RESUMO
Giardiasis represents a latent problem in public health due to the exceptionally pathogenic strategies of the parasite Giardia lamblia for evading the human immune system. Strains resistant to first-line drugs are also a challenge. Therefore, new antigiardial therapies are urgently needed. Here, we tested giardial arginine deiminase (GlADI) as a target against giardiasis. GlADI belongs to an essential pathway in Giardia for the synthesis of ATP, which is absent in humans. In silico docking with six thiol-reactive compounds was performed; four of which are approved drugs for humans. Recombinant GlADI was used in enzyme inhibition assays, and computational in silico predictions and spectroscopic studies were applied to follow the enzyme's structural disturbance and identify possible effective drugs. Inhibition by modification of cysteines was corroborated using Ellman's method. The efficacy of these drugs on parasite viability was assayed on Giardia trophozoites, along with the inhibition of the endogenous GlADI. The most potent drug against GlADI was assayed on Giardia encystment. The tested drugs inhibited the recombinant GlADI by modifying its cysteines and, potentially, by altering its 3D structure. Only rabeprazole and omeprazole decreased trophozoite survival by inhibiting endogenous GlADI, while rabeprazole also decreased the Giardia encystment rate. These findings demonstrate the potential of GlADI as a target against giardiasis.
Assuntos
Giardia lamblia/efeitos dos fármacos , Giardíase/tratamento farmacológico , Hidrolases/metabolismo , Animais , Antiprotozoários/farmacologia , Simulação por Computador , Cisteína/química , Avaliação Pré-Clínica de Medicamentos/métodos , Reposicionamento de Medicamentos/métodos , Giardia lamblia/patogenicidade , Giardíase/imunologia , Tiomalato Sódico de Ouro/farmacologia , Humanos , Hidrolases/efeitos dos fármacos , Hidrolases/ultraestrutura , Omeprazol/farmacologia , Inibidores da Bomba de Prótons/farmacologia , Rabeprazol , Tiamina/análogos & derivados , Tiamina/farmacologia , Trofozoítos/efeitos dos fármacosRESUMO
Dysfunctions in NAD+ metabolism are associated with neurodegenerative diseases, acute brain injury, diabetes, and aging. Loss of NAD+ levels results in impairment of mitochondria function, which leads to failure of essential metabolic processes. Strategies to replenish depleted NAD+ pools can offer significant improvements of pathologic states. NAD+ levels are maintained by two opposing enzymatic reactions, one is the consumption of NAD+ while the other is the re-synthesis of NAD+. Inhibition of NAD+ degrading enzymes, poly-ADP-ribose polymerase 1 (PARP1) and ectoenzyme CD38, following brain ischemic insult can provide neuroprotection. Preservation of NAD+ pools by administration of NAD+ precursors, such as nicotinamide (Nam) or nicotinamide mononucleotide (NMN), also offers neuroprotection. However, NMN treatment demonstrates to be a promising candidate as a therapeutic approach due to its multi-targeted effect acting as PARP1 and CD38 inhibitor, sirtuins activator, mitochondrial fission inhibitor, and NAD+ supplement. Many neurodegenerative diseases or acute brain injury activate several cellular death pathways requiring a treatment strategy that will target these mechanisms. Since NMN demonstrated the ability to exert its effect on several cellular metabolic pathways involved in brain pathophysiology it seems to be one of the most promising candidates to be used for successful neuroprotection.
Assuntos
Encéfalo/efeitos dos fármacos , Hidrolases/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mononucleotídeo de Nicotinamida/farmacologia , Animais , Encéfalo/metabolismo , Humanos , Hidrolases/metabolismo , Mitocôndrias/metabolismo , NAD/efeitos dos fármacos , NAD/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Niacinamida/metabolismo , Niacinamida/farmacologia , Mononucleotídeo de Nicotinamida/metabolismoRESUMO
OBJECTIVES: Exogenous phytase improved the activity of hydrolases to decrease the malting time. RESULTS: Treatment with phytase during barley steeping increased activity of hydrolases (α-/ß-amylase, proteinase, ß-glucanase and xylanase) in green malt. Maximal activity was observed for α-/ß-amylase, ß-glucanase and xylanase with 0.8 U phytase/g and proteinase with 1.2 U phytase/g. Phytase promoted acrospire growth of green malt and reduced malting process with 0.8 U phytase/g in 96 h, which is 24 h less than the control. No significant variation of malt quality was found between control malt and malt treated with 0.8 U/g or 1.2 U phytase/g (P > 0.05), which makes application of exogenous phytase during steeping process as a good option for reducing malting time. CONCLUSION: Adding phytase during steeping process increases the activity of hydrolases, which reduces malting time without impacting on malt quality.
