RESUMO
The characteristics of bacteria take up mercury into cells via membrane potential-dependent sequence-divergent members of the mercuric ion (Mer) superfamily, i.e., a periplasmic mercuric ion scavenging protein (MerP) and one or more inner membrane-spanning proteins (MerC, MerE, MerF, and MerT), which transport mercuric ions into the cytoplasm, have been applied in engineering of bioreactor used for mercurial bioremediation. We engineered bacteria to express MerC, MerE, MerF, or MerT with or without MerP to clarify their individual role and potential in transport of mercurial. By immunoblot analysis using specific polyclonal antibody, the proteins encoded by merC, merE, merF, merT or merP, were certainly expressed and identified in the membrane fraction. Bacteria expressing MerC, MerE, MerF or MerT in the absence of MerP transported significantly more C6H5Hg(I) and Hg(II) across bacterial membrane than their isogenic strain. In vivo expression of MerP in the presence of all the transporters did not cause apparent difference to the C6H5Hg(I) transport, but gives an apparently higher Hg(II) transport than that did by MerE, MerF or MerT but not by MerC. Among the four transporters studied, MerC showed more potential to transport Hg(II) across bacterial membrane than MerE, MerF and MerT. Together these findings, we demonstrated for the first time that in addition to MerE and MerT, MerF and MerC are broad-spectrum mercury transporters that mediate both Hg(II) and phenylmercury transport into cells. Our results suggested that MerC is the most efficient tool for designing mercurial bioremediation systems, because MerC is sufficient for mercurial transport into cells.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Escherichia coli/metabolismo , Mercúrio/metabolismo , Proteínas de Bactérias/genética , Biodegradação Ambiental , Transporte Biológico , Proteínas de Transporte de Cátions/genética , Escherichia coli/genética , Compostos de Fenilmercúrio/metabolismoRESUMO
Methylmercury is a highly toxic, organic derivative found in mercury-polluted wetlands and coastal sediments worldwide. Though commonly present at low concentrations in the substrate, methylmercury can biomagnify to concentrations that poison predatory animals and humans. In the interest of developing an in situ detoxification strategy, a model plant system was transformed with bacterial genes (merA for mercuric reductase and merB for organomercurial lyase) for an organic mercury detoxification pathway. Arabidopsis thaliana plants expressing both genes grow on 50-fold higher methylmercury concentrations than wild-type plants and up to 10-fold higher concentrations than plants that express merB alone. An in vivo assay demonstrated that both transgenes are required for plants to detoxify organic mercury by converting it to volatile and much less toxic elemental mercury.
Assuntos
Arabidopsis/genética , Engenharia Genética/métodos , Substâncias Perigosas/metabolismo , Compostos Organomercúricos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Poluentes Atmosféricos/metabolismo , Arabidopsis/enzimologia , Biodegradação Ambiental , Resistência a Medicamentos , Ecologia , Gases , Liases/genética , Liases/metabolismo , Mercúrio/metabolismo , Compostos de Metilmercúrio/metabolismo , Compostos de Metilmercúrio/farmacologia , Modelos Biológicos , Compostos Organomercúricos/farmacologia , Oxirredutases/genética , Oxirredutases/metabolismo , Fenótipo , Compostos de Fenilmercúrio/metabolismo , Compostos de Fenilmercúrio/farmacologia , Poluentes do Solo/metabolismo , Poluentes Químicos da Água/metabolismoRESUMO
Mercury compounds are well-known toxic environmental pollutants and potently induce severe neurotoxicological effects in human and experimental animals. Previous studies showed that one of the mechanisms of mercury compounds neurotoxicity arose from the over-activation of the N-methyl d-aspartate (NMDA)-type glutamate receptor induced by increased glutamate release. In this work, we aimed to investigate the molecular mechanisms of Hg compounds neurotoxicities by identifying their biological targets in cells. Firstly, the inhibitory effects of four Hg compounds, including three organic (methyl-, ethyl- and phenyl-mercury) and one inorganic (Hg2+) Hg compounds, on the activity of arginine decarboxylase (ADC), a key enzyme in the central agmatinergic system, were evaluated. They were found to inhibit the ADC activity significantly with methylmercury (MeHg) being the strongest (IC50=7.96nM). Furthermore, they showed remarkable inhibitory effects on ADC activity in PC12 cells (MeHg>EtHg>PhHg>HgCl2), and led to a marked loss in the level of agmatine, an endogenous neuromodulatory and neuroprotective agent that selectively blocks the activation of NMDA receptors. MeHg was detected in the immunoprecipitated ADC from the cells, providing unequivocal evidence for the direct binding of MeHg with ADC in the cell. Molecular dynamics simulation revealed that Hg compounds could form the coordination bond not only with cofactor PLP of ADC, but also with substrate arginine. Our finding indicated that MeHg could attenuate the neuroprotective effects of agmatine by the inhibition of ADC, a new cellular target of MeHg, which might be implicated in molecular mechanism of MeHg neurotoxicity.
