RESUMEN
A novel DTPA-tris(amide) derivative ligand, DTPA-N,N''-bis[bis(n-butyl)]-N'-methyl-tris(amide)(H2L3) was synthesized. With Gd3+, it forms a positively charged [Gd(L3)]+ complex, whereas with Cu2+ and Zn2+ [ML3], [MHL3]+ and [M2L3]2+ species are formed. The protonation constants of H2L3 and the stability constants of the complexes were determined by pH potentiometry. The stability constants are lower than those for DTPA-N,N''-bis[bis(n-butyl)amide)](H3L2), due to the lower negative charge and reduced basicity of the amine nitrogens in (L3)2-. The kinetic stability of [Gd(L3)]+ was characterised by the rates of metal exchange reactions with Eu3+, Cu2+ and Zn2+. The exchange reactions, which occur via proton and metal ion assisted dissociation of [Gd(L3)]+, are significantly slower than for [Gd(DTPA)]2-, since the amide groups cannot be protonated and interact only weakly with the attacking metal ions. The relaxivities of [Gd(L2)] and [Gd(L3)]+ are constant between 10-20 degrees C, indicating a relatively slow water exchange. Above 25 degrees C, the relaxivities decrease, similarly to other Gd3+ DTPA-bis(amide) complexes. The pH dependence of the relaxivities for [Gd(L3)]+ shows a minimum at pH approximately 9, thus differs from the behaviour of Gd3+-DTPA-bis(amides) which have constant relaxivities at pH 3-8 and an increase below and above. The water exchange rates for [Gd(L2)(H2O)] and [Gd(L3)(H2O)]+, determined from a variable temperature (17)O NMR study, are lower than that for [Gd(DTPA)(H2O)]2-. This is a consequence of the lower negative charge and decreased steric crowding at the water binding site in amides as compared to carboxylate analogues. Substitution of the third acetate of DTPA5- with an amide, however, results in a less pronounced decrease in kex than substitution of the first two acetates. The activation volumes derived from a variable pressure (17)O NMR study prove a dissociative interchange and a limiting dissociative mechanism for [Gd(L2)(H2O)] and [Gd(L3)(H2O)]+, respectively.
Asunto(s)
Medios de Contraste/síntesis química , Gadolinio DTPA/síntesis química , Gadolinio/química , Agua/química , Quelantes/química , Medios de Contraste/metabolismo , Estabilidad de Medicamentos , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Imagen por Resonancia MagnéticaRESUMEN
Double fluorescent and spin sensors were recently used to detect transient oxidants via simultaneous fluorescence change and production of the nitroxide radical detected by electron paramagnetic resonance. One such oxidant, singlet molecular oxygen ((1)O(2)), was detected in thylakoid membrane using these probes. In the present study, we investigated the total (physical and chemical) quenching of (1)O(2) phosphorescence by sensors composed of the 2,5-dihydro-2,2,5,5-tetramethyl-1H-pyrrole moiety attached to xanthene or dansyl fluorophores. We found that the quenching rate constants were in the range (2-7) x 10(7) M(-1)s(-1) in acetonitrile and D(2)O. Quenching of (1)O(2) is usually an additive process in which different functional groups may contribute. We estimated that the (1)O(2) quenching by the amine fragments was ca. one to two orders of magnitude lower than that for the complete molecules. Our data suggest that the incorporation of a fluorescent chromophore results in additional strong quenching of (1)O(2), which may in turn decrease the nitroxide yield via the (1)O(2) chemical path, possibly having an effect on quantitative interpretations. We have also found that probes with the dansyl fluorophore photosensitized (1)O(2) upon UV excitation with the quantum yield of 0.087 in acetonitrile at 366 nm. This result shows that care must be taken when the dansyl-based sensors are used in experiments requiring UV irradiation. We hope that our results will contribute to a better characterization and wider use of these novel double sensors.
