RESUMO
Formaldehyde-inactivated toxoid vaccines have been in use for almost a century. Despite formaldehyde's deceptively simple structure, its reactions with proteins are complex. Treatment of immunogenic proteins with aqueous formaldehyde results in heterogenous mixtures due to a variety of adducts and cross-links. In this study, we aimed to further elucidate the reaction products of formaldehyde reaction with proteins and report unique modifications in formaldehyde-treated cytochrome c and corresponding synthetic peptides. Synthetic peptides (Ac-GDVEKGAK and Ac-GDVEKGKK) were treated with isotopically labeled formaldehyde (13CH2O or CD2O) followed by purification of the two main reaction products. This allowed for their structural elucidation by (2D)-nuclear magnetic resonance and nanoscale liquid chromatography-coupled mass spectrometry analysis. We observed modifications resulting from (i) formaldehyde-induced deamination and formation of α,ß-unsaturated aldehydes and methylation on two adjacent lysine residues and (ii) formaldehyde-induced methylation and formylation of two adjacent lysine residues. These products react further to form intramolecular cross-links between the two lysine residues. At higher peptide concentrations, these two main reaction products were also found to subsequently cross-link to lysine residues in other peptides, forming dimers and trimers. The accurate identification and quantification of formaldehyde-induced modifications improves our knowledge of formaldehyde-inactivated vaccine products, potentially aiding the development and registration of new vaccines.
Assuntos
Citocromos c/química , Formaldeído/farmacologia , Lisina/química , Peptídeos/química , Aldeídos/química , Cromatografia Líquida de Alta Pressão/métodos , Reagentes de Ligações Cruzadas/química , Desaminação/efeitos dos fármacos , Cinética , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Metilação/efeitos dos fármacos , Estrutura Molecular , Vacinas de Produtos Inativados/químicaRESUMO
Metformin is an antidiabetic drug with a major impact on regulating blood glucose levels by decreasing hepatic gluconeogenesis, but also by affecting other pathways, including glucose transport and energy/lipid metabolism. Carnivorous fish are considered glucose intolerant, as they exhibit poor ability in using dietary carbohydrates. To increase the current knowledge about the molecular mechanisms by which metformin can improve glucose homeostasis in carnivorous fish, we addressed the effect of intraperitoneal administration of metformin, in the presence or absence of a glucose load, on metabolic rate-limiting enzymes and lipogenic factors in the liver of gilthead sea bream ( Sparus aurata). Hyperglycemia markedly upregulated the expression of glycolytic enzymes (glucokinase and 6-phosphofructo-1-kinase, PFK1) 5 h following glucose administration, while at 24 h posttreatment, it increased isocitrate dehydrogenase (IDH) activity, a key enzyme of the tricarboxylic acid cycle, and the expression of lipogenic factors (PGC1ß, Lpin1, and SREBP1). Metformin counteracted glucose-dependent effects, and downregulated glutamate dehydrogenase, alanine aminotransferase, and mammalian target of rapamycin 5 h posttreatment in the absence of a glucose load, leading to decreased long-term activity of PFK1 and IDH. The results of the present study suggest that hyperglycemia enhances lipogenesis in the liver of S. aurata and that metformin may exert specific metabolic effects in fish by decreasing hepatic transdeamination and suppressing the use of amino acids as gluconeogenic substrates. Our findings highlight the role of amino acid metabolism in the glucose-intolerant carnivorous fish model.
