RESUMO
The primary structure of pseudo-hevein, a minor hevein component from the latex of the rubber tree, Hevea brasiliensis, was determined. Six differences with the sequence of the major hevein component were found, one of which is a replacement of tryptophan by tyrosine in the carbohydrate binding region of the molecule. Analysis by ion-spray mass spectrometry showed that pseudo-hevein has a heterogeneous C-terminal extension of several glycine residues and that hevein itself also contains minor components with additional C-terminal amino-acid residues. A seventh difference between the two sequences occurs in these extensions.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Lectinas/química , Proteínas de Plantas/química , Sequência de Aminoácidos , Espectrometria de Massas , Dados de Sequência Molecular , Lectinas de Plantas , Homologia de Sequência de Aminoácidos , Árvores/químicaRESUMO
Low molecular weight proteins (LMWPs) are known to be reabsorbed and catabolized primarily by the proximal tubular cells of the kidneys. As such, LMWPs might serve as drug carriers that release drugs site-specifically in the kidney. We tested this concept in vitro by coupling different drugs to the LMWP lysozyme both directly (amide bond) and via different spacers: oligopeptides (amide bond), (poly-)alpha-hydroxy acids (ester bond), and a pH sensitive cis-aconityl spacer (amide bond). The capability of the kidney to release the parent drug from such drug-spacer derivatives and drug-LMWP conjugates by enzymatic or chemical hydrolysis of the bond was tested by incubation experiments in renal cortex homogenates and lysosomal lysates. Directly coupled conjugates of terminal carboxyl group containing drugs and lysozyme were catabolized to single amino acids, but did not result in release of the parent drug. The amide bond between the drug and the final amino acid (lysine) appeared to be stable in the incubation milieu. Different oligopeptide spacers coupled to the drugs showed similar results: the oligopeptide itself was cleaved but the amide bond between the drug and different single amino acids remained untouched. Only amide bonds of derivatives of carboxylic drugs with peptide structures themselves were cleaved. Some of the directly coupled conjugates of terminal amino drugs and oligopeptides showed clear release of the parent drug whereas others were stable. Terminal amino drugs were rapidly released from an acid-sensitive cis-aconityl spacer. Terminal carboxyl group containing drugs were enzymatically released from their glycolic and lactic ester spacers at different rates. These kinds of drugs were also released as parent drug from LMWP conjugates with ester spacers like L-lactic acid. Increasing spacer length by intercalating a tetra(L-lactic acid) molecular between the drug and the protein further increased the extent and rate of drug release, indicating increased accessibility of the bond to the enzymes. Terminal amino group containing drugs were rapidly generated as parent drug from LMWP conjugates using an acid-sensitive spacer. In addition the conjugates were found to be adequately stable in plasma, considering their rapid clearance from the bloodstream. It is concluded that LMWPs may indeed be of use as carriers for specific renal delivery of drugs, since renal cortex homogenates and lysosomal lysates are able to catabolize the protein and generate the parent drug from drug-LMWP conjugates bearing suitable spacers. The option of enzymatic release is limited by the narrow specificity of the lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Portadores de Fármacos/química , Córtex Renal/metabolismo , Lisossomos/metabolismo , Proteínas/química , Aminoácidos/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Endopeptidases/metabolismo , Concentração de Íons de Hidrogênio , Lactatos/química , Ácido Láctico , Leucina/química , Masculino , Peso Molecular , Muramidase/química , Muramidase/metabolismo , Naproxeno/administração & dosagem , Naproxeno/farmacocinética , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Proteínas/metabolismo , Ratos , Ratos Endogâmicos , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , SuccinimidasRESUMO
Solid-phase extraction (SPE) was directly coupled to mass spectrometry (MS) to assess the feasibility of the system for the rapid determination of prednisolone in serum. A C(18) stationary phase allowed washing of the cartridge with 25% methanol. Elution was performed by switching the methanol percentage from 25% in the washing step to 50% during elution. The high flow-rates during the extraction (5.0 ml/min) combined with ion-trap MS detection resulted in a total analysis time of 4 min. Some tailing of the prednisolone peak was observed. However, the tailing was found acceptable, since by this elution procedure most matrix compounds were prevented from eluting from the cartridge. Some matrix interference was still observed with a triple-quadrupole MS, even in the multiple reaction monitoring mode. This resulted in a detection limit (LOD) of about 10 ng/ml. The matrix interference and the LOD were similar for atmospheric pressure chemical ionisation and atmospheric pressure photo ionisation. Applying an ion-trap MS in the MS-MS mode resulted in cleaner chromatograms. Due to extensive fragmentation of prednisolone, the LOD was not lower than about 5 ng/ml prednisolone in serum, and a limit of quantitation of about 10 ng/ml (relative standard deviation <15%) was observed.