Asunto(s)
Colorantes Fluorescentes , Oxígeno Singlete/química , Marcadores de Spin , Acetonitrilos , Compuestos de Dansilo , Deuterio , Espectroscopía de Resonancia por Spin del Electrón , Indicadores y Reactivos , Mediciones Luminiscentes , Fotoquímica , Análisis Espectral , XantenosRESUMEN
Magnetic dipolar interactions between pairs of solvent-exposed nitroxide side chains separated by approximately one to four turns along an alpha-helix in T4 lysozyme are investigated. The interactions are analyzed both in frozen solution (rigid lattice conditions) and at room temperature as a function of solvent viscosity. At room temperature, a novel side chain with hindered internal motion is used, along with a more commonly employed nitroxide side chain. The results suggest that methods developed for rigid lattice conditions can be used to analyze dipolar interactions between nitroxides even in the presence of motion of the individual spins, provided the rotational correlation time of the interspin vector is sufficiently long. The distribution of distances observed for the various spin pairs is consistent with rotameric equilibria in the nitroxide side chain, as observed in crystal structures. The existence of such distance distributions places important constraints on the interpretation of internitroxide distances in terms of protein structure and structural changes.
Asunto(s)
Aminoácidos/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Muramidasa/química , Marcadores de Spin , Temperatura , Aminoácidos/genética , Bacteriófago T4/enzimología , Congelación , Modelos Químicos , Muramidasa/genética , Mutagénesis Sitio-Dirigida , Soluciones , Solventes , Sacarosa , ViscosidadRESUMEN
Oxygen free radicals play an important role in several physiologic and pathophysiologic processes. In pathophysiologic circumstances they can modify and damage biologic systems. Their functional properties (exposed to high oxygen tension) place red blood cells among the most susceptible cells to the harmful effect of free radicals. Because oxygen free radicals are involved in a wide range of diseases, scavenging these radicals should be an important therapeutic approach. In this study the antioxidant capacities of experimental and clinically used cardiovascular drugs were investigated. Phenazine methosulfate was used to generate free radicals and thus harden red blood cells. Filtration technique and potassium leaking were used to detect the scavenging effect of the examined drugs. The experimental drug H-2545 provided 43% protection against phenazine methosulfate-induced changes in red blood cell filterability (p < 0.001). Although some of the examined, clinically used cardiovascular drugs (carvedilol, metoprolol, verapamil, trimetazidine) also showed significant (p < 0.05) antioxidant effect, they were less efficient than H-2545. The scavenger effect of this novel drug exceeded the antioxidant properties of vitamin E. Modification of mexiletine with a pyrroline ring significantly improved its antioxidant capacity, suggesting that this molecular segment is responsible for the antioxidant effect.
Asunto(s)
Antiarrítmicos/farmacología , Antioxidantes/farmacología , Fármacos Cardiovasculares/farmacología , Eritrocitos/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Mexiletine/análogos & derivados , Antiarrítmicos/sangre , Carbazoles/farmacología , Fármacos Cardiovasculares/sangre , Fármacos Cardiovasculares/clasificación , Carvedilol , Humanos , Indoles/farmacología , Masculino , Metosulfato de Metilfenazonio , Mexiletine/farmacología , Propanolaminas/farmacología , Pirroles/farmacología , Sotalol/farmacologíaRESUMEN
The regulatory domain of the myosin head is believed to serve as a lever arm that amplifies force generated in the catalytic domain and transmits this strain to the thick filament. The lever arm itself either can be passive or may have a more active role storing some of the energy created by hydrolysis of ATP. A structural correlate which might distinguish between these two possibilities (a passive or an active role) is the stiffness of the domain in question. To this effect we have examined the motion of the proximal (ELC) and distal (RLC) subdomains of the regulatory domain in reconstituted myosin filaments. Each subdomain was labeled with a spin label at a unique cysteine residue, Cys-136 of ELC or Cys-154 of mutant RLC, and its mobility was determined using saturation transfer electron paramagnetic resonance spectroscopy. The mobility of the two domains was similar; the effective correlation time (tau(eff)) for ELC was 17 micros and that for RLC was 22 micros. Additionally, following a 2-fold change of the global dynamics of the myosin head, effected by decreasing the interactions with the filament surface (or the other myosin head), the coupling of the intradomain dynamics remained unchanged. These data suggest that the regulatory domain of the myosin head acts as a single mechanically rigid body, consistent with the regulatory domain serving as a passive lever.