Assuntos
Desaminação/efeitos dos fármacos , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Lipogênese/efeitos dos fármacos , Fígado/metabolismo , Metformina/farmacologia , Dourada/metabolismo , Aminoácidos/metabolismo , Animais , Desaminação/genética , Glucoquinase/genética , Glucoquinase/metabolismo , Gluconeogênese/efeitos dos fármacos , Glucose/farmacologia , Hiperglicemia/metabolismo , Lipogênese/genética , Fígado/efeitos dos fármacos , Fosfofrutoquinase-2/metabolismoRESUMO
The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain alpha-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain alpha-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5'-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5'-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5'-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Glutamato Desidrogenase/metabolismo , Metaboloma , Mitocôndrias/enzimologia , Transaminases/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Biocatálise/efeitos dos fármacos , Cromatografia de Afinidade , Reagentes de Ligações Cruzadas/farmacologia , Desaminação/efeitos dos fármacos , Descarboxilação/efeitos dos fármacos , Masculino , Metaboloma/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Piridoxamina/análogos & derivados , Piridoxamina/metabolismo , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual/efeitos dos fármacosRESUMO
Semicarbazide-sensitive amine oxidase (SSAO) is a copper-containing enzyme that catalyzes the oxidative deamination of endogenous and exogenous primary amines. SSAO exists in mammals both as a plasma-soluble and as a membrane-bound form, and its active site is able to come into contact with numerous xenobiotic, amine-containing compounds. The kinetic studies performed in this work showed that caffeine inhibition of bovine serum amine oxidase was noncompetitive when benzylamine was used as substrate and mixed when the substrate used was methylamine. Since caffeine contains an imidazole ring, it cannot be excluded that it might bind to an inhibitory imidazoline-binding site on SSAO.
Assuntos
Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Amina Oxidase (contendo Cobre)/sangue , Cafeína/farmacologia , Desaminação/efeitos dos fármacos , Aminas/química , Animais , Benzilaminas/química , Sítios de Ligação/efeitos dos fármacos , Bovinos , Cinética , Metilaminas/química , Oxirredução , Ligação Proteica/efeitos dos fármacos , Especificidade por SubstratoRESUMO
Glypican-1 is a glycosylphosphatidylinositol anchored cell surface S-nitrosylated heparan sulfate proteoglycan that is processed by nitric oxide dependent degradation of its side chains. Cell surface-bound glypican-1 becomes internalized and recycles via endosomes, where the heparan sulphate chains undergo nitric oxide and copper dependent autocleavage at N-unsubstituted glucosamines, back to the Golgi. It is not known if the S-nitrosylation occurs during biosynthesis or recycling of the protein. Here we have generated a recombinant human glypican-1 lacking the glycosylphosphatidylinositol-anchor. We find that this protein is directly secreted into the culture medium both as core protein and proteoglycan form and is not subjected to internalization and further modifications during recycling. By using SDS-PAGE, Western blotting and radiolabeling experiments we show that the glypican-1 can be S-nitrosylated. We have measured the level of S-nitrosylation in the glypican-1 core protein by biotin switch assay and find that the core protein can be S-nitrosylated in the presence of copper II ions and NO donor. Furthermore the glypican-1 proteoglycan produced in the presence of polyamine synthesis inhibitor, alpha-difluoromethylornithine, was endogenously S-nitrosylated and release of nitric oxide induced deaminative autocleavage of the HS side chains of glypican-1. We also show that the N-unsubstituted glucosamine residues are formed during biosynthesis of glypican-1 and that the content increased upon inhibition of polyamine synthesis. It cannot be excluded that endogenous glypican-1 can become further S-nitrosylated during recycling.
