RESUMEN
Oxygen free radicals have been indirectly implicated in reperfusion injury following ischemia in the isolated rabbit heart. The authors moved to detect free radicals in an isolated rat heart model as a prerequisite to studying its effects during ischemia and reperfusion. Several different spin trapping agents and electron paramagnetic resonance (EPR) spectroscopy were used to detect free radicals being generated during ischemia and reperfusion. The possible roles of ferrous iron and hydrogen peroxide generation in reperfusion injury were also investigated. No free radical "bursts" were detected with any of the spin traps used in this model during ischemia or reperfusion. Hydrogen peroxide and hydroxyl free radicals do not appear to be involved in tissue reperfusion injury. This study suggests that free radicals are not produced in clinically significant quantities under these conditions to account for ischemic myocardial damage.
Asunto(s)
Daño por Reperfusión Miocárdica/metabolismo , Miocardio/química , Oxígeno/metabolismo , Animales , Espectroscopía de Resonancia por Spin del Electrón , Compuestos Ferrosos/metabolismo , Radicales Libres/metabolismo , Ratas , Ratas Sprague-Dawley , Organismos Libres de Patógenos Específicos , Detección de SpinRESUMEN
The 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adduct of myoglobin (Mb) or hemoglobin (Hb) was formed when metmyoglobin (MetMb) or methemoglobin (MetHb) reacted with H2O2 in the presence of DMPO, and both decayed with half-life of a few minutes. The DMPO spin adduct of Mb decayed with biphasic kinetics with k1 = 0.645 min-1 and k2 = 0.012 min-1, indicating that the spin adduct consisted of two kinetically heterogeneous species, stable and unstable ones. The DPMO spin adduct of Hb, however, was homogeneous. Decay of both spin adducts was accelerated in the presence of tyrosine, tryptophan or cysteine, but not phenylalanine, methionine or histidine. The decay obeyed the first order kinetics at varying concentrations of the spin adducts. The decay was accelerated by denaturation and proteolysis of protein moiety. The decay rate was not affected by the extra addition of MetMb or MetHb to each spin adduct. The decay rate of the spin adduct of Mb was increased by hematin in the presence of H2O2 and decreased by catalase. Decay of stable spin adduct of Mb, however, was not significantly changed under any experimental conditions used. These results led us to conclude that instability of the DMPO-spin adducts of Mb and Hb is due to intramolecular redox reactions between the spin adducts and amino acid residues and/or products of the reaction between heme and H2O2.
Asunto(s)
Óxidos N-Cíclicos/química , Hemoproteínas/química , Peróxido de Hidrógeno/química , Metahemoglobina/química , Metamioglobina/química , Marcadores de Spin , Animales , Bovinos , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Semivida , CaballosRESUMEN
When Escherichia coli was incubated with xanthine oxidase and acetaldehyde, the killing of E. coli was accelerated by iron-EDTA but inhibited by hematin or hemoglobin. On the other hand, when E. coli was incubated with human neutrophils in the presence of phorbol myristate acetate (PMA), all of these iron species at concentrations of a few micromolar accelerated the inactivation of neutrophils and in so doing protected the E. coli from being killed by the neutrophils. The inactivation of the neutrophils was accompanied by an increase in lipid peroxidation and by a decrease in viability measured with trypan blue. This inactivation was inhibited by scavengers such as deoxyribose, mannitol, or thiourea. Desferrioxamine B and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) both inhibited the inactivation mediated by iron-EDTA, but had no effect on the hematin- or hemoglobin-mediated inactivation. Vanadium (vanadyl ion), an effective Fenton reagent, behaved in the same way as iron-EDTA relative to the effects of DMPO on neutrophil inactivation. These results led us to conclude that neutrophils were inactivated during PMA stimulation by OH radicals in the presence of iron-EDTA and by some other oxidizing species when hematin or Hb is present. Ascorbate enhanced the inactivation of neutrophils mediated by these iron species. Catalase was very effective in inhibiting neutrophil inactivation. Superoxide dismutase was not as effective but the combination with catalase was most effective.
