RESUMEN
NADH dehydrogenase-2 (NDH-2) from Escherichia coli respiratory chain is a membrane-bound cupric-reductase encoded by ndh gene. Here, we report that the respiratory system of a ndh deficient strain suffered a faster inactivation than that of the parental strain in the presence of tert-butyl hydroperoxide due to endogenous copper. The inactivation was similar for both strains when copper concentration increased in the culture media. Furthermore, several ndh deficient mutants grew less well than the corresponding parental strains in media containing either high or low copper concentrations. A mutant strain complemented with ndh gene almost recovered the parental phenotype for growing in copper limitation or excess. Then, NDH-2 gives the bacteria advantages to diminish the susceptibility of the respiratory chain to damaging effects produced by copper and hydroperoxides and to survive in extreme copper conditions. These results suggest that NDH-2 contributes in the bacterial oxidative protection and in the copper homeostasis.
Asunto(s)
Cobre/toxicidad , Escherichia coli/efectos de los fármacos , Homeostasis/efectos de los fármacos , NADH Deshidrogenasa/fisiología , Estrés Oxidativo/efectos de los fármacos , Oxidorreductasas/fisiología , terc-Butilhidroperóxido/toxicidad , Cobre/metabolismo , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Transporte de Electrón/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Homeostasis/fisiología , Membranas/enzimología , Mutación , NADH Deshidrogenasa/metabolismo , Estrés Oxidativo/fisiología , Oxidorreductasas/metabolismo , Fenotipo , Factores de Tiempo , terc-Butilhidroperóxido/metabolismoRESUMEN
NADH dehydrogenase-2 (NDH-2) from Escherichia coli is a membrane-bound flavoprotein linked to the respiratory chain. We have previously shown that this enzyme has cupric reductase activity that is involved in hydroperoxide-induced oxidative stress. In this paper we present spectroscopic evidence that NDH-2 contains thiolate-bound Cu(I) with luminescence properties. Purified NDH-2 exhibits an emission band at 670nm with excitation wavelengths of 280 and 580nm. This emission is quenched by the specific Cu(I) chelator bathocuproine disulfonate, but not by EDTA. The luminescence intensity is sensitive to the enzyme substrates and, thus, the Cu(I)-thiolate chromophore reflects the redox and/or conformational states of the protein. There is one copper atom per polypeptide chain of the purified NDH-2, as determined by atomic absorption spectroscopy. Bioinformatics allowed us to recognize a putative copper-binding site and to predict four structural/functional domains in NDH-2: (I) the FAD-binding domain, (II) the NAD(H)-binding domain, (III) the copper-binding domain, and (IV) the domain of anchorage to the membrane containing two transmembrane helices, at the C-terminus. A NDH-2 topology model, based on the secondary structure prediction, is proposed. This is the first description of a copper-containing NADH dehydrogenase. Comparative sequence analysis allowed us to identify a branch of homologous dehydrogenases that bear a similar metal-binding motif.
Asunto(s)
Cobre/metabolismo , Compuestos Organometálicos/química , Compuestos Organometálicos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Citoplasma/metabolismo , Transporte de Electrón , Escherichia coli/enzimología , Modelos Genéticos , Datos de Secuencia Molecular , NADH Deshidrogenasa/química , NADH Deshidrogenasa/metabolismo , Estrés Oxidativo , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrofotometría , Factores de TiempoRESUMEN
In this paper we report the up to now ignored fluorescence properties of the specific Cu(I)-chelator bathocuproine disulfonate and their application in assays of total copper and Cu(I). The method is based on the linear quenching of the bathocuproine disulfonate emission at 770 nm (lambda(ex)580 nm) by increasing concentrations of Cu(I), at pH 7.5. Copper concentrations as low as 0.1 microM can be determined. Other metal ions (iron, manganese, zinc, cadmium, cobalt, nickel) do not interfere. The procedure for total copper determination in proteins includes HCl treatment to release the copper, neutralization to pH 7.5 in the presence of citrate to stabilize the copper, and reduction of the copper to Cu(I) by ascorbate in the presence of the chelator. This assay gave results coincident with the analysis by atomic absorption spectroscopy in two selected proteins. In addition, conditions are described (omitting HCl treatment and reduction by ascorbate) for direct measurement of Cu(I) in native proteins, as illustrated for the Escherichia coli NADH dehydrogenase-2. Data show that the fluorometric assays described in this paper are simple and convenient procedures for total copper and direct Cu(I) quantification in determined biological samples.
Asunto(s)
Cobre/análisis , Compuestos Organometálicos/química , Fenantrolinas/química , Animales , Sitios de Unión , Bovinos , Quelantes/química , Eritrocitos/enzimología , Escherichia coli/enzimología , Fluorescencia , Ácido Clorhídrico/metabolismo , Indicadores y Reactivos , NADH Deshidrogenasa/química , NADH Deshidrogenasa/metabolismo , Oxidación-Reducción , Fenantrolinas/metabolismo , Espectrofotometría Atómica , Superóxido Dismutasa/metabolismoRESUMEN
Con el objetivo de determinar las principales caracteristicas de la muerte del medico en la República Dominicana, se revisaron los certificados de defución depositados en los archivos de la Asociación Médica Dominicana (AMD). De los médicos fallecidos durante los últimos 12 años (1978-1990), encontramos un total de 81 médicos fallecidos, 92.6 por ciento corresponden al sexo masculino, 82.7 por ciento eran casados y mayores de 60 años, el 90.1 por ciento de los decesos corresponde a muerte natural predominando las enfermedades cardiovasculares (42.5 por ciento ), neoplasias malignas (20.5 por ciento ). Los decesos por accidentes obtuvieron 62.5 por ciento , se concluye que la esperanza de vida del médico dominicano (61.2 años) es inferior a la de la población general (66 años)
Asunto(s)
Humanos , Masculino , Femenino , Mortalidad , Médicos , Certificado de DefunciónRESUMEN
The accurate evaluation of cardiovascular reactions to psychological challenge requires stable baselines against which change can be evaluated. When more than one challenge is employed, the recovery of this baseline becomes important in order to avoid carryover effects. Resting periods, even those of 20 min or more, do not guarantee baseline stability. We compared a 20-min resting condition and a new form of baseline condition in 48 college men using video tasks as the psychological challenges. The new form was a minimally demanding color detection task, termed the "vanilla" baseline condition. A 10-min version and a 20-min version of this condition were tested. Comparisons to 10-min resting baselines were made using our prior work and values from the literature. Vanilla baseline conditions were shown to be equal to or better than resting baseline conditions using criteria of between- and within-baseline stability, amplitude and significance of responsivity, and generalizability between sessions on separate days. Ten-minute resting baselines also showed acceptable stability, questioning the value of lengthy baselines. The good performance of the 10-min vanilla baseline in initial and replication samples supported its utility for estimating baselines for many purposes.