Assuntos
6-Fitase , Manipulação de Alimentos/métodos , Germinação/efeitos dos fármacos , Hordeum , Hidrolases , 6-Fitase/metabolismo , 6-Fitase/farmacologia , Grão Comestível , Hordeum/efeitos dos fármacos , Hordeum/enzimologia , Hordeum/metabolismo , Hidrolases/efeitos dos fármacos , Hidrolases/metabolismo , Fatores de TempoRESUMO
AIMS: The tryptophan metabolites 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HAA) inhibit the liver mitochondrial low Km aldehyde dehydrogenase and possess alcohol-aversive and immunosuppressant properties. As the disulfiram (DS) metabolite carbon disulphide activates enzymes forming 3-HK and 3-HAA, we investigated if repeated disulfiram treatment increases the hepatic and serum levels of these 2 metabolites. METHODS: Livers and sera of male Wistar rats were analysed for tryptophan and kynurenine metabolites after repeated DS treatment for 7 days. RESULTS: DS increased liver and serum [3-HK] and [3-HAA] possibly by increasing the flux of tryptophan down the hepatic kynurenine pathway and activation of kynurenine hydroxylase and kynureninase. CONCLUSIONS: We provisionally suggest that elevation of some kynurenine metabolites may be an additional mechanism of the alcohol-aversive and anticancer effects of disulfiram.
Assuntos
Ácido 3-Hidroxiantranílico/metabolismo , Dissuasores de Álcool/farmacologia , Dissulfiram/farmacologia , Cinurenina/análogos & derivados , Fígado/efeitos dos fármacos , Animais , Cromatografia Líquida de Alta Pressão , Hidrolases/efeitos dos fármacos , Hidrolases/metabolismo , Cinurenina/efeitos dos fármacos , Cinurenina/metabolismo , Quinurenina 3-Mono-Oxigenase/efeitos dos fármacos , Quinurenina 3-Mono-Oxigenase/metabolismo , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar , Transaminases/efeitos dos fármacos , Transaminases/metabolismo , Triptofano/efeitos dos fármacos , Triptofano/metabolismoRESUMO
The effect of postharvest 1-methylcyclopropene and/or cold storage application on texture quality parameters during storage was determined. The changes in fruit quality (including weight loss, firmness, total soluble solids content, and ethylene production), cell wall material (including water-soluble fraction, ethylenediaminetetraacetic acid-soluble fraction, Na2CO3-soluble fraction, 4% KOH-soluble fraction, and 14% KOH-soluble fraction), and cell wall hydrolase activities (including polygalacturonase, endo-1,4-beta-D-glucanase, pectinesterase, alpha-L-arabinofuranosidase, and beta-galactosidase) were periodically measured up to 25 days after postharvest treatments. The application of cold storage reduced weight loss, ethylene production, and delayed ripening of blueberry fruit. The inhibition of senescence was associated with suppressed increase in cell wall hydrolase activities and retarded solubilization of pectins and hemicelluloses. Furthermore, no obvious differences in firmness, weight loss, ethylene production, and cell wall hydrolase activities between fruits with or without 1-methylcyclopropene application were observed, while significant lower levels of the detected parameters were found in cold storage fruit compared with fruit stored in room temperature. Thus, cold storage can be viewed as an effective means to extend the shelf life of blueberry fruit.
Assuntos
Mirtilos Azuis (Planta)/química , Parede Celular/química , Temperatura Baixa , Ciclopropanos/farmacologia , Qualidade dos Alimentos , Armazenamento de Alimentos/métodos , Análise de Variância , Mirtilos Azuis (Planta)/efeitos dos fármacos , Hidrolases de Éster Carboxílico/análise , Hidrolases de Éster Carboxílico/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Etilenos/análise , Hidrolases/análise , Hidrolases/efeitos dos fármacos , Pectinas/análise , Poligalacturonase/análise , Poligalacturonase/efeitos dos fármacos , Polissacarídeos/análise , Fatores de TempoRESUMO
The chemical properties of the B(6) vitamers are uniquely suited for wide use as cofactors in essential reactions, such as decarboxylations and transaminations. This review addresses current efforts to explore vitamin B(6) dependent enzymatic reactions as drug targets. Several current targets are described that are found amongst these enzymes. The focus is set on diseases caused by protozoan parasites. Comparison across a range of these organisms allows insight into the distribution of potential targets, many of which may be of interest in the development of broad range anti-protozoan drugs. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.