Assuntos
Carboxiliases/antagonistas & inibidores , Poluentes Ambientais/toxicidade , Inibidores Enzimáticos/toxicidade , Compostos de Metilmercúrio/toxicidade , Modelos Moleculares , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Absorção Fisiológica , Agmatina/antagonistas & inibidores , Agmatina/metabolismo , Animais , Arginina/metabolismo , Sítios de Ligação , Biocatálise/efeitos dos fármacos , Carboxiliases/química , Carboxiliases/genética , Carboxiliases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/antagonistas & inibidores , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Descarboxilação/efeitos dos fármacos , Poluentes Ambientais/antagonistas & inibidores , Poluentes Ambientais/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Cloreto Etilmercúrico/antagonistas & inibidores , Cloreto Etilmercúrico/metabolismo , Cloreto Etilmercúrico/toxicidade , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Cloreto de Mercúrio/antagonistas & inibidores , Cloreto de Mercúrio/metabolismo , Cloreto de Mercúrio/toxicidade , Compostos de Metilmercúrio/antagonistas & inibidores , Compostos de Metilmercúrio/metabolismo , Simulação de Dinâmica Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Neurônios/metabolismo , Compostos de Fenilmercúrio/antagonistas & inibidores , Compostos de Fenilmercúrio/metabolismo , Compostos de Fenilmercúrio/toxicidade , RatosRESUMO
We have previously indicated that bovine pulmonary artery smooth muscle plasma membrane possesses a complex of 72-kDa gelatinase and TIMP-2 (MMP-2/TIMP-2 complex) [Mol. Cell. Biochem. 258 (2004) 73]. In this paper, we described isolation of MMP-2 from the MMP-2/TIMP-2 complex, characterizations of the isolated MMP-2 and also the complex. MMP-2/TIMP-2 complex was purified from bovine pulmonary vascular smooth muscle plasma membrane using a combination of purification steps. Heparin-sepharose (100 mM NaCl eluate)-purified preparation contained the MMP-2/TIMP-2 complex. The MMP-2/TIMP-2 complex, which was electrophoresed under reducing condition on the SDS-PAGE and immunobloted with a mixture of polyclonal MMP-2 and TIMP-2 antibodies, revealed two separate immunoreactive bands at their respective electrophoretic migration. Continuous elution electrophoresis of the complex resulted to MMP-2 free of any detectable TIMP-2. The homogeneity of the isolated MMP-2 and the complex was demonstrated by SDS-PAGE under nonreducing condition and also by nondenaturing native-PAGE. The purified TIMP-2 free enzyme electrophoresed as a single band of 72-kDa, which could be activated rapidly and fully by aminophenylmercuric acetate (APMA) with the formation of 62-kDa and 45-kDa active species like native MMP-2 purified from the same source (bovine pulmonary artery smooth muscle). Identical treatment of the MMP-2/TIMP-2 complex with APMA resulted to significantly slower and partial conversion of the active species. Addition of pure TIMP-2 to the TIMP-2 free MMP-2 formed a complex with the progelatinase and prevented the rapid autolytic conversion induced by APMA. Immunoblot study with polyclonal MMP-2 antibody suggested that the isolated 72-kDa gelatinase is the MMP-2. We have also presented additional data indicating that the isolated preparation of 72-kDa gelatinase exhibited properties that are identical with MMP-2 obtained from different sources.