Assuntos
Glucocorticoides/sangue , Espectrometria de Massas/métodos , Prednisolona/sangue , Humanos , Sensibilidade e EspecificidadeRESUMO
Rats which had received N-hydroxy-2-acetylaminofluorene (N-OH-AAF) excreted two glutathione conjugates in the bile. Pretreatment with the sulphotransferase inhibitor pentachlorophenol reduced the amounts excreted by 50% suggesting that these conjugates were formed in vivo from the highly reactive N-O-sulphate ester of N-OH-AAF (AAF-N-sulphate). In order to identify these biliary conjugates synthetic GSH conjugates were prepared by the reaction of N-acetoxy-AAF, (a model compound for AAF-N-sulphate), with GSH. Four conjugates were isolated and characterized by ultraviolet, proton magnetic resonance, mass spectroscopy and by the determination of their amino acid composition. These conjugates were identified as 1-, 3-, 4- and 7-(glutathion-S-yl)-N-acetyl-2-aminofluorene, respectively. The two biliary conjugates were identified as 1- and 3-(glutathion-S-yl)-N-acetyl-2-aminofluorene.
Assuntos
2-Acetilaminofluoreno/análogos & derivados , Glutationa/urina , Hidroxiacetilaminofluoreno/urina , Animais , Radioisótopos de Carbono , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Ratos , Ratos Endogâmicos , Espectrofotometria Ultravioleta , TrítioRESUMO
Liquid chromatography-ion-spray mass spectrometry was used to elucidate the structures of the decomposition products of salbutamol. The best sensitivity in mass spectrometry was achieved by using a mixture of acetonitrile and ammonium formate (10 mM, pH 3.3) as the mobile phase in liquid chromatography. Fragmentation of the compounds was obtained by increasing the nozzle voltage in the first vacuum stage of the mass spectrometer. Tentative structure elucidation showed that both acidic and basic decomposition products are formed from salbutamol.
Assuntos
Albuterol/química , Cromatografia Líquida , Espectrometria de Massas , Espectrofotometria UltravioletaRESUMO
The metabolism of the dopamine D2 agonist N-0923 was investigated by an in vitro isolated liver perfusion. Determining the metabolic profile and identity of the different metabolites was achieved by using high-performance liquid chromatography with UV detection, combined with atmospheric pressure ionization mass spectrometry. Using this technique, no extensive sample cleanup is required, and the studies can be performed without radioactivity. In addition to previously observed metabolites, nine new metabolic products were identified. All metabolites were exclusively excreted into the bile, except for the despropyl metabolite, which was also detectable in the perfusate. 5-O-Glucuronidation and N-depropylation followed by 5-O-glucuronidation are the most important metabolic routes. N-dealkylation of the thienylethyl group followed by 5-O-glucuronidation and sulfation is a second major metabolic pathway. Catechol formation of the despropyl metabolite with or without subsequent conjugation was not found. Catechol formation of the desthienylethyl metabolite occurred, but only its glucuronide conjugates were found. This study complements previous results of in vivo metabolic studies using the radiolabeled racemate N-0437, and it explains differences in bile excretion during isolated liver perfusions using N-0923 and radiolabeled N-0923.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dopaminérgicos/análise , Fígado/química , Espectrometria de Massas/métodos , Tetra-Hidronaftalenos/análise , Tiofenos/análise , Animais , Bile/química , Dopaminérgicos/metabolismo , Técnicas In Vitro , Masculino , Perfusão , Ratos , Ratos Wistar , Espectrofotometria Ultravioleta , Tetra-Hidronaftalenos/metabolismo , Tiofenos/metabolismoRESUMO
The identification of residues of polychlorinated biphenyls in a test sample of Flores Chamomillae could be achieved by the retention behavior at gas chromatographic analyses on packed and capillary columns compared with reference standard Clophen A 60, respectively as well as well by capillary GC/MS using single ion monitoring of substance-characteristic ion mass.