Assuntos
Enzimas/metabolismo , Infecções por Protozoários/tratamento farmacológico , Fosfato de Piridoxal/metabolismo , Animais , Aspartato Aminotransferases/efeitos dos fármacos , Aspartato Aminotransferases/metabolismo , Liases de Carbono-Enxofre/efeitos dos fármacos , Liases de Carbono-Enxofre/metabolismo , Cisteína Sintase/efeitos dos fármacos , Cisteína Sintase/metabolismo , Glicina Hidroximetiltransferase/efeitos dos fármacos , Glicina Hidroximetiltransferase/metabolismo , Humanos , Hidrolases/efeitos dos fármacos , Hidrolases/metabolismo , Ornitina Descarboxilase/efeitos dos fármacos , Ornitina Descarboxilase/metabolismo , Infecções por Protozoários/enzimologia , Infecções por Protozoários/metabolismo , Trypanosoma cruzi/enzimologiaRESUMO
BACKGROUND: Aim of this study is investigates the influence of spiperone on hydrolase activity pathway in chronic obstructive pulmonary disease (COPD). PATIENTS AND METHODS: Differentially expressed genes (DEGs) were calculated by the limma package from microarray data GSE20257, and analysed via gene set enrichment analysis (GSEA) for identifying COPD related pathways. The regulation of hydrolase activity pathway related drugs was predicted by connectivity Map analysis (CMap). Western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR) were used to investigate the effect of spiperone on regulation of hydrolase activity pathway in vitro experiment. RESULTS: A total of 378 DEGs were identified by the limma package. GSEA suggested that the regulation of hydrolase activity pathway was involved in the development of COPD. CMap of hub genes of regulation of hydrolase activity pathwayshown the most significant compound was spiperone. Results of vitro experiment verify that cigarette smoke extract (CSE) can increase the expression of fibronectin 1 (FN1) and epidermal growth factor (EGF), coinsided with decrease the expression of chemokine (C-X3-C motif) ligand 1 (CX3CL1), chemokoine (C-C motif) ligand 20 (CCL20), complement component 3 (C3) and slithomolog 2 (SLIT2) in BESA-2B cells and U937 cells. Spiperone can reverse the effect of CSE in BESA-2B cells and U937 cells. CONCLUSION: Regulation of hydrolase activity pathway was involved in the occurrence of COPD, spiperone was a potential drug for the treatment of COPD by affecting the regulation of hydrolase activity pathway. This study had provided new insights into the potential pathogenesis and treatment of COPD.
Assuntos
Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Espiperona/uso terapêutico , Adulto , Western Blotting , Feminino , Humanos , Hidrolases/efeitos dos fármacos , Hidrolases/metabolismo , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma , Células U937RESUMO
The selective serotonin reuptake inhibitor fluoxetine has been used for the treatment of depression. Although sexual disorders have been reported in male patients, few studies have demonstrated the fluoxetine effect on the reproductive histophysiology, and the target of this antidepressant in testes is unknown. We evaluated the impact of short-term treatment with fluoxetine on the adult rat testes, focusing on steroidogenesis by Leydig cells (LC) and androgen-dependent testicular parameters, including Sertoli cells (SC) and peritubular myoid cells (PMC). Since UCHL1 (ubiquitincarboxyl-terminal hydrolase L1) seems to control spermatogenesis, the immunoexpression of this hydrolase was also analyzed. Adult male rats received 20 mg/kg BW of fluoxetine (FG) or saline (CG) for eleven days. In historesin-embedded testis sections, the seminiferous tubule (ST) and epithelial (Ep) areas, and the LC nuclear diameter (LCnu) were measured. The number of abnormal ST, androgen-dependent ST, SC and PMC was quantified. Testicular ß-tubulin levels and peritubular actin immunofluorescence were evaluated. Serum testosterone levels (STL) and steroidogenesis by 17ß-HSD6 immunofluorescence were analyzed, and either UCHL1-immunolabeled or TUNEL-positive germ cells were quantified. In FG, abnormal ST frequency increased whereas ST and Ep areas, androgen-dependent ST number, LCnu, 17ß-HSD6 activity and STL reduced significantly. TUNEL-positive PMC and SC was related to decreased number of these cells and reduction in peritubular actin and ß-tubulin levels. In FG, uncommon UCHL1-immunoexpression was found in spermatocytes and spermatids, and the number of UCHL1-immunolabeled and TUNEL-positive germ cells increased in this group. These findings indicate that LC may be a fluoxetine target in testes, impairing PMC-SC integrity and disturbing spermatogenesis. The increase of UCHL1 in the damaged tubules associated with high incidence of cell death confirms that this hydrolase regulates germ cell death and may be controlled by androgens. The fertility in association with the androgenic status of patients treated with fluoxetine should be carefully evaluated.
