RESUMEN
The 28 kDa peroxiredoxin from rat exhibited peroxidase activity only in the presence of dithiothreitol. Both organic and nonorganic peroxidases were found to be substrates for the 28-kDa peroxiredoxin activity. Analysis of the protective antioxidant activity of the 28-kDa peroxiredoxin revealed that it is accounted for by its peroxidase activity.
Asunto(s)
Antioxidantes/metabolismo , Peroxidasas/metabolismo , Animales , Ditiotreitol/metabolismo , Peroxirredoxinas , Ratas , Especificidad por SustratoRESUMEN
Peroxiredoxins are a novel family of antioxidant proteins that specifically prevent enzymes from metal-catalyzed oxidation. The localization of a member of the mono-cystein subfamily of peroxiredoxins, the 28-kDa protein, in different rat tissues and its antioxidant properties were investigated. By immunoblotting, the 28-kDa peroxiredoxin was found to be most highly concentrated in olfactory epithelium and present in all tissues tested (skin, lung, trachea, kidney, womb, and brain). Immunostaining with rabbit polyclonal antibody raised against the 28-kDa peroxiredoxin revealed the particularly high level of the 28-kDa peroxiredoxin immunoreactivity in air-contacting areas (apical regions and mucus of the olfactory and respiratory epithelium and skin epidermis), which are continually exposed to numerous air-borne reactive oxygen species. In the apical regions of the olfactory and respiratory epithelium, the 28-kDa-peroxiredoxin immunogold labeling outlined microvilli and cilia and was mainly located in sustentacular cells and in respiratory and goblet cells, as electron-microscopic analysis revealed. In skin epidermis, the 28-kDa peroxiredoxin immunoreactivity was confined to the granular layer and specifically concentrated in sebaceous glands of hair follicle. In situ hybridization with 33P-labeled antisense RNA probe revealed the expression of the 28-kDa peroxiredoxin mRNA in tissues with a high level of the 28-kDa peroxiredoxin immunoreactivity. Immunodepletion of the 28-kDa peroxiredoxin profoundly decreased the antioxidant activity of the olfactory tissue extract.
Asunto(s)
Antioxidantes/análisis , Mucosa Olfatoria/química , Peroxidasas/análisis , Animales , Antioxidantes/metabolismo , Dendritas/química , Dendritas/metabolismo , Expresión Génica/fisiología , Hibridación in Situ , Masculino , Microscopía Inmunoelectrónica , Mucosa Olfatoria/metabolismo , Mucosa Olfatoria/ultraestructura , Oxidación-Reducción , Peroxidasas/genética , Peroxidasas/metabolismo , Peroxirredoxinas , ARN Mensajero/análisis , Conejos , Ratas , Ratas WistarRESUMEN
Conformation behavior of phase T2 DNA in the process of its interaction with it E. coli RNA polymerase was studied using spin labeling technique. T2 DNA was modified by the spin-labeled imidazole at OH-groups of glucosylated cytidine residues. It was shown that the binding of RNA polymerase under the conditions favoring the formation of open promoter complexes induces specific conformational changes in the spin-labeled DNA. The observed conformational changes encompass not only the promoter regions of DNA which are involved in direct contacts with RNA polymerase molecules but extend over remote DNA sites (long-range effect). In relation to this effect, current theoretical models of DNA dynamics are discussed.
RESUMEN
The 28 kDa secretory protein is one of the abundant water-soluble proteins in olfactory epithelium of mammals. Analysis of partial amino acid sequence of the 28 kDa protein strongly suggested that it belongs to a new family of highly conserved antioxidant proteins requiring thiol for their antioxidant activity (TSA/AhpC family). In the present study, we found the 28 kDa protein to have thiol-dependent antioxidant activity, thereby protecting radical-sensitive proteins such as glutamine synthetase and hemoglobin from oxidative modification caused by thiol-dependent metal ion-catalyzed oxidation system. The purified 28 kDa protein did not possess catalase or glutathione peroxidase activities, and required thiols to exhibit its antioxidant activity. The 28 kDa protein is the first member of the family of thiol-specific antioxidants identified in olfactory epithelium and the first secretory protein shown to be thiol-specific antioxidant.
Asunto(s)
Antioxidantes/química , Proteínas de Neoplasias , Mucosa Olfatoria/química , Proteínas/química , Compuestos de Sulfhidrilo/metabolismo , Animales , Glutamato-Amoníaco Ligasa/metabolismo , Hemoglobinas/metabolismo , Peróxido de Hidrógeno/metabolismo , Hierro/metabolismo , Estrés Oxidativo/fisiología , Peroxidasas/farmacología , Peroxirredoxinas , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The secretory 28 kD protein, an abundant water-soluble protein from rat olfactory epithelium, belongs to the 1-Cys subfamily of thiol-specific antioxidants (peroxiredoxins). The 28 kD protein contains a single cysteine residue at the position 46 which accounts for the antioxidant activity. Here we studied the effects of N-ethyilmaleimide and t-butyl hydroperoxide on the antioxidant activity of the 28 kD protein and that of the 23 kD protein from rat erythrocyte which is a member of 2-Cys subfamily of peroxiredoxines. N-ethylmaleimide, modifier for cysteine residues, had no effect on antioxidant activity of the dithiothreitol-treated 28 kD protein but irreversibly inhibited activity of the 23 kD protein under reducing conditions. The 28 kD protein was sensitive to treatment with peroxides: t-butyl hydroperoxide at micromolar concentrations was shown to irreversibly inactivate 28 kD protein. In the presence of dithiothreitol, the lower level of peroxide concentrations was required to inhibit 28 kD protein activity. The mechanism of this effect may be mediated through conversion of sulfhydryl group of 46Cys to oxidized states (46Cys-SO2H and 46Cys-SO3H). Antioxidant property of 23 kD protein was impaired by t-butyl hydroperoxide only in the presence of dithiothreitol. The concentrations of t-butyl hydroperoxide needed to affect the 23 kD protein were at least one order of magnitude higher than were required for the 28 kD protein inhibition. The given results suggest the essential differences between catalytic site of 28 kD protein and that of 2-Cys peroxiredoxins.
Asunto(s)
Mucosa Olfatoria/metabolismo , Peroxidasas/metabolismo , Animales , Catálisis , Etilmaleimida/farmacología , Peroxirredoxinas , Ratas , Ratas WistarRESUMEN
Conformational behaviour of T2 DNA during its complex formation with E.coli RNA polymerase was studied by spin label technique. T2 DNA was selectively modified at its readily melting AT-rich sites by bromoacetooxypiperidine-1-oxyl-radical. Specific conformational changes are induced in DNA structure by RNA polymerase attachment. The changes are observed under the conditions when open transcriptionally competent complexes are formed. The readily melting sites of T2 DNA were shown to be involved in the formation of functionally active promoters.