Asunto(s)
Ácido Edético/farmacología , Compuestos Férricos/farmacología , Hemina/farmacología , Hemoglobinas/farmacología , Neutrófilos/fisiología , Superóxidos/metabolismo , Adulto , Animales , Ácido Ascórbico/farmacología , Actividad Bactericida de la Sangre , Catalasa/farmacología , Bovinos , Óxidos N-Cíclicos/farmacología , Escherichia coli , Depuradores de Radicales Libres , Humanos , Peróxido de Hidrógeno/metabolismo , Radical Hidroxilo/farmacología , Peroxidación de Lípido , Neutrófilos/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
BACKGROUND: Several investigators have indirectly implicated oxygen free radicals in reperfusion injury following ischemia in the isolated rabbit heart. METHODS: A quantitative assessment of free-radical production during ischemia and reperfusion was made using electron paramagnetic resonance (EPR) spectroscopy. Serial frozen (77 degrees K) tissue biopsies of the left ventricular wall in isolated rabbit hearts were performed during perfusion, ischemia, and reperfusion. These were pulverized into a fine powder that was filled into EPR tubes. EPR spectra of the tissue were recorded at 77 degrees K. RESULTS: Three predominant signals were seen on EPR analysis: signal A, g = 2.028; signal B, g = 2.005, and signal C, g = 1.940. All three signals were present during the perfusion period and decreased in size during the period of ischemia. After reperfusion, all three signals doubled in size. CONCLUSION: This study lends direct support to the theory that free radicals are generated during myocardial reperfusion and may result in reperfusion injury.
Asunto(s)
Ventrículos Cardíacos/metabolismo , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Oxígeno/metabolismo , Animales , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/metabolismo , Técnicas In Vitro , Isquemia Miocárdica/complicaciones , Daño por Reperfusión Miocárdica/etiología , ConejosRESUMEN
The mouse embryo fibroblast cell line, C3H/10T1/2, synthesized and deposited a large amount of fibronectin especially in the pericellular matrix. Confluent cultures of these cells cultured in the presence of 0.3 micrograms/ml of retinyl acetate released cell surface fibronectin and the extracellular matrix fibronectin fibrils were disorganized. The immunoblot analysis demonstrated that the number of the fibronectin receptor was decreased in the prolonged culturing of retinyl acetate-treated cells. Immunoprecipitation of 35S-methionine pulse-chase labeled cell extracts by antifibronectin receptor antibody indicated that about one-half of the pre-beta subunit was processed and converted to the mature form in control cells, and only about one-fourth of the pre-beta subunit was processed in the retinyl acetate-treated confluent cells. 1-deoxymannojirimycin (MNJ), which is an inhibitor of oligosaccharide processing, induced disorganization of the extracellular matrix fibronectin assembly similar to that observed with retinyl acetate. The results of this study suggest that a mechanism of action of retinyl acetate is inhibition of the glycosylation during processing of the fibronectin receptor, a step necessary for fibronectin binding and for assembly of the extracellular matrix.
Asunto(s)
Matriz Extracelular/efectos de los fármacos , Fibronectinas/efectos de los fármacos , Receptores de Fibronectina/efectos de los fármacos , Vitamina A/análogos & derivados , Animales , Línea Celular , Diterpenos , Embrión de Mamíferos , Matriz Extracelular/metabolismo , Fibroblastos , Fibronectinas/metabolismo , Ratones , Ratones Endogámicos C3H , Receptores de Fibronectina/análisis , Ésteres de Retinilo , Vitamina A/farmacologíaRESUMEN
OBJECTIVE: The aim was to determine whether mannitol, previously shown to have several myocardial protective properties, could reduce the myocardial infarct size after coronary occlusion in the baboon. METHODS: Anaesthetised baboons underwent a 2 h transient coronary artery occlusion. Each was randomised into one of two groups receiving either mannitol (n = 6) or no adjunct (n = 8). Mannitol (20%) was given at a rate of 0.4 ml.min-1 x kg-1 starting at 105 min postocclusion until reperfusion was allowed at 2 h, and at a rate of 0.2 ml.min-1 x kg-1 thereafter, until a total of 500 ml had been delivered. Changes in the ST segments were recorded with epicardial wires. The animals were killed at 24 h postocclusion and the hearts excised. Silicone microvascular dye was injected into the previously occluded coronary artery to delineate the perfusion bed. The hearts were fixed in formalin, sliced, and mounted on slides. Using planimetry the ratios of the mean volume of infarct to the mean volume of the perfusion bed (VI/VPB) were calculated and compared. RESULTS: The VI/VPB for the mannitol treated group was 71.7(SEM 14.0)% and for the control group, 65.6(6.9)% (NS). No significant difference was noted in the mean summated ST segment elevations between the two groups. CONCLUSIONS: Mannitol does not reduce myocardial infarct size or ischaemia in the baboon.