Assuntos
Androgênios/metabolismo , Morte Celular/efeitos dos fármacos , Fluoxetina/farmacologia , Células Germinativas/efeitos dos fármacos , Túbulos Seminíferos/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Animais , Células Germinativas/metabolismo , Hidrolases/efeitos dos fármacos , Hidrolases/metabolismo , Marcação In Situ das Extremidades Cortadas/métodos , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Ratos , Túbulos Seminíferos/metabolismo , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/metabolismo , Ubiquitinas/metabolismoRESUMO
Altered membrane integrity and inflammation play a key role in cardiovascular damage. We investigated the salubrious effect of exogenously administered alpha-mangostin against beta-adrenergic cathecolamine-induced cardiovascular toxicity with special reference to membrane ATPases, lysosomal hydrolases and inflammatory mediators TNF-alpha and Cyclooxygenase-2 (COX-2) expressions in albino rats. Induction of rats with isoproterenol (150mg/kg body wt, i.p.) for 2 days resulted in a significant increase in the activities of serum and cardiac lysosomal hydrolases (beta-d-glucuronidase, beta-d-galactosidase, beta-d-N-acetylglucosaminidase, acid phosphatase and cathepsin-D). A significant increase in cardiac levels of sodium, calcium with a decrease in the level of potassium paralleled by abnormal activities of membrane-bound phosphatases (Na(+)-K(+) ATPase, Ca(2+) ATPase and Mg(2+) ATPase) were observed in the heart of ISO-administered rats. Cardiac TNF-alpha and COX-2 expressions were assessed by Western blotting. Cardiac TNF-alpha and COX-2 expressions were significantly elevated in ISO-intoxicated rats. Pre-co-treatment with alpha-mangostin (200mg/kg body wt.) orally for 8 days significantly attenuated these abnormalities and restored the levels to near normalcy when compared to ISO intoxicated group of rats. In conclusion, alpha-mangostin preserves the myocardial membrane integrity and extenuates anomalous TNF-alpha and COX-2 expressions by mitigating ISO-induced oxidative stress and cellular damage effectively. Restoration of cellular normalcy accredits the cytoprotective role of alpha-mangostin.
Assuntos
Ciclo-Oxigenase 2/efeitos dos fármacos , Garcinia mangostana , Isquemia Miocárdica/prevenção & controle , Fitoterapia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Xantonas/farmacologia , Agonistas Adrenérgicos beta/toxicidade , Animais , Western Blotting , Ciclo-Oxigenase 2/biossíntese , Garcinia mangostana/química , Coração/efeitos dos fármacos , Hidrolases/análise , Hidrolases/efeitos dos fármacos , Isoproterenol/toxicidade , Masculino , Isquemia Miocárdica/induzido quimicamente , Extratos Vegetais/farmacologia , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Aframomum melegueta (alligator pepper (AP)) and Aframomum danielli (bastered melegueta (BM)) seeds have been known to improve sexual function in folkloric medicine. This study investigates the effects of AP and BM seeds' alkaloid extracts on the activities of enzymes (acetylcholinesterase (AChE), angiotensin-1-converting enzyme (ACE), phosphodiesterase-5 (PDE-5), and arginase) relevant to erectile dysfunction (ED). Alkaloids from the seeds were prepared by the solvent extraction method and their interactions with AChE, ACE, PDE-5, and arginase were assessed. Gas chromatographic (GC) analyses of the extracts were also performed. The results revealed that the extracts inhibited the enzymes in a concentration-dependent manner. However, alkaloid extract from AP seed had higher AChE (IC50 = 5.42 µg/mL) and ACE (IC50 = 12.57 µg/mL) but lower PDE-5 (IC50 = 33.80 µg/mL) and arginase (IC50 = 31.36 µg/mL) inhibitory effects when compared to that of BM extract (AChE, IC50 = 42.00; ACE, IC50 = 60.67, PDE-5, IC50 = 7.24; and arginase, IC50 = 2.53 µg/mL). The GC analyses revealed the presence of senkirkine, angustifoline, undulatine, myristicin, safrole, lupanine, powelle, and indicine-N-oxide, among others. The inhibition of these enzymes could be the possible mechanisms by which the studied seeds were being used in managing ED in folklores. Nevertheless, the seed of AP exhibited higher potentials.