Assuntos
Membrana Celular/enzimologia , Metaloproteinase 2 da Matriz/isolamento & purificação , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/citologia , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Animais , Bovinos , Ativação Enzimática , Proteínas da Matriz Extracelular/metabolismo , Gelatina/metabolismo , Substâncias Macromoleculares , Metaloproteinase 2 da Matriz/metabolismo , Peso Molecular , Miócitos de Músculo Liso/enzimologia , Oxidantes/metabolismo , Compostos de Fenilmercúrio/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Artéria Pulmonar/anatomia & histologia , Inibidor Tecidual de Metaloproteinase-2/isolamento & purificaçãoRESUMO
Nucleosides cross the human erythrocyte membrane by a facilitated-diffusion process which is selectively inhibited by nanomolar concentrations of nitrobenzylthioinosine (NBMPR). The chemical asymmetry of the transporter was investigated by studying the effects of p-chloromercuriphenyl sulphonate (PCMBS) on uridine transport and high-affinity NBMPR binding in inside-out and right-side-out membrane vesicles, unsealed erythrocyte ghosts and intact cells. PCMBS was an effective inhibitor of the transporter (50% inhibition at 30 microM), but only when the organomercurial had access to the cytoplasmic membrane surface. PCMBS inhibition of NBMPR binding to ghosts was reversed by incubation with dithiothreitol. Both uridine and NBMPR were able to protect the transporter against PCMBS inhibition.
Assuntos
4-Cloromercuriobenzenossulfonato/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas de Membrana/metabolismo , Compostos de Fenilmercúrio/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Ditiotreitol/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Guanosina/análogos & derivados , Guanosina/metabolismo , Humanos , Proteínas de Transporte de Nucleosídeos , Relação Estrutura-Atividade , Tioinosina/análogos & derivados , Tioinosina/metabolismo , Tionucleosídeos/metabolismo , Uridina/metabolismoRESUMO
Organomercurial pollution occurring in the Rhine river in 1986 led us to study the possibility of depollution by mercury-resistant environmental aquatic strains. Four species of Pseudomonas were investigated for their ability to biotransform phenylmercuric acetate (PMA). Such biological depollution was demonstrated to be due to an enzymatic activity in whole cells and in cell-free extracts from Pseudomonas fluorescens and other Pseudomonas species. PMA biotransformation was followed by high-performance liquid chromatography. Some of those bacteria growing between 4 and 41 degrees C probably represent a natural means of organomercurial depollution, which acts slowly in interaction with other organisms and non-organic porous surfaces.
Assuntos
Compostos de Fenilmercúrio/metabolismo , Pseudomonas/metabolismo , Poluição Química da Água/análise , Biodegradação Ambiental , Cromatografia Líquida de Alta Pressão , Cisteína/metabolismo , Resistência Microbiana a Medicamentos , Técnicas In Vitro , Compostos de Fenilmercúrio/análise , Estimulação QuímicaRESUMO
1-Octanol/water partition coefficients, [HgII]octanol/[HgII]water, provide a simple but limited model system for aspects of the biological behavior of methylmercury(II) and commonly used organomercury(II) medicinal compounds. In an octanol/water system some widely studied antidotes for mercury poisoning at least partly displace the biological thiols L-cysteine and glutathione from binding to MeHgII at pH 6.9. Addition of the antidote meso-dimercaptosuccinic acid to MeHgII in the presence of glutathione results in formation of metallic mercury. For RHgII derivatives of L-cysteine and glutathione, octanol/water partition coefficients follow the order Ph greater than Et greater than Me. An exceptionally high value for diphenylmercury, compared with PhHgII derivatives of L-cysteine and glutathione, is consistent with reported results of the distribution of mercury compounds in rats. Ethylmercury(II) is partly displaced from thimerosal by L-cysteine and glutathione in the octanol/water system, indicating that the active form of thimerosal in vivo may involve binding of EtHgII to biological ligands.
Assuntos
Antídotos , Compostos de Metilmercúrio/intoxicação , Modelos Biológicos , Octanóis , Compostos Organomercúricos , Água , Animais , Antídotos/metabolismo , Fenômenos Químicos , Físico-Química , Cisteína/metabolismo , Compostos de Etilmercúrio/metabolismo , Glutationa/metabolismo , Compostos de Metilmercúrio/metabolismo , Compostos Organomercúricos/metabolismo , Compostos de Fenilmercúrio/metabolismo , Ratos , Succímero/metabolismoRESUMO
Averaged ERGs of rats treated with phenylmercury borate and methylmercury chloride were recorded. Amplitude differences were seen only with the latter treatment. A high Hg tot. level was found in eyes of animals treated with methylmercury.