Asunto(s)
Miosinas Cardíacas , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/fisiología , Miosinas/química , Miosinas/fisiología , Animales , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Proteínas Motoras Moleculares/síntesis química , Proteínas Motoras Moleculares/genética , Cadenas Ligeras de Miosina/química , Cadenas Ligeras de Miosina/genética , Miosinas/síntesis química , Estructura Terciaria de Proteína/genética , Conejos , Proteínas Recombinantes/química , Marcadores de SpinRESUMEN
Two single cysteine substitution mutants at helix surface sites in T4 lysozyme (D72C and V131C) have been modified with a series of nitroxide methanethiosulfonate reagents to investigate the structural and dynamical origins of their electron paramagnetic resonance spectra. The novel reagents include 4-substituted derivatives of either the pyrroline or pyrrolidine series of nitroxides. The spectral line shapes were analyzed as a function of side chain structure and temperature using a simulation method with a single order parameter and diffusion rates about three orthogonal axes as parameters. Taken together, the results provide strong support for an anisotropic motional model of the side chain, which was previously proposed from qualitative features of the spectra and crystal structures of spin labeled T4 lysozyme. Site-specific differences in apparent order parameter are interpreted in terms of backbone dynamics modes with characteristic correlation times in the nanosecond or faster time scale. The saturated 4-substituted pyrrolidine nitroxides are shown to be a suitable template for novel "functionalized" side chains designed to mimic salient features of the native side chains they replace.
Asunto(s)
Bacteriófago T4/enzimología , Muramidasa/química , Muramidasa/genética , Marcadores de Spin , Detección de Spin , Sustitución de Aminoácidos/genética , Anisotropía , Arginina/genética , Ácido Aspártico/genética , Bacteriófago T4/genética , Cisteína/genética , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Óxidos de Nitrógeno/química , Conformación Proteica , Estructura Secundaria de Proteína/genética , Marcadores de Spin/síntesis química , Detección de Spin/métodos , Temperatura , Valina/genéticaRESUMEN
We compared the effect of photoinhibition by excess photosynthetically active radiation (PAR), UV-B irradiation combined with PAR, low temperature stress and paraquat treatment on photosystem (PS) II. Although the experimental conditions ensured that the four studied stress conditions resulted in approximately the same extent of PS II inactivation, they clearly followed different molecular mechanisms. Our results show that singlet oxygen production in inactivated PS II reaction centres is a unique characteristic of photoinhibition by excess PAR. Neither the accumulation of inactive PS II reaction centres (as in UV-B or chilling stress), nor photo-oxidative damage of PS II (as in paraquat stress) is able to produce the special oxidizing conditions characteristic of acceptor-side-induced photoinhibition.
Asunto(s)
Estrés Oxidativo , Fotosíntesis/fisiología , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Transporte de Electrón , Luz , Estructura Molecular , Oxígeno/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/efectos de la radiación , Complejo de Proteína del Fotosistema II , Hojas de la Planta/metabolismo , Plantas Tóxicas , Oxígeno Singlete , Nicotiana , Rayos UltravioletaRESUMEN
The efficacy and mechanism of protection of a new 2,2,5, 5-tetramethylpyrroline derivative of mexiletine, MEX-NH, against ischemia/reperfusion-induced cardiac dysfunction are reported. The MEX-NH and its nitroxide metabolite are membrane-permeable antioxidants. Studies were performed in an isolated rat heart model to measure the efficacy of MEX-NH in preventing postischemic injury. Serial measurements of contractile function and coronary flow were performed on hearts subjected to 30 min of global 37 degrees C ischemia followed by 45 min of reperfusion. Hearts were either untreated or treated with 25 microM MEX-NH or MEX for 1 min before ischemia. The hearts treated with MEX-NH showed marked recovery of left ventricular developed pressure (96.3 +/- 2.7% of preischemic value) compared with untreated (13.7 +/- 1.0%) or MEX-treated (19.9 +/- 2.7%) hearts. The cardiac sarcolemmal Na(+),K(+)-ATPase activity showed that the enzyme activity was fully restored in hearts treated with MEX-NH compared with 65 +/- 5.3% inhibition in the untreated hearts. Competitive inhibition of [(3)H]ouabain binding revealed that the MEX-NH binds at the K(+)-binding site of the enzyme. The present study establishes that the compound MEX-NH provides marked protection against ischemia/reperfusion-induced contractile dysfunction in isolated hearts. A combination of reversible inhibition of Na(+)/K(+)-ATPase activity during ischemia and site-targeted antioxidative effect upon reperfusion seems to contribute to this cardioprotection.