Assuntos
Alcaloides/farmacologia , Disfunção Erétil/tratamento farmacológico , Hidrolases/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/farmacologia , Zingiberaceae/química , Acetilcolinesterase/efeitos dos fármacos , Animais , Arginase/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/efeitos dos fármacos , Disfunção Erétil/enzimologia , Masculino , Peptidil Dipeptidase A/efeitos dos fármacos , Extratos Vegetais/química , Ratos , Ratos Wistar , Zingiberaceae/classificaçãoRESUMO
BACKGROUND: Arginine deiminase (ArcA) has been speculated to facilitate the intracellular survival of Streptococcus suis under acidic conditions. However, the physical and biological properties and function of SS2-ArcA have not yet been elucidated. METHODS: Recombinant SS2-ArcA (rSS2-ArcA) was expressed and purified using Ni-NTA affinity chromatography. Under various pH and temperature conditions, the enzymatic properties of purified rSS2-ArcA and crude native SS2-ArcA were determined. RESULTS: The SS2-arcA-deduced amino acid sequence contained a conserved catalytic triad (Cys399-His273-Glu218). The optimum temperature and pH of 47-kDa rSS2-ArcA and crude native SS2-ArcA were 42°C and pH 7.2. The rSS2-ArcA and crude native SS2-ArcA were stable for 3 h at 4 and 25°C, respectively. The pH stability and dependency tests suggested that rSS2-ArcA and crude native SS2-ArcA were functionally active in acidic conditions. The L-arginine substrate binding affinity (Km) values of rSS2-ArcA (specific activity 16.00 U/mg) and crude native SS2-ArcA (specific activity 0.23 U/mg) were 0.058 and 0.157 mM, respectively. rSS2-ArcA exhibited a weak binding affinity with the common ArcA inhibitors L-canavanine and L-NIO. Furthermore, the partial inactivation of SS2-ArcA significantly impaired the viability and growth of SS2 at pH 4.0, 6.0, and 7.5. CONCLUSIONS: This study profoundly demonstrated the involvement of ArcA enzymatic activity in S. suis survival under acidic conditions.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Hidrolases/química , Hidrolases/genética , Sorogrupo , Streptococcus suis/enzimologia , Streptococcus suis/genética , Sequência de Aminoácidos , Arginina/metabolismo , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Canavanina/antagonistas & inibidores , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Concentração de Íons de Hidrogênio , Hidrolases/efeitos dos fármacos , Hidrolases/metabolismo , Cinética , Ornitina/análogos & derivados , Ornitina/antagonistas & inibidores , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Streptococcus suis/metabolismo , TemperaturaRESUMO
Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA). The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection.
Assuntos
Proteínas de Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Endopeptidase Clp/antagonistas & inibidores , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Hidrolases/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Transativadores/efeitos dos fármacos , Animais , Autólise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Modelos Animais de Doenças , Endopeptidase Clp/genética , Endopeptidase Clp/fisiologia , Genes Bacterianos/genética , Hidrolases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/fisiologia , Transativadores/metabolismo , Virulência/genéticaRESUMO
BACKGROUND: Tetrachlorohydroquinone dehalogenase catalyzes the reductive dehalogenation of tetrachlorohydroquinone to trichlorohydroquinone and then to 2,6-dichlorohydroquinone. This enzyme undergoes oxidative damage during purification which causes it to form aberrant products. The damage is reversible by treatment with dithiothreitol. Possible types of oxidative damage include an inappropriate disulfide bond, a cysteine sulfenic acid, or a methionine sulfoxide. RESULTS: Using electrospray liquid chromatography / mass spectrometry, we have demonstrated that oxidation of tetrachlorohydroquinone dehalogenase with H2O2 results in formation of a sulfenic acid at Cys13. Further oxidation to a sulfinic acid was also observed. CONCLUSIONS: Oxidation of Cys 13 to a sulfenic acid prevents the normal reductive dehalogenation reaction from being completed. This finding is consistent with previous work which suggested that Cys 13 acts as a nucleophile during the conversion of tetrachlorohydroquinone to trichlorohydroquinone. The technique described for identification and localization of the cysteine sulfenic acid should be applicable to a wide variety of biological systems.