Assuntos
Glipicanas/metabolismo , Óxido Nítrico/metabolismo , Proteínas Recombinantes/metabolismo , Linhagem Celular , Desaminação/efeitos dos fármacos , Eflornitina/farmacologia , Vetores Genéticos , Glucosamina/metabolismo , Glipicanas/isolamento & purificação , Proteoglicanas de Heparan Sulfato/química , Humanos , Poliaminas/metabolismo , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Glutamate dehydrogenase (GDH) plays a major role in amino acid catabolism. To increase the current knowledge of GDH function, we analysed the effect of GDH silencing on liver intermediary metabolism from gilthead sea bream (Sparus aurata). Sequencing of GDH cDNA from S. aurata revealed high homology with its vertebrate orthologues and allowed us to design short hairpin RNAs (shRNAs) to knockdown GDH expression. Following validation of shRNA-dependent downregulation of S. aurata GDH in vitro, chitosan-tripolyphosphate (TPP) nanoparticles complexed with a plasmid encoding a selected shRNA (pCpG-sh2GDH) were produced to address the effect of GDH silencing on S. aurata liver metabolism. Seventy-two hours following intraperitoneal administration of chitosan-TPP-pCpG-sh2GDH, GDH mRNA levels and immunodetectable protein decreased in the liver, leading to reduced GDH activity in both oxidative and reductive reactions to about 53-55 % of control values. GDH silencing decreased glutamate, glutamine and aspartate aminotransferase activity, while increased 2-oxoglutarate content, 2-oxoglutarate dehydrogenase activity and 6-phosphofructo-1-kinase/fructose-1,6-bisphosphatase activity ratio. Our findings show for the first time that GDH silencing reduces transdeamination and gluconeogenesis in the liver, hindering the use of amino acids as gluconeogenic substrates and enabling protein sparing and metabolisation of dietary carbohydrates, which would reduce environmental impact and production costs of aquaculture.
Assuntos
Quitosana/análogos & derivados , Desaminação/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Glutamato Desidrogenase/antagonistas & inibidores , RNA Interferente Pequeno/farmacologia , Dourada/genética , Animais , Ciências Biocomportamentais , Quitosana/química , Clonagem Molecular , Proteínas de Peixes/antagonistas & inibidores , Proteínas de Peixes/genética , Glutamato Desidrogenase/genética , Células Hep G2 , Humanos , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Fígado/metabolismo , Nanopartículas/química , Plasmídeos/administração & dosagem , RNA Interferente Pequeno/química , Dourada/metabolismoRESUMO
3'-ethynyladenosine (EAdo) was an adenosine analog with potent antitumor activity against various human tumor cells in vitro. However, EAdo was enzymatically inactivated by adenosine deaminase (ADA) in vitro and in vivo. Therefore, we synthesized two ADA-resistant EAdo derivatives (2-F-EAdo and EAdo-5'-monophosphate, EAMP) and examined their antitumor activities.
Assuntos
Adenosina/análogos & derivados , Adenosina/síntese química , Antineoplásicos/síntese química , Adenosina/química , Adenosina/farmacologia , Adenosina Desaminase/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desaminação/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , HumanosRESUMO
Hypoxanthine (Hx) is a major DNA lesion generated by deamination of adenine during chronic inflammatory conditions, which is an underlying cause of various diseases including cancer of colon, liver, pancreas, bladder and stomach. There is evidence that deamination of DNA bases induces mutations, but no study has directly linked Hx accumulation to mutagenesis and strand-specific mutations yet in human cells. Using a site-specific mutagenesis approach, we report the first direct evidence of mutation potential and pattern of Hx in live human cells. We investigated Hx-induced mutations in human nonmalignant HEK293 and cancer HCT116 cell lines and found that Hx is mutagenic in both HEK293 and HCT116 cell lines. There is a strand bias for Hx-mediated mutations in both the cell lines; the Hx in lagging strand is more mutagenic than in leading strand. There is also some difference in cell types regarding the strand bias for mutation types; HEK293 cells showed largely deletion (>80%) mutations in both leading and lagging strand and the rest were insertions and A:TâG:C transition mutations in leading and lagging strands, respectively, whereas in HCT116 cells we observed 60% A:TâG:C transition mutations in the leading strand and 100% deletions in the lagging strand. Overall, Hx is a highly mutagenic lesion capable of generating A:TâG:C transitions and large deletions with a significant variation in leading and lagging strands in human cells. In recent meta-analysis study AâG (TâC) mutations were found to be a prominent signature in a variety of cancers, including a majority types that are induced by inflammation. The deletions are known to be a major cause of copy-number variations or CNVs, which is a major underlying cause of many human diseases including mental illness, developmental disorders and cancer. Thus, Hx, a major DNA lesion induced by different deamination mechanisms, has potential to initiate inflammation-driven carcinogenesis in addition to various human pathophysiological consequences.