Asunto(s)
Mexiletine/farmacología , Daño por Reperfusión Miocárdica/prevención & control , Pirroles/farmacología , Disfunción Ventricular Izquierda/prevención & control , Animales , Antiarrítmicos/farmacología , Antioxidantes/farmacología , Unión Competitiva , Circulación Coronaria/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Radical Hidroxilo/metabolismo , Mexiletine/análogos & derivados , Mexiletine/metabolismo , Contracción Miocárdica/efectos de los fármacos , Isquemia Miocárdica/enzimología , Miocardio/enzimología , Miocardio/metabolismo , Ouabaína/metabolismo , Oxidación-Reducción , Pirroles/metabolismo , Ratas , Ratas Sprague-Dawley , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Disfunción Ventricular Izquierda/etiología , Presión Ventricular/efectos de los fármacosRESUMEN
A series of nitroxide spin-labeled alpha- or beta-galactopyranosides and a nitroxide spin-labeled beta-glucopyranoside have been synthesized and examined for binding to the lactose permease of Escherichia coli. Out of the twelve nitroxide spin-labeled galactopyranosides synthesized, 1-oxyl-2, 5, 5-trimethyl-2-[3-nitro-4-N-(hexyl-1-thio-beta-D-galactopyranosid-1 -yl )]aminophenyl pyrrolidine (NN) exhibits the highest affinity for the permease based on the following observations: (a) the analogue inhibits lactose transport with a K(I) about 7 microM; (b) NN blocks labeling of single-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 microM; (c) electron paramagnetic resonance demonstrates binding of the spin-labeled sugar by purified wild-type permease in a manner that is reversed by nonspin-labeled ligand. The equilibrium dissociation constant (K(D)) is about 23 microM and binding stoichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of single-Cys148 permease with MIANS. It is concluded that NN binds specifically to lac permease with an affinity in the low micromolar range. Furthermore, affinity of the permease for the spin-labeled galactopyranosides is directly related to the length, hydrophobicity, and geometry of the linker between the galactoside and the nitroxide spin-label.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/enzimología , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos , Pirrolidinas/metabolismo , Marcadores de Spin , Simportadores , Tiogalactósidos/metabolismo , Alquilantes/metabolismo , Naftalenosulfonatos de Anilina/metabolismo , Sitios de Unión , Transporte Biológico , Cisteína/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Galactosa/metabolismo , Lactosa/metabolismo , Proteínas de Transporte de Membrana/química , Estructura Secundaria de Proteína , Pirrolidinas/química , Detección de Spin , Tiogalactósidos/síntesis química , Tiogalactósidos/químicaAsunto(s)
Aminas/metabolismo , Oxígeno/análisis , Plantas/metabolismo , Aminas/química , Óxidos N-Cíclicos/química , Espectroscopía de Resonancia por Spin del Electrón/métodos , Luz , Fotoquímica , Plantas/efectos de la radiación , Oxígeno Singlete , Espectrometría de Fluorescencia/métodos , Marcadores de SpinRESUMEN
We investigated the cardioprotective efficacy of a new compound based on 2,2,5,5-tetramethyl-3-pyrroline-3-carboxamide (TPC-NH). Biochemical studies using electron paramagnetic resonance (EPR) spectroscopy suggest that TPC-NH is a scavenger of reactive oxygen species. In vitro cellular studies show that TPC-NH protects isolated cardiomyocytes against oxidative damage caused by superoxide radicals. Ex vivo EPR studies on the isolated rat heart indicate that the TPC-NH is metabolically oxidized to the nitroxide form. Studies were also performed in the isolated rat heart model to measure the efficacy of TPC-NH and its metabolites in preventing postischemic reperfusion injury. Serial measurements of contractile function were performed on hearts subjected to ischemia-reperfusion. Hearts were either untreated or treated with 50 microM TPC-NH or with its metabolites for 1 min before ischemia and during the first 5 min of reflow. TPC-NH showed marked protection with a more than 3-fold increased recovery of contractile function compared with control hearts, whereas its oxidative metabolites exhibited significant but lower protection. Thus, TPC-NH and, to a lesser extent, its oxidation metabolites exhibit potent membrane-targeted antioxidant action and exert marked protection against myocardial injury in the postischemic heart.