Assuntos
Peróxido de Hidrogênio/farmacologia , Hidrolases/química , Espectrometria de Massas/métodos , Ácidos Sulfênicos/análise , Sequência de Aminoácidos , Catálise , Hidrolases/efeitos dos fármacos , Hidrolases/metabolismo , Dados de Sequência MolecularRESUMO
The enzyme S-adenosyl-homocysteine hydrolase (AdoHcyase) which catalyzes the reversible hydrolysis of AdoHcy to adenosine and homocysteine is an adenosine binding protein. In the present study we examined the characteristics of [(3)H]cAMP binding to purified AdoHcyase from bovine kidney in comparison with the high affinity adenosine binding site of AdoHcyase. AdoHcyase exhibits one [(3)H]cAMP binding site with an affinity of K(d)=23.1+/-1.1nM and a B(max) of 116.6+/-3.8pmol/mg protein. Binding of [(3)H]cAMP obeyed a monophasic reaction with a k(+1) value of 0.035min/M. The dissociation of AdoHcyase-[(3)H]cAMP complex exhibited a time- and temperature-dependent character. After a 240min incubation at 0 degrees only 5-10%, however, at 20 degrees 90% were displaceable. Adenosine and cAMP displace each other with similar affinities of EC(50) 57nM vs. EC(50) 65nM. 2'-Deoxyadenosine, N(6)-methyladenosine, and NECA displace 25nM [(3)H]cAMP and 10nM [(3)H]adenosine with EC(50) values of 94, 90 and 80nM, respectively. All other nucleosides studied, adenine, inosine, adenosine-2',3'-dialdehyde, 2-chloroadenosine, aristeromycin, and adenine nucleotides were only week competitors for [(3)H]cAMP and [(3)H]adenosine. These compounds displace [(3)H]cAMP and [(3)H]adenosine with equal potencies. Our data indicate that the binding site for nanomolar concentrations of cAMP and adenosine at the AdoHcyase appears to be identical. The physiological implications of a cAMP binding site at the AdoHcyase remain to be established.
Assuntos
AMP Cíclico/metabolismo , Hidrolases/metabolismo , Rim/enzimologia , Adenosil-Homocisteinase , Animais , Sítios de Ligação , Bovinos , Hidrolases/efeitos dos fármacos , Hipoxantina/metabolismo , Teofilina/farmacologia , Trítio , Xantinas/farmacologiaRESUMO
In mammals, S-adenosylhomocysteine hydrolase (AdoHcyase) is the only known enzyme to catalyze the breakdown of S-adenosylhomocysteine (AdoHcy) to homocysteine and adenosine. AdoHcy is the product of all adenosylmethionine (AdoMet)-dependent biological transmethylations. These reactions have a wide range of products, and are common in all facets of biometabolism. As a product inhibitor, elevated levels of AdoHcy suppress AdoMet-dependent transmethylations. Thus, AdoHcyase is a regulator of biological transmethylation in general. The three-dimensional structure of AdoHcyase complexed with reduced nicotinamide adenine dinucleotide phosphate (NADH) and the inhibitor (1'R, 2'S, 3'R)-9-(2',3'-dihyroxycyclopenten-1-yl)adenine (DHCeA) was solved by a combination of the crystallographic direct methods program, SnB, to determine the selenium atom substructure and by treating the multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. The enzyme architecture resembles that observed for NAD-dependent dehydrogenases, with the catalytic domain and the cofactor-binding domain each containing a modified Rossmann fold. The two domains form a deep active site cleft containing the cofactor and bound inhibitor molecule. A comparison of the inhibitor complex of the human enzyme and the structure of the rat enzyme, solved without inhibitor, suggests that a 17 degrees rigid body movement of the catalytic domain occurs upon inhibitor/substrate binding.
Assuntos
Hidrolases/química , Hidrolases/metabolismo , Adenosil-Homocisteinase , Sequência de Aminoácidos , Animais , Antiparasitários/farmacologia , Antivirais/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Hidrolases/efeitos dos fármacos , Mamíferos , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
His20 of L-2-halo acid dehalogenase from Pseudomonas cepacia MBA4 was suggested to serve as a catalytic base [Biochem. J. (1993) 292, 69-74]. In this study, we substituted Asn or Leu for His19 of L-2-halo acid dehalogenase from Pseudomonas sp. YL, which corresponds to His20 of the P. cepacia enzyme. Although the substrate specificity was affected by the substitution, the susceptibilities of substrate halo acids were not substantially diminished, and the Km and kcat values of the mutant enzymes for L-2-chloropropionate were not significantly different from those of the wild-type enzyme. In addition, the wild-type and mutant enzymes showed the same pH optimum. Accordingly, His19 is not essential for catalysis of L-2-halo acid dehalogenase.