Assuntos
Contaminação de Medicamentos , Plantas Medicinais/análise , Bifenilos Policlorados/análise , Cromatografia Gasosa , Cromatografia Gasosa-Espectrometria de MassasRESUMO
For the GC analysis of DDT isomers and metabolites in extracts of Flores Chamomillae end Radix Valerianae the separation on a packed QF-1/OV-17 column was compared with various capillary columns of the CP-Sil type. Identification of the individual compounds could be achieved by comparing the retention behavior, chemical transformation of DDT and DDE, as well as by capillary GC-MS using single ion monitoring of substance-characteristic ion mass. In this way, residues of p,p'-DDT, o,p'-DDE and p,p'-TDE could be identified.
Assuntos
DDT/análise , Contaminação de Medicamentos/análise , Resíduos de Praguicidas/análise , Plantas Medicinais/análise , Cromatografia Gasosa , Cromatografia Gasosa-Espectrometria de Massas , Extratos Vegetais/análiseRESUMO
Electrospray and ionspray techniques use samples that exist as ions or ion-molecule complexes in solution. After the dispersion of the solution into an electrically charged aerosol, the sample ions may escape from the solution into the gas phase in a region that is at atmospheric pressure. The sample ions are transported into the mass analyser which is operated under a high vacuum. Liquid chromatographs can be coupled to electrospray and ionspray interfaces. Flow injection or continuous infusion of a sample solution (both without the use of a separating column) may be preferred over on-line liquid chromatography-mass spectometry in certain applications. Electrospray or ionspray is applicable to polar or ionic samples. Weakly polar and apolar samples are not ionized under electrospray or ionspray conditions. Applications of the techniques are in the fields of drug metabolism, natural product analysis and the determination of high molecular weights through the observation of multiply charged ions.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , 1-Metil-4-fenilpiridínio/química , Aerossóis , Dextrometorfano/química , Dextrorfano/química , Peso Molecular , Porfirinas/química , Brometo de Vecurônio/químicaRESUMO
The aim of these studies was to investigate the scope and limitations of electrochemistry on-line with mass spectrometry as a quick and convenient way to mimic phase I oxidative reactions in drug metabolism. A compound with previously reported in vitro and in vivo metabolism, the dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin, was examined in an electrochemistry/mass spectrometry (EC/MS) system. The previously reported N-dealkylation was mimicked by the electrochemical cell while the oxidation of the phenol function was not fully mimicked by the EC/MS system, since the catechol and p-hydroquinone formed were immediately oxidized to the corresponding quinones. Since cytochrome P450 isoenzymes are the most important enzymes in phase I oxidative metabolism, two standard substrates used for the characterization of those enzymes, lidocaine and 7-ethoxycoumarin, were tested in the EC/MS system. The electrochemical cell was capable of mimicking the N-dealkylation of lidocaine but, under the conditions used in our experiments, the O-deethylation of 7-ethoxycoumarin could not be simulated in the electrochemical cell.