Assuntos
Compostos de Metilmercúrio/toxicidade , Compostos de Fenilmercúrio/toxicidade , Retina/efeitos dos fármacos , Animais , Eletrorretinografia , Exposição Ambiental , Feminino , Masculino , Compostos de Metilmercúrio/metabolismo , Compostos de Fenilmercúrio/metabolismo , Ratos , Retina/fisiologiaRESUMO
The therapeutic use of stem cells to treat diseases and injuries is a promising tool in regenerative medicine. The umbilical cord provides a rich source of stem cells; we have previously reported a population of stem cells isolated from Wharton's jelly. In this report, we aimed to isolate a novel cell population that was different than those found in Wharton's jelly. We isolated stem cells from the subepithelial layer of the umbilical cord; the cells could be expanded for greater than 90 population doubling and had mesenchymal stem cell characteristics, expressing CD9, SSEA4, CD44, CD90, CD166, CD73, and CD146 but were negative for STRO-1. The cells can be directionally differentiated and undergo osteo-, chondro-, adipo-, and cardiogenesis. In addition, we have identified for the first time that mesenchymal stem cells isolated from umbilical cord can produce microvesicles, termed exosomes. This is the first report describing a stem cell population isolated from the subepithelial layer of the umbilical cord. Given the growth capacity, multilineage potential, and most importantly the low levels of HLA-ABC, we propose that this novel cell isolated from the subepithelial layer of umbilical cord is an ideal candidate for allogeneic cell-based therapy.
Assuntos
Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Adipogenia , Antígenos CD/metabolismo , Antígenos de Superfície/metabolismo , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Separação Celular , Células Cultivadas , Condrogênese , Dexametasona/análogos & derivados , Dexametasona/metabolismo , Combinação de Medicamentos , Células Epiteliais/citologia , Humanos , Cariotipagem , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Compostos de Fenilmercúrio/metabolismo , Antígenos Embrionários Estágio-Específicos/metabolismo , Cordão Umbilical/patologiaRESUMO
Studies were carried out to characterize organomercurial lyase genes from wild type mercury-resistant Escherichia coli isolates, previously collected from five geographically distinct regions of the Indian subcontinent. PCR amplification followed by DNA sequencing of amplified fragments showed three merB identical to the previously characterized mer B from E. coli pR831b that were thus considered as the same gene. The remaining two genes derived from E. coli isolates of an almost mercury-free site (Dal lake, Kashmir) and designated as pIAAD3 merB and pIAAD14 merB showed slight variation (2%) at base. However, this variation in pIAAD3 due to the absence of base "T" at 479 position results in complete frame shift and the predicted MerB-like polypeptide derived from it showed 21.53% divergent at its C terminal end from the previously characterized pR831b MerB. The expression profile of pIAAD3 merB in pQE30 and pUC18 vectors each demonstrated 22.2 kDa proteins. The induced DH5alpha E. coli cells possessing pIAAD3 merB cloned in pUC18 vector split phenyl mercuric acetate (PMA) into benzene and inorganic mercury efficiently, thus giving a clue that the expressed gene product is biologically active. The current study suggests that such genetic changes may take place in the continued absence of mercury pressure, and with such modifications, they finally break down to act as vestigial remnants. Further work is going on in our lab to exploit pIAAD3 merB for the bioremediation of mercury-polluted sites.
Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Liases/genética , Compostos de Mercúrio/metabolismo , Compostos de Mercúrio/farmacologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Benzeno/metabolismo , DNA Bacteriano/análise , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Índia , Liases/química , Dados de Sequência Molecular , Peso Molecular , Compostos de Fenilmercúrio/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Human kidney prolinase, assayed with Pro-Ala, and non-specific dipeptidase, assayed with Gly-Leu, were purified by using DEAE-cellulose, gel-filtration, metal-ion-chelate, hydrophobic and adsorption chromatography and chromatofocusing. Both enzymes gave single peaks of activity that were congruent and the ratio of their activities was constant throughout the purification. Gel filtration indicated an Mr of 100 000 and chromatofocusing a pI of 5.4. Ni2+-chelate chromatography demonstrated the presence of exposed histidine residues on the enzyme and was an effective separative procedure. Polyacrylamide-gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. Both enzyme activities decayed at the same rate at 53 degrees C and were inhibited to the same extent by p-hydroxymercuribenzoate. Of six non-specific dipeptidase substrates tested Gly-Leu gave the highest activity, and of six prolinase substrates Pro-Leu had the highest activity. Gly-Leu was hydrolysed at double the rate of Pro-Leu. Pro-Ala was a competitive inhibitor of activity towards Gly-Leu, and Gly-Leu was a competitive inhibitor of activity towards Pro-Ala. Mixed-substrate studies strongly suggested that Gly-Leu and Pro-Ala were hydrolysed at a common active site. The data are consistent with prolinase and non-specific dipeptidase activity in human kidney being due to a single enzyme.