Assuntos
Histidina/metabolismo , Hidrolases/metabolismo , Ácido Aspártico , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catálise , Histidina/química , Hidrolases/efeitos dos fármacos , Hidrolases/genética , Cinética , Mutagênese Sítio-Dirigida , Pseudomonas/enzimologia , Especificidade por SubstratoRESUMO
Leupeptin is an established, reversible inhibitor of cathepsin B, a lysosomal cysteine proteinase. Yet, in rat fibroblasts as well as in foetal mouse calvaria, we observed an increase of the activity of cathepsin B in homogenates of cells and tissue harvested after culture in the presence of leupeptin. This effect was also seen for other lysosomal hydrolases, namely sphingomyelinase, N-acetyl-beta-glucosaminidase, arylsulphatase A and phospholipase A1 in fibroblasts, and beta-glucuronidase in mouse calvaria. In calvaria, antipain, another reversible cysteine proteinase inhibitor, caused a similar effect, whereas E-64, an irreversible inhibitor, was consistently inhibitory of the cathepsin B activity; yet it also caused an increase of beta-glucuronidase activity. The effect of leupeptin in fibroblasts was dose and time-dependent, required the continuous presence of the inhibitor, and was not dependent from protein synthesis. Actually, addition of cycloheximide caused a severe loss of activity of cathepsin B and of sphingomyelinase. In the presence of both cycloheximide and leupeptin, however, these two activities were retained to a value corresponding to that found in excess in cells cultivated with leupeptin alone. The data therefore suggests that leupeptin exerts the effects described in this paper by preventing the degradation of cathepsin B, sphingomyelinase and probably several other lysosomal hydrolases by cysteine proteinases. We therefore propose that cysteine proteinases play a key role in the control of the steady-state levels of these enzymes in normal conditions.
Assuntos
Catepsina B/metabolismo , Fibroblastos/efeitos dos fármacos , Leupeptinas/farmacologia , Crânio/efeitos dos fármacos , Animais , Reabsorção Óssea , Catepsina B/efeitos dos fármacos , Células Cultivadas , Técnicas de Cultura , Cicloeximida/farmacologia , Fibroblastos/metabolismo , Hidrolases/efeitos dos fármacos , Hidrolases/metabolismo , Camundongos , Hormônio Paratireóideo/antagonistas & inibidores , Hormônio Paratireóideo/farmacologia , Biossíntese de Proteínas , Proteínas/efeitos dos fármacos , Ratos , Ratos Wistar , Crânio/embriologia , Fatores de TempoRESUMO
Exposure to the trichothecene mycotoxin deoxynivalenol (DON) alters immune functions in vitro and in vivo. To gain further insight into DON's immunotoxic effects, microarrays were used to determine how acute exposure to this mycotoxin modulates gene expression profiles in murine spleen. B6C3F1 mice were treated orally with 25mg/kg body weight DON, and 2h later spleens were collected for macroarray analysis. Following normalization using a local linear regression model, expression of 116 out of 1176 genes was significantly altered compared to average expression levels in all treatment groups. When genes were arranged into an ontology tree to facilitate comparison of expression profiles between treatment groups, DON was found primarily to modulate genes associated with immunity, inflammation, and chemotaxis. Real-time polymerase chain reaction was used to confirm modulation for selected genes. DON was found to induce the cytokines interleukin (IL)-1alpha, IL-1beta, IL-6 and IL-11. In analogous fashion, DON upregulated expression of the chemokines macrophage inhibitory protein-2 (MIP-2), cytokine-induced chemoattractant protein-1 (CINC-1), monocyte chemoattractant protein (MCP)-1, MCP-3, and cytokine-responsive gene-2 (CRG-2). c-Fos, Fra-, c-Jun, and JunB, components of the activator protein-1 (AP-1) transcription factor complex, were induced by DON as well as another transcription factor, NR4A1. Four hydrolases were found to be upregulated by DON, including mitogen-activated protein kinase phosphatase 1 (MKP1), catalytic subunit beta isoform (CnAbeta), protein tyrosine phosphatase receptor type J (Ptprj), and protein tyrosine phosphatase nonreceptor type 8 (Ptpn8), whereas three other hydrolases, microsomal epoxide hydrolase (Eph) 1, histidine triad nucleotide binding protein (Hint), and proteosome subunit beta type 8 (Psmb8) were significantly decreased by the toxin. Finally, cysteine-rich protein 61 (CRP61) and heat-shock protein 40 (Hsp40), genes associated with signaling, were increased, while Jun kinase 2 (JNK2) was decreased. Taken together, data suggest that DON upregulated the expression of multiple immediate early genes, many of which are likely to contribute to the complex immunological effects reported for this and other trichothecenes.