Assuntos
Dano ao DNA/efeitos dos fármacos , Hipoxantina/toxicidade , Mutagênicos/toxicidade , Sequência de Aminoácidos , Reparo do DNA , Desaminação/efeitos dos fármacos , Células HCT116 , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Mutação , Reprodutibilidade dos TestesRESUMO
Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Fluoruracila/metabolismo , Espaço Intracelular/metabolismo , Timidilato Sintase/antagonistas & inibidores , Ativação Metabólica/efeitos dos fármacos , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Bioensaio , Desaminação/efeitos dos fármacos , Decitabina , Humanos , Células K562 , Cinética , Fosforilação/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Timidilato Sintase/metabolismo , Fatores de TempoRESUMO
The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a viral center molecule for HBV infection and persistence. However, the cellular restriction factors of HBV cccDNA are not well understood. Here, we show that TGF-ß can induce nuclear viral cccDNA degradation and hypermutation via activation-induced cytidine deaminase (AID) deamination activity in hepatocytes. This suppression by TGF-ß is abrogated when AID or the activity of uracil-DNA glycosylase (UNG) is absent, which indicates that AID deamination and the UNG-mediated excision of uracil act in concert to degrade viral cccDNA. Moreover, the HBV core protein promotes the interaction between AID and viral cccDNA. Overall, our results indicate a novel molecular mechanism that allows cytokine TGF-ß to restrict viral nuclear cccDNA in innate immunity, thereby suggesting a novel method for potentially eliminating cccDNA.
Assuntos
Citidina Desaminase/metabolismo , DNA Circular/metabolismo , DNA Viral/metabolismo , Vírus da Hepatite B/metabolismo , Hepatócitos/virologia , Fator de Crescimento Transformador beta/metabolismo , Uracila-DNA Glicosidase/metabolismo , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Imunoprecipitação da Cromatina , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/genética , DNA Circular/isolamento & purificação , DNA Viral/isolamento & purificação , Desaminação/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/imunologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/imunologia , Hepatócitos/metabolismo , Humanos , Hidrólise/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Mutação , Interferência de RNA , RNA Interferente Pequeno , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Uracila-DNA Glicosidase/antagonistas & inibidores , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismoRESUMO
We have developed a rapid, accurate, and quantitative method for the detection of methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by primer extension and ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). The application of the method is illustrated by analysis of differentially imprinted alleles arising from Prader-Willi and Angelman syndromes. In order to convert unmethylated cytosines to uracil, plasmid and genomic DNA samples were treated with sodium bisulfite and the targeted sequence was then amplified using oligodeoxynucleotide primers specific for the bisulfite-deaminated DNA. The PCR product(s) from this step was used as a template for a primer extension reaction and the products were subsequently analyzed chromatographically using IP RP HPLC. This method eliminates the need to use restriction enzymes to determine the methylation status of the amplicon and circumvents the need for radio labeling for the quantitative measurements. Finally, this method removes the need for nucleotide sequencing because it is not solely reliant on the presence or absence of one or more PCR products, as is the case with related methods.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Metilação de DNA , Marcadores Genéticos/genética , Resistência a Ampicilina/genética , Síndrome de Angelman/genética , Sequência de Bases/genética , Ilhas de CpG/efeitos dos fármacos , Ilhas de CpG/genética , Citosina/química , DNA/análise , DNA/química , Metilação de DNA/efeitos dos fármacos , Primers do DNA/genética , Primers do DNA/metabolismo , Desaminação/efeitos dos fármacos , Marcadores Genéticos/efeitos dos fármacos , Impressão Genômica/efeitos dos fármacos , Impressão Genômica/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Síndrome de Prader-Willi/genética , Sulfitos/química , Sulfitos/metabolismo , Uracila/químicaRESUMO