Asunto(s)
Manitol/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Infarto del Miocardio/patología , Miocardio/patología , Papio , Insuficiencia del TratamientoRESUMEN
ESR spin-labeling studies designed to yield information regarding the relationship between function and conformation of rat liver NADPH-cytochrome P450 reductase (EC 1.6.4.2) were carried out. The purified enzyme was spin labeled by a nitroxide derivative of p-chloromercuribenzoate. Two conditions for spin labeling were employed: (i) the presence of NADP+, yielding an active site-protected spin-labeled reductase, and (ii) the absence of NADP+, yielding completely spin-labeled reductase. Reductase in which the active site was protected by binding NADP+ and then spin-labeled retains most of its enzymatic activity; on the other hand, completely spin-labeled reductase is devoid of any enzymatic activity. Completely spin-labeled reductase yields a two-component resolved ESR spectrum that reflects two classes of spin-labeled binding sites, a strongly immobilized (S) and a weakly immobilized (W) site. The ratio of W/S provides a valuable parameter for studying the relationship between function and conformation. Structural perturbants, such as urea, KCl, and pH, were employed to determine their effects on the activity of the enzyme and their relationship to changes in the conformational state of the reductase. It was further observed that the enzymatically active spin-labeled derivative generated superoxide radical in the presence of NADPH and cytochrome c, which in turn reduced completely the attached spin-label.
Asunto(s)
Microsomas Hepáticos/enzimología , NADPH-Ferrihemoproteína Reductasa/química , NADPH-Ferrihemoproteína Reductasa/metabolismo , Acetilación , Animales , Sitios de Unión , Ditiotreitol/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Mononucleótido de Flavina/análisis , Flavina-Adenina Dinucleótido/análisis , Concentración de Iones de Hidrógeno , Cinética , Masculino , Microsomas Hepáticos/efectos de los fármacos , NADP/metabolismo , NADPH-Ferrihemoproteína Reductasa/aislamiento & purificación , Oxidación-Reducción , Fenobarbital/farmacología , Cloruro de Potasio/farmacología , Unión Proteica , Conformación Proteica , Ratas , Ratas EndogámicasRESUMEN
Using ESR spin-trapping techniques with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we confirmed the 1:1 stoichiometry for the formation of hydroxyl radicals with Fe2+ in the Fenton reaction under experimental conditions wherein [H2O2] is 90 microM and [Fe2+] is very low, 1 microM or less. The stoichiometry decreased markedly as the Fe2+ concentration was increased. The efficiency of hydroxyl radical generation varied with the nature of the iron chelators used and increased in the order of phosphate alone approximately ADP less than EDTA less than diethylenetriaminepentaacetic acid (DETAPAC). The second order rate constant for the Fenton reaction was measured to be 2.0 x 10(4) M-1 s-1 for phosphate alone, 8.2 x 10(3) M-1 s-1 for ADP, 1.4 x 10(4) M-1 s-1 for EDTA, and 4.1 x 10(2) M-1 s-1 for DETAPAC. Measuring the radicals formed as spins trapped in the presence of ethanol, we estimated the amount of total oxidizing intermediates formed in the Fenton reaction, which we concluded consists of hydroxyl radicals and an iron species. The oxidizing species of iron which might be assigned as ferryl, FeO2+, or Fe(IV) = O was generated effectively in the presence of ADP even at low Fe2+ concentrations. In general, as the Fe2+ concentration was increased, the ferryl species predominated over the hydroxyl radical except for the case of Fe(II)-DETAPAC, which generated only hydroxyl radicals as the oxidizing species. Three possible pathways are proposed for the Fenton reaction, the dominant ones depending very much on the nature of the iron chelator being used.