Asunto(s)
Antioxidantes/farmacología , Isquemia Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/prevención & control , Ftalimidas/farmacología , Animales , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Radicales Libres , Corazón/efectos de los fármacos , Corazón/fisiopatología , Isquemia Miocárdica/fisiopatología , Estrés Oxidativo , Ratas , Ratas Sprague-DawleyRESUMEN
KcsA is a prokaryotic potassium channel. The present study employs cysteine scanning mutagenesis and site-directed spin labeling to investigate the structure of the second transmembrane segment (residues 82-120) in functional tetrameric channels reconstituted in lipid bilayers. Spin-spin interactions are observed between nitroxide side chains at symmetry-related sites close to the 4-fold axis of symmetry. To aid in quantitative analysis of these interactions, a new diamagnetic analogue of the nitroxide side chain is used to prepare magnetically dilute samples with constant structure. Using constraints imposed by the spin-spin interactions, a packing model for this segment is deduced that is in excellent agreement with the recently reported crystal structure [Doyle, D., et al. (1998) Science 280, 69-77]. The relatively immobilized state of the nitroxide side chains suggests that the channel is rigid on the electron paramagnetic resonance time scale. Moreover, the poor sulfhydryl reactivity of the cysteine at many locations indicates that the channel is not subject to the low-frequency fluctuations that permit reaction of buried cysteines. At sites expected to be located in the pore, the accessibility of the side chains to collision with O(2) or nickel(II) ethylenediaminediacetate is low. This inaccessibility, together with the generally low mobility of the side chains throughout the sequence, makes it difficult to detect the presence of the pore based on these measurements. However, the presence of a solvated pore can be directly demonstrated using a polarity parameter deduced from the EPR spectra recorded at low temperature. These measurements also reveal the presence of a polarity gradient in the phospholipid bilayer.
Asunto(s)
Proteínas Bacterianas/química , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Canales de Potasio/química , Marcadores de Spin , Streptomyces/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Cristalografía por Rayos X , Cisteína/genética , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Estructura Secundaria de Proteína , Streptomyces/genéticaRESUMEN
The synthesis of new bifunctional spin-labeled cross-linking reagents is described. Covalent attachment to papain was achieved via a thiol-specific thiosulfonate residue and, for the second anchor point, via a nonspecific photoreactive azido function. The thiosulfonate formed a reversible disulfide linkage, which could be cleaved again reductively by dithiothreitol. The spin label, a pyrroline-1-oxyl radical, was highly immobilized after attachment to papain by both functional groups and showed little if any relative motion with respect to the protein.
Asunto(s)
Marcadores de Spin/síntesis química , Reactivos de Enlaces Cruzados , Ditiotreitol/química , Espectroscopía de Resonancia por Spin del Electrón , Indicadores y Reactivos , Modelos Moleculares , Papaína/química , Fotólisis , Reactivos de Sulfhidrilo/químicaRESUMEN
Previous studies have shown that the mobility of nitroxide side chains in a protein, inferred from the electron paramagnetic resonance (EPR) spectra, can be used to classify particular sites as helix surface sites, tertiary contact sites, buried sites, or loop sites. In addition, the sequence dependence of mobility can identify regular secondary structure. However, in the most widely used side chain, an apparent interaction of the nitroxide ring with the protein at some helix surface sites gives rise to EPR spectra degenerate with those at tertiary contact sites. In the present study, we use selected sites in T4 lysozyme to evaluate novel nitroxide side chains designed to resolve this degeneracy. The results indicate that the reagent 3-(methanesulfonylthiomethyl)-2,2, 5,5-tetramethylpyrrolidin-1-yloxy reacts with cysteine to give a nitroxide side chain that has a high contrast in mobility between helix surface and tertiary contact sites, effectively resolving the degeneracy. The reagent 3-(iodomercuriomethyl)-2,2,5,5-tetramethyl-2, 5-dihydro-1H-pyrrol-1-yloxy reacts with cysteine to provide a mercury-linked nitroxide that also shows reduced interaction with the protein at most helix surface sites. Thus, these new side chains may be the preferred choices for structure determination using site-directed spin labeling.