To test the hypothesis that analyses of drug targets for polymorphism will help to establish gene-based information for the treatment of cancer patients, we investigated the functional single-nucleotide polymorphisms in the human cytidine deaminase (HDCA) gene. The cDNAs from 52 leukaemia/lymphoma samples and 169 control blood samples were direct-sequenced and analysed for the polymorphisms. Three different polymorphisms (A79C, G208A and T435C) were identified in the coding region of the HDCA gene and displayed allelic frequencies of 20.1%, 4.3% and 70.1%, respectively. No association with susceptibility to disease was observed. A novel polymorphism, G208A produced an alanine to threonine substitution (A70T) within the conserved catalytic domain. By introduction of the polymorphic HCDA genes into the yeast CDA-null mutants, the HCDA-70T showed 40% and 32% activity of prototype for cytidine and ara-C substrates, respectively (P < 0.01). The ara-C IC50 value of the yeast transformants carrying HCDA-70T was 757 +/- 33 micromol and was significantly lower (P < 0.01) than that of prototype (941 +/- 58 micromol). This study demonstrated a population characterized with 208A genotype for, which potentially leads one more sensitive to ara-C treatment than prototype. Accumulation of polymorphisms in the genes responsible for drug metabolism and determination of polymorphism-induced biological variations could provide the additional therapeutic strategies in risk-stratified protocols for the treatment of childhood malignancies.
Assuntos
Citarabina/farmacologia , Citidina Desaminase/genética , Polimorfismo de Nucleotídeo Único , Saccharomyces cerevisiae/efeitos dos fármacos , Pré-Escolar , Citidina Desaminase/biossíntese , Citidina Desaminase/química , DNA Complementar/genética , Desaminação/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia/genética , Testes de Sensibilidade Microbiana , Proteínas Recombinantes/química , Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
The effects of 2-chloro-2'-deoxyadenosine (2CdA) on the activity of enzymes important for the metabolism of deoxyadenosine were studied in lysates prepared from human primary central nervous system (CNS) lymphomas and normal human lymphocytes. Strong inhibition (approximately 100%) of the phosphorylation of deoxyadenosine to its deoxynucleotide phosphate derivatives was produced in both systems in the presence of 2CdA, which was phosphorylated concomitantly to 2-chloro-2'-deoxyAMP. Interestingly, 2CdA was also found to be an inhibitor of the deamination of both deoxyadenosine (over 50%) and AMP (70%). These findings add to our understanding of the mechanisms of toxicity of this drug, especially considering that 2CdA is resistant to deamination by adenosine deaminase. These results challenge the existing theories of 2CdA toxicity, which have been limited to the formation of phosphate derivatives of 2CdA. The present in vitro studies have demonstrated that 2CdA also inhibits both phosphorylation and deamination of deoxyadenosine (dAdo), suggesting that its mechanism of toxicity includes a block in dAdo metabolic pathways. This has important implications for the perturbation of cell methylation, a functionality associated with, for example, apoptosis.
Assuntos
Neoplasias do Sistema Nervoso Central/metabolismo , Cladribina/farmacologia , Desoxiadenosinas/metabolismo , Inibidores Enzimáticos/farmacologia , Linfoma/metabolismo , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/metabolismo , Adenina Fosforribosiltransferase/metabolismo , Adenosina Desaminase/metabolismo , Monofosfato de Adenosina/metabolismo , Adenosil-Homocisteinase , Apoptose/efeitos dos fármacos , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Neoplasias do Sistema Nervoso Central/enzimologia , Desaminação/efeitos dos fármacos , Ativação Enzimática , Humanos , Hidrolases/antagonistas & inibidores , Hidrolases/metabolismo , Linfoma/tratamento farmacológico , Linfoma/enzimologia , Fosforilação/efeitos dos fármacosRESUMO
Monoamine oxidase type B (MAO-B) activity is elevated in certain neurological diseases such as Alzheimer's and Parkinson's disease with respect to age-matched controls; the cause of this elevation is unknown. The documented accumulation of aluminum in certain neurodegenerative diseases prompted us to test the effect of Al3+ on the activity of MAO-B in rat brain homogenate. Results showed that the metal ion significantly increased MAO-B enzymatic activity in a dose-dependent manner, yielding a K(M) of 5.69 microM compared with 34.45 microM in the absence of the metal ion. The Vmax of 45.34 micromol/min was unchanged in the presence of the metal ion.