Asunto(s)
Peróxido de Hidrógeno , Hidróxidos , Hierro , Oxígeno/toxicidad , Adenosina Difosfato , Óxidos N-Cíclicos , Ácido Edético , Espectroscopía de Resonancia por Spin del Electrón/métodos , Etanol , Radicales Libres , Radical Hidroxilo , Cinética , Ácido Pentético , Marcadores de SpinRESUMEN
Electron spin resonance (ESR) studies on spin trapping of superoxide and hydroxyl radicals by 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were performed in NADPH-cytochrome P-450 reductase-paraquat systems at pH 7.4. Spin adduct concentrations were determined by comparing ESR spectra of the adducts with the ESR spectrum of a stable radical solution. Kinetic analysis in the presence of 100 microM desferrioxamine B (deferoxamine) showed that: 1) the oxidation of 1 mol of NADPH produces 2 mol of superoxide ions, all of which can be trapped by DMPO when extrapolated to infinite concentration; 2) the rate constant for the reaction of superoxide with DMPO was 1.2 M-1 s-1; 3) the superoxide spin adduct of DMPO (DMPO-OOH) decays with a half-life of 66 s and the maximum level of DMPO-OOH formed can be calculated by a simple steady state equation; and 4) 2.8% or less of the DMPO-OOH decay occurs through a reaction producing hydroxyl radicals. In the presence of 100 microM EDTA, 5 microM Fe(III) ions nearly completely inhibited the formation of the hydroxyl radical adduct of DMPO (DMPO-OH) as well as the formation of DMPO-OOH and, when 100 microM hydrogen peroxide was present, produced DMPO-OH exclusively. Fe(III)-EDTA is reduced by superoxide and the competition of superoxide and hydrogen peroxide in the reaction with Fe(II)-EDTA seems to be reflected in the amounts of DMPO-OOH and DMPO-OH detected. These effects of EDTA can be explained from known kinetic data including a rate constant of 6 x 10(4) M-1 s-1 for reduction of DMPO-OOH by Fe(II)-EDTA. The effect of diethylenetriamine pentaacetic acid (DETAPAC) on the formation of DMPO-OOH and DMPO-OH was between deferoxamine and EDTA, and about the same as that of endogenous chelator (phosphate).
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Reductasas del Citocromo/metabolismo , Hidróxidos/metabolismo , NADP/metabolismo , Paraquat/metabolismo , Superóxidos/metabolismo , Animales , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Radical Hidroxilo , Quelantes del Hierro , CinéticaRESUMEN
The nonenzymatic oxidation of NADH was studied spectrophotometrically in the presence of two vanadium compounds, sodium orthovanadate and vanadyl sulfate. At physiological pH 7.4, in 25 mM sodium phosphate buffer, addition of the synthetic thiol, dithioerythritol (DTE) results in a marked increase of NADH oxidation in the presence of sodium orthovanadate, but not in the presence of vanadyl sulfate. Other reductants, such as dithiothreitol and cysteine, can also increase NADH oxidation, whereas glutathione and ascorbate cannot. In all reactions, superoxide dismutase and catalase completely inhibit the vanadium-stimulated oxidation of NADH. Inhibition occurs in a concentration-dependent manner, and the boiled enzymes do not inhibit the thiol reaction. The hydroxyl radical scavenger, thiourea, inhibits the reaction, whereas urea cannot. ESR studies show that the ability of the thiol to reduce vanadate can be correlated with the degree of NADH oxidation. Using spin trapping techniques, hydroxyl radicals are detected during the course of the reaction. Addition of hydrogen peroxide to vanadyl in the presence of DTE greatly increases NADH oxidation; however, no NADH oxidation occurs when hydrogen peroxide is added to vanadyl and ascorbic acid. These results provide a partial explanation for the ability of vanadium compounds to both decrease cellular reducing equivalents and promote lipid peroxidation.