Asunto(s)
Bacteriófago T4/enzimología , Muramidasa/química , Marcadores de Spin , Sustitución de Aminoácidos/genética , Arginina/genética , Ácido Aspártico/genética , Sitios de Unión/genética , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Muramidasa/genética , Mutagénesis Sitio-Dirigida , Óxidos de Nitrógeno/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Valina/genéticaRESUMEN
In plants experiencing environmental stress, the formation of reactive oxygen is often presumed. In this study, singlet oxygen was detected in broad bean (Vicia faba) leaves that were photoinhibited in vivo. Detection was based on the reaction of singlet oxygen with DanePy (dansyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole) yielding a nitroxide radical (DanePyO) which is EPR active and also features lower fluorescence compared to DanePy. The two (fluorescent and spin) sensor fuctions of DanePy are commensurate, which makes detecting singlet oxygen possible with a spectrofluorimeter in samples hard to measure with EPR spectroscopy [Kálai, T., Hideg, E., Vass, I., and Hideg, K. (1998) Free Radical Biol. Med. 24, 649-652]. We found that in leaves saturated with DanePy, the fluorescence of this double sensor was decreased when the leaves were photoinhibited by 1500 micromol m-2 s-1 photosynthetically active radiation. This fluorescence quenching is the first direct experimental evidence that photoinhibition of photosynthesis in vivo is accompanied by 1O2 production and is, at least partly, governed by the process characterized as acceptor side-induced photoinhibition in vitro.
Asunto(s)
Óxidos de Nitrógeno/farmacología , Oxígeno/metabolismo , Fotosíntesis , Hojas de la Planta/metabolismo , Compuestos de Dansilo/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Fabaceae , Polarización de Fluorescencia , Radicales Libres/farmacología , Fotoquímica , Fotosíntesis/efectos de los fármacos , Proteínas del Complejo del Centro de Reacción Fotosintética/antagonistas & inhibidores , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Piperidinas/metabolismo , Hojas de la Planta/efectos de los fármacos , Plantas Medicinales , Oxígeno Singlete , Espectrometría de Fluorescencia , Marcadores de Spin , Factores de TiempoRESUMEN
The protective effects of stable nitroxides, as well as their hydroxylamine and amine precursors, have been tested in Chinese hamster V79 cells subjected to H2O2 exposure at fixed concentration or exposure to ionizing radiation. Cytotoxicity was evaluated by monitoring the viability of the cells assessed by the clonogenic assay. The compounds tested at fixed concentration varied in terms of ring size, oxidation state, and ring substituents. Electrochemical studies were carried out to measure the redox midpoint potentials. The studies show that in the case of protection against H2O2 exposure, the protection was determined by the ring size, oxidation state, and redox midpoint potentials. In general the protection factors followed the order nitroxides > hydroxylamines > amines. Both the six-membered ring nitroxides and substituted five-membered ring nitroxides were efficient protectors. For six-membered ring nitroxides, the compounds exhibiting the lowest midpoint potentials exhibited maximal protection. In the case of X-radiation, nitroxides were the most protective though some hydroxylamines were also efficient. The amines were in some cases found to sensitize the toxicity of aerobic radiation exposure. The protection observed by the nitroxides was not dependent on the ring size. However, the ring substituents had significant influence on the protection. Compounds containing a basic side chain were found to provide enhanced protection. The results in this study suggest that these compounds are novel antioxidants which can provide cytoprotection in mammalian cells against diverse types of oxidative insult and identify structural determinants optimal for protection against individual types of damage.