Assuntos
Alumínio/farmacologia , Encéfalo/enzimologia , Monoaminoxidase/metabolismo , Compostos de Alumínio/farmacologia , Doença de Alzheimer/enzimologia , Doença de Alzheimer/etiologia , Animais , Encéfalo/citologia , Extratos Celulares , Desaminação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Cinética , Lactatos/farmacologia , Masculino , Doença de Parkinson/enzimologia , Doença de Parkinson/etiologia , Fenetilaminas/metabolismo , Ratos , Ratos Wistar , Lactato de Sódio/farmacologia , TermodinâmicaRESUMO
Deamination of the nucleoside analogues ARA-C and 5-AZA-CdR by CR deaminase results in a loss of antileukemic activity. To prevent the inactivation of these analogues, inhibitors of CR deaminase may prove to be useful agents. In the present study we investigated the effects of the deaminase inhibitors Zebularine, 5-F-Zebularine, and diazepinone riboside on the deamination of CR, ARA-C, and 5-AZA-CdR using highly purified human CR deaminase (EC 3.5.4.5). These inhibitors produced a competitive type of inhibition with each substrate, the potency of which followed the patterns diazepinone riboside greater than 5-F-Zebularine and THU greater than Zebularine. 5-AZA-CdR was more sensitive than ARA-C to the inhibition produced by these deaminase inhibitors. The inhibition constants for diazepinone riboside lay in the range of 5-15 nM, suggesting that this inhibitor could be an excellent candidate for use in combination chemotherapy with either ARA-C or 5-AZA-CdR in patients with leukemia.
Assuntos
Antineoplásicos/metabolismo , Azacitidina/análogos & derivados , Citarabina/metabolismo , Citidina Desaminase/antagonistas & inibidores , Azacitidina/metabolismo , Azepinas/farmacologia , Citidina/análogos & derivados , Desaminação/efeitos dos fármacos , Decitabina , Humanos , Cinética , Nucleosídeos de Pirimidina/farmacologia , Tetra-Hidrouridina/farmacologiaRESUMO
The colourimetric assay of monoamine oxidase activity, as hydrogen peroxide production, normally requires the use of sodium azide to inhibit breakdown of hydrogen peroxide by catalase. Sodium azide was shown to act as an uncompetitive inhibitor of benzylamine deamination with an inhibitor constant of 1.5 mM. Catalase activity of isolated rat liver mitochondria could be eliminated with the irreversible inhibitor of catalase, 3-amino-1,2,4-triazole. The treatment did not affect benzylamine deaminating activity. The catalase-free preparation could be used to assay monoamine oxidase activity colourimetrically, as hydrogen peroxide production, in the absence of sodium azide.
Assuntos
Azidas/farmacologia , Monoaminoxidase/análise , Amitrol (Herbicida)/farmacologia , Animais , Benzaldeídos/metabolismo , Benzilaminas/metabolismo , Catalase/antagonistas & inibidores , Colorimetria , Desaminação/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Masculino , Mitocôndrias Hepáticas/enzimologia , Ratos , Ratos Wistar , Azida SódicaRESUMO
Titration of monoamine oxidase activity in isolated guinea pig liver mitochondria with clorgyline and assay of remaining activity with tyramine yielded biphasic inhibition curves. The position of the plateaus obtained with mitochondria from four animals, indicated that the B form of monoamine oxidase accounted for 30% to 70% of the tyramine deaminating activity. Benzylamine deamination was selectively inhibited by (-)-deprenyl. However, benzylamine and other amines which are selective substrates for the B form of monoamine oxidase from the rat, were deaminated at only low rates by the guinea pig liver enzyme. Guinea pig liver contains a monoamine oxidase-B which is unusual in that although it exhibits apparently normal sensitivity to selective irreversible inhibitors, it has a low catalytic activity with substrates which the enzyme from rat liver deaminates rapidly.