Assuntos
Dipeptidases/metabolismo , Rim/enzimologia , Dipeptidases/antagonistas & inibidores , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Humanos , Hidrólise , Cinética , Peptídeos/metabolismo , Compostos de Fenilmercúrio/metabolismoRESUMO
Methylmercury labelled with mercury-203 was used for the investigation of the uptake and the release of methylmercury in fish. It has been found that methylmercury compounds adsorbed on fish food remain completely in fish and that they are released with the biological half-time of 110 +/- 20 days. The cumulation of methylmercury from water is very rapid: the cumulation constant is 237 +/- 67 days-1. Equations for the calculation of the concentration of methylmercury in fish were derived and compared with the uptake of phenylmercury and inorganic mercury.
Assuntos
Peixes/metabolismo , Compostos de Metilmercúrio/metabolismo , Ração Animal , Animais , Mercúrio/metabolismo , Compostos de Fenilmercúrio/metabolismo , Poluentes Químicos da Água/metabolismoRESUMO
The pyruvate dehydrogenase kinase consists of a catalytic subunit (Kc) and a basic subunit (Kb) which appear to be anchored to the dihydrolipoyl transacetylase core component (E2) by another subunit, referred to as protein X (Rahmatullah, M., Jilka, J. M., Radke, G. A., and Roche, T. E. (1986) J. Biol. Chem. 261, 6515-6523). We determined the catalytic requirements for reduction and acetylation of the lipoyl moiety in protein X and linked those changes in protein X to regulatory effects on kinase activity. Using fractions prepared by resolution and proteolytic treatments, we evaluated which subunits are required for regulatory effects on kinase activity. With X-KcKb fraction (treated to remove the mercurial agent used in its preparation), we found that the resolved pyruvate dehydrogenase component, the isolated inner domain of E2 (lacking the lipoyl-bearing region of E2), and the dihydrolipoyl dehydrogenase component directly utilize protein X as a substrate. The resulting reduction and acetylation of protein X occurs in association with enhancement of kinase activity. Following tryptic cleavage of E2 and protein X into subdomains, full acetylation of the lipoyl-bearing subdomains of these proteins is retained along with the capacity of acetylating substrates to stimulate kinase activity. All kinase-containing fractions, including those in which the Kb subunit was digested, were inhibited by pyruvate or ADP, alone, and synergistically by the combination suggesting that pyruvate and ADP bind to Kc. Our results suggest that the Kb subunit of the kinase does not contribute to the observed regulatory effects. A dynamic role of protein X in attenuating kinase activity based on changes in the mitochondrial redox and acetylating potentials is considered.
Assuntos
Proteínas Quinases/metabolismo , Acetilação , Difosfato de Adenosina/metabolismo , Alquilação , Animais , Rim/enzimologia , Cinética , Substâncias Macromoleculares , NAD/metabolismo , Oxirredução , Compostos de Fenilmercúrio/metabolismo , Proteínas Serina-Treonina Quinases , Piruvato Desidrogenase Quinase de Transferência de Acetil , Piruvatos/metabolismo , Ácido PirúvicoRESUMO
To investigate the individual role of MerT and MerP encoded by Pseudomonas K-62 pMR26 in the transport of phenylmercury, a series of mutants with a specific point mutation in merT and/or genetic deletion in merP were constructed and transformed into Escherichia coli XL1-Blue. Transport of phenylmercury across the cytoplasmic membrane of E. coli mediated by MerT and MerP was inhibited by NaCN and by cold temperatures. Deletion of merP reduced, but did not completely abolish the C6H5Hg+-hyperuptake and -hypersensitive phenotypes suggesting that transport of phenylmercury into the cytoplasm of E. coli is still occurring. Mutations of the vicinal cysteine residues (Cys24 and Cys25) in the first transmembrane region of MerT to serine caused complete loss of Hg2+-hyperuptake and -hypersensitivity, whereas the mutations did not affect the C6H5Hg+-hyperuptake and -hypersensitive phenotypes. In addition, no additive effect on the C6H5Hg+-hyperuptake and -hypersensitive phenotypes was found, when mutations of the two cysteines in MerT to serine were further introduced in the merP-deleted mutants. These results clearly demonstrated that the vicinal cysteine residues of MerT are not involved in the transport of C6H5Hg+, but indeed are involved in the transport of Hg2+ as previously reported.
