the inhibitions produced by ursodeoxycholic and tauroursodeoxycholic acids were competitive toward both bilirubin and UDP-glucuronic acid. The estimated Ki(app) for both substrates did not differ statistically between ursodeoxycholic and tauroursodeoxycholic acids. Both bile salts were weak inhibitors toward bilirubin but rather strong inhibitors toward UDP-glucuronic acid.
Assuntos
Glucuronosiltransferase/antagonistas & inibidores , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Ácido Ursodesoxicólico/farmacologia , Animais , Ácidos e Sais Biliares/farmacologia , Digitonina/farmacologia , Ativação Enzimática , Glucuronatos/metabolismo , Glucuronosiltransferase/metabolismo , Cinética , Masculino , Membranas/efeitos dos fármacos , Membranas/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Ratos , Ratos Wistar , Ácido Tauroquenodesoxicólico/farmacologiaRESUMO
Lectins from Lens culinaris and Arachis hypogaea immobilized on polyacrylamide beads were used for selective isolation of glycosylated surface membrane domains of adult Schistosoma mansoni worms, and the method was compared with the membrane isolation procedure developed with polycationic (Affi-Gel) beads. The lentil lectin proved to be suitable for interaction with surface membrane components: an increment in the specific activities of tegumental phosphohydrolases was observed in the bound fraction with respect to that observed in a total worm homogenate. A characteristic polypeptide pattern on gel electrophoresis was also seen, more restricted than that obtained with the bound Affi-Gel fraction. Immobilized peanut lectin was not successful as a method for isolating membrane material from the tegument of adult worms. Solubilization and dissociation of the lentil lectin-bound enzyme markers was achieved after addition of detergent and competing sugars. Glycosylation of the solubilized enzymes was further confirmed by affinity chromatography with fresh lentil lectin-coated beads. These results, together with histochemical evidences, suggest that the active sites of some of these enzymes are located within or close to the cytoplasmic leaflet of the surface tegumental membranes, and allow us to propose a model for the double surface membrane complex where some proteins may be crossing the two bilayers.
Assuntos
Lectinas/metabolismo , Membranas , Schistosoma mansoni/anatomia & histologia , Animais , Fracionamento Químico/instrumentação , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Enzimas Imobilizadas , Membranas/anatomia & histologia , Membranas/enzimologia , Membranas/metabolismo , Camundongos , Microesferas , Proteínas/análise , Schistosoma mansoni/metabolismoRESUMO
Canatoxin, a toxic protein isolated from the seeds of Canavalia ensiformis, was studied for its effects on the sarcoplasmic reticulum vesicles obtained from rabbit skeletal muscle. Canatoxin inhibited Ca2+ accumulation catalysed by the Ca(2+)-ATPase, without affecting the hydrolytic activity of this enzyme or membrane permeability to Ca2+. The effects of canatoxin were dose dependent, but not time dependent. It is concluded that canatoxin interacts with the Ca2+ pump and uncouples Ca2+ uptake from Ca(2+)-dependent ATP hydrolysis.
Assuntos
ATPases Transportadoras de Cálcio/antagonistas & inibidores , Lectinas/farmacologia , Proteínas de Plantas , Retículo Sarcoplasmático/enzimologia , Toxinas Biológicas , Trifosfato de Adenosina/metabolismo , Animais , ATPase de Ca(2+) e Mg(2+)/metabolismo , Cálcio/metabolismo , Técnicas In Vitro , Membranas/efeitos dos fármacos , Membranas/enzimologia , Músculos/efeitos dos fármacos , Músculos/metabolismo , Coelhos , Retículo Sarcoplasmático/efeitos dos fármacosRESUMO
1. Matrix-induced alkaline phosphatase prepared from rat osseous plate was solubilized with polidocanol and purified on a Sephacryl S-300 column. 2. Purified solubilized alkaline phosphatase has a molecular weight of ca 115,000 and bind one magnesium and two zinc ions. At least 110 detergent molecules are bound to each enzyme molecule. 3. Solubilization and purification procedures did not destroy the ability of the enzyme to hydrolyze adenosine-5'-triphosphate, p-nitrophenylphosphate, pyrophosphate and bis p-nitrophenylphosphate. 4. Magnesium, manganese and cobalt ions are stimulators of PNPPase activity of solubilized enzyme whereas calcium and zinc ions are inhibitors.