Assuntos
Fígado/enzimologia , Monoaminoxidase/metabolismo , Animais , Benzilaminas/metabolismo , Clorgilina/farmacologia , Desaminação/efeitos dos fármacos , Cobaias , Cinética , Masculino , Mitocôndrias Hepáticas/enzimologia , Ratos , Ratos Wistar , Tiramina/metabolismoRESUMO
The relation of ascorbic acid to the ultraviolet light-induced deamination of cytosine (to uracil) and 5-methylcytosine (to thymine) was examined using gas chromatography-mass spectrometry. There was an inverse relation between the uracil content measured in solutions of cytosine after exposure to short wavelength (254 nm) ultraviolet light and the ascorbic acid concentration of the solution indicating inhibition of deamination of cytosine by ascorbic acid. Ascorbic acid also inhibited ultraviolet light-induced deamination of 5-methylcytosine. The ascorbic acid was only partially consumed during the exposure suggesting that protection from deamination was not due entirely to the antioxidant properties of ascorbic acid. Mercaptoethanol did not prevent ultraviolet light-induced deamination of either cytosine or 5-methylcytosine. However, near identical protection from ultraviolet light-induced deamination was provided by 4-aminobenzoic acid and ascorbic acid at equivalent ultraviolet light absorbing concentrations. This observation suggests that ascorbic acid prevents short wavelength ultraviolet light-induced deamination through absorbance of ultraviolet light rather than through antioxidant mechanisms.
Assuntos
Ácido Ascórbico/farmacologia , Citosina/análogos & derivados , Citosina/química , Compostos de Organossilício , Protetores contra Radiação/farmacologia , Raios Ultravioleta , 5-Metilcitosina , Citosina/efeitos da radiação , Desaminação/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas , Silício/farmacologiaRESUMO
E. coli single-stranded binding protein (SSB) has been examined for its ability to modulate bisulfite-induced cytosine deamination rates in single-stranded DNA (ssDNA). We used a lacZ alpha-complementation reversion assay to detect C-->U rates at a single codon in M13mp2 DNA, whether in free ssDNA or in an SSB:ssDNA complex. When incubated at 37 degrees C, the average bisulfite-induced reversion rate constant was four-fold less in SSB:ssDNA complexes than in ssDNA, at a single codon. Across a 250 base pair target and over 23 scorable C-->U sites, the forward rate constant was 4.9-fold less in SSB:ssDNA complexes than in ssDNA alone. After treatment with N-uracil glycosylase, ssDNA incubated with bisulfite had reversion frequencies at the background rate of ssDNA incubated without bisulfite, indicating that virtually all mutations scored were due to C-->U events. The decrease in cytosine deamination rates occurred both in a single codon and over a 250 bp target, indicating that interactions between SSB and ssDNA reduce bisulfite-catalyzed mutations. The structural role of SSB is well recognized in multiple cellular processes; SSB can also function to minimize bisulfite-induced ssDNA mutations.
Assuntos
Citosina/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Sulfitos/farmacologia , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/farmacologia , Desaminação/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Frequência do Gene , Substâncias Macromoleculares , MutaçãoRESUMO
Rat aorta semicarbazide-sensitive amine oxidase (SSAO) exhibits very high affinity in the deamination of an homologous series of aliphatic amines of 1 to 18 straight chain carbon atoms. The Km value decreases substantially as the chain length of these amines increases. The Vmax values are higher for the short chain amines. Diamines are poor substrates for SSAO or are not acted upon by the enzyme. The substrate preference for SSAO differs from that for monoamine oxidase.