Assuntos
Proteínas de Bactérias/fisiologia , Proteínas de Transporte/fisiologia , Proteínas de Transporte de Cátions , Proteínas de Membrana/fisiologia , Compostos de Fenilmercúrio/metabolismo , Plasmídeos , Proteínas , Pseudomonas/genética , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/genética , Primers do DNA , Proteínas de Membrana/genética , Mutagênese Sítio-Dirigida , Mutação PuntualRESUMO
Effects of inorganic ions on the uptake of chloromercuribenzene-p-sulphonic acid (CMBS) were studied in microdissected pancreatic islets of non-inbred ob/ob-mice. Na2SO4 stimulated the total islet cell uptake of CMBS but decreased the amount of CMBS remaining in islets after brief washing with L-cysteine. CaCl2 stimulated both the total and the cysteine-nondisplaceable uptake; the stimulatory effect of CaCl2 on the cysteine-non-displaceable CMBS uptake was counteracted by Na2SO4. NaCl, KCl or choline chloride had no significant effect on the total islet cell uptake of CMBS, whereas LiCl was stimulatory. It is concluded that beta-cells resemble erythrocytes in having a permeation path for CMBS that is inhibited by SO4(2-). By analogy with existing models of the erythrocyte membrane, it is suggested that the SO4(2-) sensitive path leads to sulphydryl groups controlling monovalent cationic permeability in beta-cells.
Assuntos
4-Cloromercuriobenzenossulfonato/metabolismo , Cátions Monovalentes/farmacologia , Ilhotas Pancreáticas/metabolismo , Compostos de Fenilmercúrio/metabolismo , Animais , Cloreto de Cálcio/farmacologia , Camundongos , Cloreto de Potássio/farmacologia , Cloreto de Sódio/farmacologia , Sulfatos/farmacologiaRESUMO
An enzyme (splitting enzyme 2) which catalyzes the splitting of carbon-mercury linkage of arylmercury compounds was found in extracts of mercury-resistant Pseudomonas K-62. This enzyme was purified about 725-fold by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-75 and diethylaminoethyl-cellulose. A purified preparation of the enzyme showed a single band in electrophoresis either on polyacrylamide or sodium dodecyl sulfate-containing polyacrylamide gels. The molecular weight of the enzyme was estimated to be 20,000 (determined by Sephadex G-75 gel filtration) 17,000 (determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis). The enzyme showed a Km of 180 micron and a Vmax of 3.1 mumol/min per mg for p-chloromercuribenzoic acid and a Km of 250 micron and a Vmax of 20 mumol/min per mg for phenylmercuric acetate. The optimum temperature and pH for the reaction were 40 degrees C and 5.0, respectively.
Assuntos
Hidrolases , Compostos de Fenilmercúrio/metabolismo , Pseudomonas/enzimologia , Cloromercurobenzoatos/metabolismo , Resistência Microbiana a Medicamentos , Temperatura Alta , Concentração de Íons de Hidrogênio , Hidrolases/isolamento & purificação , Mercúrio/farmacologia , Peso Molecular , Oxirredutases/metabolismo , Pseudomonas/efeitos dos fármacos , Compostos de Sulfidrila/farmacologiaRESUMO
Mercuric chloride, phenylmercuric chloride, ethylmercuric chloride /0,23 mg Hg/kg/ and methylmercurycyan guanidine /0,46 mg Hg/kg/ were orally administered to rats every second day for 14 weeks. The same doses of the above mentioned mercury compounds were administered alternately with sodium selenite /0,18 mg Se/kg/ to parallel groups of rats at the same time. The level of total and inorganic mercury and of metallothionein was determined. All mercury compounds increased the level of metallothionein in rat kidneys. In rats which received only selenium the level of metallothionein was twice lower in the kidneys in relation to the physiological level of this protein. Selenium eliminated the stimulation of biosynthesis of metallothionein by mercury.