Assuntos
Preparações Farmacêuticas/metabolismo , Anestésicos Locais/metabolismo , Animais , Remoção de Radical Alquila , Agonistas de Dopamina/metabolismo , Eletroquímica , Técnicas In Vitro , Lidocaína/metabolismo , Fígado/metabolismo , Espectrometria de Massas , Oxirredução , Ratos , Tetra-Hidronaftalenos/metabolismo , Tiofenos/metabolismoRESUMO
It is demonstrated that the combination of positive ion chemical ionization and negative ion chemical ionization GC-MS analyses of herb and root extracts of EUPATORIUM CANNABINUM L. offers a rapid, tentative structure elucidation of pyrrolizidine alkaloids (PAs). Compounds identified in aerial parts of E. CANNAHIMUM in this way are four echinatine isomers, like lycopsamine and intermedine, and a number of their beta-acetyl, beta-angelyl/tiglyl and beta-(iso)valeryl esters. PAs without a substituent at C-7 were tentatively identified as supinine and amabiline. In addition to a number of these alkaloids, some beta-(iso)butyryl, beta-angelyl/tiglyl, and beta-(iso)valeryl esters of supinine or amabiline were detected in subterranean parts of the plant. PAS with a saturated necine base like the three trachelanthamine isomers and some beta-anglyl/tiglyl esters could be detected in the root material only. A C-9-viridifloryl/trachelanthyl ester of a saturated amino-alcohol like turneforcidine and one of its beta-angelyl/tiglyl esters have also been found. The latter 2 compounds, the beta-(iso)butyryl, the beta-(iso)valeryl, and the beta-angelyl/tiglyl esters of supinine or amabiline and the beta-(iso)valeryl ester of an echinatine isomer have not been described in nature before.
RESUMO
2,3-Dihydroxybutane, a compound not observed in normal urines, was identified in the urine of two patients by combined gas chromatography-mass spectrometry. It is suggested, that this 2,3-dihydroxybutane is derived from pyruvate by bacterial metabolism.
Assuntos
Butileno Glicóis/urina , Cromatografia Gasosa , Humanos , Recém-Nascido , Masculino , Espectrometria de Massas , Métodos , VolatilizaçãoRESUMO
Haloalkane dehalogenase (DhlA) from Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic cleavage of carbon-halogen bonds in a broad range of halogenated aliphatic compounds. Previous work has shown that Asp124, which is located close to the internal substrate-binding cavity, carries out a nucleophilic attack on the C-alpha of the alkylhalide, displacing the halogen. The resulting alkyl-enzyme intermediate is subsequently hydrolyzed. In order to study the role of His289 in the hydrolysis of the intermediate, a His289-->Gln mutant was constructed by site-directed mutagenesis. The purified mutant enzyme was not catalytically active with haloalkanes, but a halide burst stoichiometric to the amount of enzyme was observed with 1,2-dibromoethane. Using ion spray mass spectrometry, accumulation of the covalent alkyl-enzyme and binding of the alkyl moiety of the substrate to an Asp124-containing tryptic peptide were shown. Fluorescence-quenching experiments indicated that halide ions are strongly bound by the alkyl-enzyme but not by the substrate-free enzyme. The results show that His289 is the base catalyst for the dealkylation of the covalent intermediate, but that it is not essential for the initial nucleophilic attack of Asp124 on the C-1 atom of the haloalkane. Furthermore, the halide ion that is released in the first step probably leaves the active site only after hydrolysis of the alkyl-enzyme.
Assuntos
Bactérias Anaeróbias Gram-Negativas/enzimologia , Hidrolases/química , Alquilação , Proteínas de Bactérias/química , Sequência de Bases , Catálise , Primers do DNA/química , Histidina/química , Cinética , Espectrometria de Massas , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/análise , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The peptides released from beta-casein by the action of PI-type proteinase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry. After 24 h of incubation of beta-casein with purified PrtP, a stable mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this beta-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface. Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH+ mass of each eluted peptide. All peptides corresponding to each of the MH+ calculated masses were determined. In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions. Hydrolysis of beta-casein by PrtP was observed to proceed much further than reported previously. More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides. With the exception of Phe, significant release of amino acids or di- and tripeptides could not be observed. Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system. Uptake of these peptides could supply L. lactis with all amino acids, including the essential ones, indicating that growth of L. lactis might be possible on peptides released from beta-casein by proteinase only.
Assuntos
Proteínas de Bactérias/metabolismo , Caseínas/metabolismo , Endopeptidases/metabolismo , Lactococcus lactis/metabolismo , Oligopeptídeos/metabolismo , Serina Endopeptidases , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Transporte Biológico , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Endopeptidases/isolamento & purificação , Hidrólise , Lactococcus lactis/enzimologia , Espectrometria de Massas , Dados de Sequência MolecularRESUMO
A determination of histamine in urine by selected ion monitoring, using (15N2)histamine as internal standard, is described. Three different ionization methods were used, chemical ionization with ammonia as reactant gas offering the highest sensitivity (detection limit 40 fmol of histamine on column). The 24 h urinary excretions of 10 normal adults ranged from 142-1100 nmol (mean 321 nmol). Patients with an anaphylactoid reaction and patients with mastocytosis showed above normal values.