Asunto(s)
Antioxidantes/farmacología , Óxidos N-Cíclicos/farmacología , Protectores contra Radiación/farmacología , Animales , Antioxidantes/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cricetinae , Cricetulus , Óxidos N-Cíclicos/química , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Radicales Libres/química , Radicales Libres/farmacología , Peróxido de Hidrógeno/toxicidad , Oxidantes/toxicidad , Oxidación-Reducción , Protectores contra Radiación/química , Relación Estructura-ActividadRESUMEN
A series of dansylated sterically hindered amines designed to trapping reactive oxygen species, were synthesized. Compounds were tested in isolated thylakoid membranes subjected to photoinhibition by excess photosynthetically active radiation (400-700 nm). DanePy showed good selectivity for singlet oxygen and the formation of nitroxide was detected by appearance of ESR signal and quenching fluorescence.
Asunto(s)
Cloroplastos/química , Compuestos de Dansilo/síntesis química , Oxígeno/análisis , Piperidinas/síntesis química , Especies Reactivas de Oxígeno/metabolismo , Marcadores de Spin , Cloroplastos/ultraestructura , Óxidos N-Cíclicos/química , Espectroscopía de Resonancia por Spin del Electrón , Membranas Intracelulares/química , Luz , Óxidos de Nitrógeno/metabolismo , Oxígeno Singlete , Espectrometría de Fluorescencia , Spinacia oleraceaRESUMEN
alpha-Aryl N-adamant-1-yl nitrones were synthesized and evaluated with respect to the stability of the hydroxyl radical adduct. The polarity and water solubility of nitrones were altered with changing the alpha-aryl groups. Introduction of adamantane ring instead of tert-butyl group resulted in a reasonable good stability of hydroxyl radical adduct for biological measurements.
Asunto(s)
Radical Hidroxilo/química , Óxidos de Nitrógeno/síntesis química , Marcadores de Spin/síntesis química , Espectroscopía de Resonancia por Spin del Electrón , Depuradores de Radicales Libres/síntesis química , Depuradores de Radicales Libres/farmacología , Óxidos de Nitrógeno/farmacologíaRESUMEN
The recent determination of the myosin head atomic structure has led to a new model of muscle contraction, according to which mechanical torque is generated in the catalytic domain and amplified by the lever arm made of the regulatory domain [Fisher, A. J., Smith, C. A., Thoden, J., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995) Biochemistry 34, 8960-8972]. A crucial aspect of this model is the ability of the regulatory domain to move independently of the catalytic domain. Saturation transfer-EPR measurements of mobility of these two domains in myosin filaments give strong support for this notion. The catalytic domain of the myosin head was labeled at Cys-707 with indane dione spin label; the regulatory domain was labeled at the single cysteine residue of the essential light chain and exchanged into myosin. The mobility of the regulatory domain in myosin filaments was characterized by an effective rotational correlation time (tauR) between 24 and 48 micros. In contrast, the mobility of the catalytic domain was found to be tauR = 5-9 micros. This difference in mobility between the two domains existed only in the filament form of myosin. In the monomeric form, or when bound to actin, the mobility of the two domains in myosin was indistinguishable, with tauR = 1-4 micros and >1,000 micros, respectively. Therefore, the observed difference in filaments cannot be ascribed to differences in local conformations of the spin-labeled sites. The most straightforward interpretation suggests a flexible hinge between the two domains, which would have to stiffen before force could be generated.
Asunto(s)
Miosinas/química , Animales , Espectroscopía de Resonancia por Spin del Electrón , Conformación Proteica , Marcadores de SpinRESUMEN
The contributions of intramembranous and extramembranous segments of transmembrane proteins to frictional forces have been studied by covalently attached 14N- and 15N-indane dione and maleimide spin labels using saturation transfer electron spin resonance spectroscopy. The role of molecular size and membrane viscosity is discussed in determining rotational mobilities of proteins. By comparing the measured rotational correlation times with the predictions of hydrodynamic models the aggregation states of transmembrane proteins is estimated. On increasing the viscosity of the aqueous phase by polyols the viscous drag of the extramembranous segments of proteins is increased and from systematic hydrodynamic measurements the size of the protruding segments can be estimated. The role of slowed molecular diffusion is briefly discussed in the inhibition of enzymatic activity.