Assuntos
Rim/metabolismo , Metaloproteínas/metabolismo , Metalotioneína/metabolismo , Compostos Organomercúricos/metabolismo , Selênio/farmacologia , Animais , Exposição Ambiental , Compostos de Etilmercúrio/metabolismo , Fígado/metabolismo , Compostos de Metilmercúrio/metabolismo , Compostos de Fenilmercúrio/metabolismo , RatosRESUMO
Phenylmercuric borate (PHB) is very rapidly incorporated into the cells of Escherichia coli, and binds directly to its sites of fixation. The rapid incorporation of PHB probably results from the lipophilic character of the [phenylmercuric]+ cation. The distribution of PHB in the cell is fairly uniform if one compares with that of the proteins. This shows the low specificity of PHB with regard to proteins, all of which appear to be potential sites for fixation. However, from the structural point of view, PHB is more differentiated in its distribution. The cell wall of E. coli and the soluble cytoplasmic fraction do not constitute important sites of fixation. On the contrary, the cytoplasmic membrane appears to be a preferential site of fixation. Moreover, it is very probable that the distribution of PHB in vivo should be even more likely at the cytoplasmic membrane since the process of homogenization of the cells solubilizes a large part of the proteins associated to the membrane. On the membrane, an important part of PHB seems to be associated with the ribosomes and particularly to the ribosomal proteins.
Assuntos
Escherichia coli/efeitos dos fármacos , Compostos de Fenilmercúrio/farmacologia , Proteínas de Bactérias/metabolismo , Boratos , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Compostos de Fenilmercúrio/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Frações SubcelularesRESUMO
Using a newly identified organomercury lyase gene (merB3) expression system from Tn MERI1, the mercury resistance transposon first found in Gram-positive bacteria, a dual-purpose system to detect and remove organomercurial contamination was developed. A plasmid was constructed by fusing the promoterless luxAB genes as bioluminescence reporter genes downstream of the merB3 gene and its operator/promoter region. Another plasmid, encoding mer operon genes from merR1 to merA, was also constructed to generate an expression regulatory protein, MerR1, and a mercury reductase enzyme, MerA. These two plasmids were transformed into Escherichia coli cells to produce a biological system that can detect and remove environmental organomercury contamination. Organomercurial compounds, such as neurotoxic methylmercury at nanomolar levels, were detected using the biomonitoring system within a few minutes and were removed during the next few hours.
Assuntos
Proteínas de Bactérias/genética , Técnicas Biossensoriais/métodos , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Mercúrio/análise , Compostos Organomercúricos/análise , Elementos de DNA Transponíveis , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Liases/metabolismo , Compostos de Fenilmercúrio/metabolismo , Plasmídeos/síntese químicaRESUMO
It has been recently demonstrated that the Mg(2+)-dependent 3'-processing activity of purified human immunodeficiency virus type-1 (HIV-1) integrase is stimulated by the addition of exogenous Zn2+ [Lee, S. P., & Han, M. K. (1996) Biochemistry 35, 3837-3844]. This activation was hypothesized to result from integrase self-association. In this report, we examine the Zn2+ content of purified HIV-1 integrase by atomic absorption spectroscopy and by application of a thiol modification reagent, p-(hydroxymercuri)benzenesulfonate, with a metallochromic indicator, 4-(2-pyridylazo)resorcinol. We find that the Zn2+ content of HIV-1 integrase varies from 0.1 to 0.92 equiv of Zn2+ per monomer depending on the conditions of protein purification. In vitro activity assays, time-resolved fluorescence emission anisotropy, and gel filtration chromatographic analyses all indicate that EDTA yields an apoprotein which is predominantly monomeric and less active with Mg2+. Further, sedimentation equilibrium studies reveal that reconstitution of the apoprotein with Zn2+ results in a monomer-tetramer-octamer transition. These results suggest that Zn2+ promotes a conformation with enhanced oligomerization and thereby stimulates Mg(2+)-dependent 3'-processing. This may also imply that multimers larger than dimers (tetramers and possibly octamers) are required for in vitro activity of integrase in the presence of Zn2+ and Mg2+. It should be noted, however, that the content of Zn2+ did not significantly affect the 3'-processing and strand transfer reactions with Mn2+ in vitro.