Assuntos
Histamina/urina , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Espectrometria de Massas/métodosRESUMO
Amino acid auxotrophous bacteria such as Lactococcus lactis use proteins as a source of amino acids. For this process, they possess a complex proteolytic system to degrade the protein(s) and to transport the degradation products into the cell. We have been able to dissect the various steps of the pathway by deleting one or more genes encoding key enzymes/components of the system and using mass spectrometry to analyse the complex peptide mixtures. This approach revealed in detail how L. lactis liberates the required amino acids from beta-casein, the major component of the lactococcal diet. Mutants containing the extracellular proteinase PrtP, but lacking the oligopeptide transport system Opp and the autolysin AcmA, were used to determine the proteinase specificity in vivo. To identify the substrates of Opp present in the casein hydrolysate, the PrtP-generated peptide pool was offered to mutants lacking the proteinase, but containing Opp, and the disappearance of peptides from the medium as well as the intracellular accumulation of amino acids and peptides was monitored in peptidase-proficient and fivefold peptidase-deficient genetic backgrounds. The results are unambiguous and firmly establish that (i) the carboxyl-terminal end of beta-casein is degraded preferentially despite the broad specificity of the proteinase; (ii) peptides smaller than five residues are not formed in vivo; (iii) use of oligopeptides of 5-10 residues becomes only possible after uptake via Opp; (iv) only a few (10-14) of the peptides generated by PrtP are actually used, even though the system facilitates the transport of oligopeptides up to at least 10 residues. The technology described here allows us to monitor the fate of individual peptides in complex mixtures and is applicable to other proteolytic systems.
Assuntos
Caseínas/metabolismo , Lactococcus lactis/enzimologia , Sequência de Aminoácidos , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Proteínas de Transporte/genética , Cromatografia Líquida de Alta Pressão , Endopeptidases/metabolismo , Espaço Extracelular/enzimologia , Cinética , Lactococcus lactis/fisiologia , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Mutação/genética , Fragmentos de Peptídeos/química , Serina Endopeptidases/metabolismo , Especificidade por Substrato/fisiologiaRESUMO
A peptide comprising 37 amino acids of the antigen binding site of a monoclonal antibody directed against glycoprotein D of herpes simplex virus was synthesized. The synthetic peptide and the impurities formed in the synthesis were characterized by capillary electrophoresis/ionspray mass spectrometry and by 252Cf plasma desorption-time of flight mass spectrometry. The measured average molecular mass of the synthetic peptide was 4627.16 Da, which was only 0.08 Da higher than the calculated value (4627.08 Da). The plasma desorption mass spectrum of the synthetic peptide showed a protonated molecule at m/z 4624.1, which was 4 Da lower than the calculated one (4628.09 Da). The amino acid sequence of the peptide was confirmed in part by electrospray (ionspray) mass spectrometry using a high nozzle skimmer voltage difference. Five impurities were separated and identified by capillary electrophoresis/mass spectrometry and two of them also appeared in the plasma desorption mass spectrum.