Assuntos
Perfilação da Expressão Gênica , Genes Precoces/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Baço/efeitos dos fármacos , Tricotecenos/toxicidade , Animais , Quimiocinas/genética , Quimiocinas/imunologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/genética , Quimiotaxia/imunologia , Citocinas/efeitos dos fármacos , Citocinas/genética , Citocinas/imunologia , Avaliação Pré-Clínica de Medicamentos , Perfilação da Expressão Gênica/métodos , Genes Precoces/genética , Genes Precoces/imunologia , Hidrolases/efeitos dos fármacos , Hidrolases/genética , Hidrolases/imunologia , Inflamação , Modelos Lineares , Camundongos , Camundongos Endogâmicos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Filogenia , Reação em Cadeia da Polimerase , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Baço/imunologia , Toxicogenética , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Tricotecenos/genética , Tricotecenos/imunologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
The distribution of plasmalogenase for the hydrolysis of 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) in the subcellular fractions of guinea pig tissues was examined. Plasmalogenase activity was found in high abundance in the cytosolic fractions of the brain and the heart. Assessment of microsomal marker enzyme activities in the cytosolic fraction revealed that plasmalogenase activity in the cytosol was not due to microsomal contaminations. The characteristics of the cytosolic plasmalogenase were very similar to the microsomal enzyme with respect to the pH profile of the reaction, the presence of divalent cations and Km values for plasmenylethanolamine. However, the cytosolic enzyme was slightly less stable at 55 degrees C than the microsomal enzyme. Cytosolic enzyme activity was eluted as a broad peak in Sepharose 6B chromatography with an average molecular weight of 250,000. Our results demonstrate that most of brain plasmalogenase activity is soluble which makes the brain cytosol an excellent source to initiate the purification of this enzyme.
Assuntos
Hidrolases/análise , Frações Subcelulares/enzimologia , Animais , Encéfalo/enzimologia , Cátions/farmacologia , Citosol/enzimologia , Ácido Edético/farmacologia , Cobaias , Concentração de Íons de Hidrogênio , Hidrolases/efeitos dos fármacos , Hidrolases/isolamento & purificação , Fígado/enzimologia , Microssomos/enzimologia , Miocárdio/enzimologia , Plasmalogênios/metabolismo , Desnaturação Proteica , Distribuição TecidualRESUMO
To examine the effect of bile acids on the activity of intestinal aminopeptidase in vivo, we measured the activity of aminopeptidase in the intestinal mucosa from rats fed the diet containing cholestyramine which sequesters luminal bile acids (experiment 1) and from bile diverted rats (experiment 2). After 32 h fasting, rats were refed for 16 h either of a standard diet (25% casein diets), the same diet containing cholestyramine, or the fat-free diet in experiment 1. In the intestinal washing, the content of total bile acids was markedly decreased with feeding cholestyramine and activities of trypsin and chymotrypsin were also lowered with cholestyramine. Cholestyramine feeding decreased the specific activity of aminopeptidase in the homogenate of intestinal mucosa but increased the specific activities of sucrase and alkaline phosphatase. All these parameters were not modified by the fat-free diet. In experiment 2, bile diverted and sham operated rats were refed the standard diet for 16 h with prior 32 h fasting. Bile diversion, like cholestyramine feeding, lowered the content of total bile acids, the activities of pancreatic hydrolases in the intestinal washings, and the specific activity of aminopeptidase in the intestinal mucosa. The specific activity of sucrase in the intestinal mucosa was higher in bile diverted rats but the activity of alkaline phosphatase was not changed. These data indicate that the decreased abundance of intraluminal bile acid affects the activity of intestinal aminopeptidase not through the decreased absorption of dietary lipid.(ABSTRACT TRUNCATED AT 250 WORDS)