Assuntos
Anticorpos Monoclonais/análise , Anticorpos Antivirais/análise , Fragmentos de Imunoglobulinas/análise , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Califórnio , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Peso Molecular , Receptores de Antígenos/química , Proteínas do Envelope Viral/análiseRESUMO
The role of sulfation in the metabolic activation of the liver carcinogen N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) in male rat liver was investigated. N-OH-FAABP was a substrate for sulfotransferases in vitro and sulfation was inhibited by the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The main metabolite of N-OH-FAABP excreted in bile in vivo, and in the isolated perfused liver, was identified as the N-O-glucuronide conjugate. Inhibition of sulfation in vivo by PCP or DCNP, or in vitro by omission of inorganic sulfate, resulted in a large increase in the excretion of the N-O-glucuronide conjugate. It was estimated that at least 21% of the dose was sulfated in control animals. Inhibition of sulfation in vivo by PCP or DCNP prevented the covalent binding of N-OH-FAABP to liver (and kidney) macromolecules by 70% and 20% respectively. HPLC analysis of the fluorobiphenyl DNA and RNA adducts showed that the formation of both N-acetylated and deacetylated (deoxy)-guanosine adducts was prevented. Furthermore, omission of inorganic sulfate in the isolated perfused liver prevented the formation of all fluorobiphenyl DNA adducts by 70-80%. It is concluded that two sulfotransferase-dependent pathways exist for the metabolic activation of N-OH-FAABP in male rat liver: (i) direct sulfation of the hydroxamic acid, resulting, upon decomposition of the FAABP-N-sulfate ester, in the formation of N-acetylated DNA adducts and (ii) deacetylation followed by sulfation of the hydroxylamine to FABP-N-sulfate, leading to the formation of deacetylation DNA adducts.
Assuntos
Compostos de Aminobifenil/metabolismo , Carcinógenos/metabolismo , Clorofenóis/farmacologia , Rim/metabolismo , Fígado/metabolismo , Nitrofenóis/farmacologia , Pentaclorofenol/farmacologia , Sulfatos/metabolismo , Sulfurtransferases/antagonistas & inibidores , Animais , Biotransformação , Cromatografia Líquida de Alta Pressão , DNA/metabolismo , Glucuronidase/metabolismo , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , RNA/metabolismo , Ratos , Ratos EndogâmicosRESUMO
To gather more information on stereochemical factors in the hepatic disposition of organic cations, mass spectrometry coupled to liquid chromatography was used to determine the identity of the metabolites excreted in bile after isolated rat liver perfusions with the quaternary ammonium derivatives of the enantiomeric drugs dextrorphan and levorphanol. Ionspray mass spectrometry was chosen for its soft ionization and absence of thermal degradation of labile compounds. The drugs were labelled with a stable (2H) isotope and mixed with unlabelled drugs to create an artificial isotope pattern in the mass spectrum and facilitate the recognition of unknown metabolites. In mass spectra that were recorded under normal conditions, fragmentation was absent and metabolites of N-methyl dextrorphan and N-methyl levorphanol were visible as parent-ion 'doublets'. Collision-induced fragmentation studies were performed to support the identification of the metabolites. For N-methyl dextrorphan the glucuronide, the glutathione conjugate and the glucuronide of the N-demethylated metabolite were found in bile. For N-methyl levorphanol the glucuronide, the glutathione conjugate, the sulphate conjugate and the glucuronide of a hydroxylated N-methyl levorphanol were excreted in bile. Thus a remarkable stereoselectivity occurs in the metabolism of these quaternary ammonium compounds in the rat liver.
Assuntos
Dextrorfano/análise , Levalorfano/análogos & derivados , Fígado/metabolismo , Animais , Bile/metabolismo , Cromatografia Líquida , Dextrorfano/análogos & derivados , Técnicas In Vitro , Levalorfano/análise , Levalorfano/metabolismo , Fígado/química , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Conformação Molecular , Perfusão , Ratos , Ratos Wistar , EstereoisomerismoRESUMO
Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in a broad range of halogenated aliphatic compounds. The X-ray structure suggests that Asp124, which is located close to an internal cavity, carries out a nucleophilic attack on the C alpha of the substrate, releasing the halogen. To study the mechanism of hydrolysis, this aspartate residue was mutated to alanine, glycine, or glutamate. The mutant enzymes showed no activity toward 1,2-dichloroethane and 1,2-dibromoethane. Incubation of purified wild-type dehalogenase with 1,2-dichloroethane in the presence of H2(18)O resulted in the incorporation of 18O in 2-chloroethanol and in the carboxylate group of Asp124. This shows that the reaction proceeds by covalent catalysis with the formation of an alkyl-enzyme intermediate that is hydrolyzed by attack of solvent water on the carbonyl carbon of Asp124. On the basis of amino acid sequence similarity between haloalkane dehalogenase and epoxide hydrolases, it is proposed that a conserved aspartate residue is also involved in covalent catalysis by the latter enzymes.