Asunto(s)
NAD/análisis , Compuestos de Sulfhidrilo/farmacología , Vanadatos/farmacología , Compuestos de Vanadio , Vanadio/farmacología , Ácido Ascórbico/farmacología , Ditioeritritol/farmacología , Sinergismo Farmacológico , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Peróxido de Hidrógeno/farmacología , Hidroxilación , Cinética , Oxidación-Reducción/efectos de los fármacos , Superóxido Dismutasa/farmacologíaRESUMEN
Vanadium compounds are known to stimulate the oxidation of NAD(P)H, but the mechanism remains unclear. This reaction was studied spectrophotometrically and by electron spin resonance spectroscopy (ESR) using vanadium in the reduced state (+4, vanadyl) and the oxidized state (+5, vanadate). In 25 mM sodium phosphate buffer at pH 7.4, vanadyl was slightly more effective in stimulating NADH oxidation than was vanadate. Addition of a superoxide generating system, xanthine/xanthine oxidase, resulted in a marked increase in NADH oxidation by vanadyl, and to a lesser extent, by vanadate. Decreasing the pH with superoxide present increased NADH oxidation for both vanadate and vanadyl. Addition of hydrogen peroxide to the reaction mixture did not change the NADH oxidation by vanadate, regardless of concentration or pH. With vanadyl however, addition of hydrogen peroxide greatly enhanced NADH oxidation which further increased with lower pH. Use of the spin trap DMPO in reaction mixtures containing vanadyl and hydrogen peroxide or a superoxide generating system resulted in the detection by ESR of hydroxyl. In each case, the hydroxyl radical signal intensity increased with vanadium concentration. Catalase was able to inhibit the formation of the DMPO--OH adduct formed by vanadate plus superoxide. These results show that the ability of vanadium to act in a Fenton-type reaction is an important process in the vanadium-stimulated oxidation of NADH.
Asunto(s)
Hidróxidos/metabolismo , NAD/metabolismo , Vanadio/farmacología , Óxidos N-Cíclicos , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Peróxido de Hidrógeno/farmacología , Concentración de Iones de Hidrógeno , Radical Hidroxilo , Oxidación-Reducción , Espectrofotometría , Marcadores de Spin , Superóxidos/metabolismo , Superóxidos/farmacología , Vanadatos/farmacología , Xantina , Xantina Oxidasa/metabolismo , Xantinas/metabolismoRESUMEN
The present study was designed to determine which form of vanadium is involved in initiating conjugated diene formation in both purified and partially peroxidized fatty acids, and to determine if active oxygen radicals are involved in this process. We report that vanadyl is the active form of vanadium in initiating conjugated diene formation in micelles prepared from purified fatty acids or partially peroxidized fatty acids. Vanadate did not initiate conjugated diene formation in either case. Hydroxyl radicals were shown to be involved in the initiation of diene conjugation when vanadyl and hydrogen peroxide were added together in a reaction mixture. In this case, there was a rapid burst of conjugated diene formation which quickly leveled off. Using spin trapping techniques, hydroxyl radicals were shown to be generated in the vanadyl-catalyzed break-down of fatty acid hydroperoxides. A comparison was made between the ability of vanadyl or vanadyl chelates to decompose hydrogen peroxide and catalyze the decomposition of fatty acid hydroperoxides. It was found that strongly chelated vanadyl (vanadyl/EDTA) was much less effective in decomposing both hydrogen peroxide and fatty acid hydroperoxides than the weak vanadyl chelates (e.g., vanadyl/ADP). This study suggests a mechanism to explain the effects of vanadium on lipid peroxidation.
Asunto(s)
Peróxidos Lipídicos/biosíntesis , Vanadatos , Espectroscopía de Resonancia por Spin del Electrón , Ácidos Grasos/metabolismo , Radicales Libres , Peróxido de HidrógenoAsunto(s)
Catalasa/uso terapéutico , Hemoglobinas/uso terapéutico , Peritonitis/prevención & control , Superóxido Dismutasa/uso terapéutico , Animales , Modelos Animales de Enfermedad , Infecciones por Escherichia coli/sangre , Infecciones por Escherichia coli/fisiopatología , Manitol/uso terapéutico , Papio , Peritonitis/sangre , Ratas , Ratas EndogámicasRESUMEN
A model system has been used to study the types of radicals formed on denitrosation of N-nitroso compounds. Free radicals were formed at room temperature (22 degrees-23 degrees C) and neutral pH by photolytic cleavage of N-nitroso bonds and were partially characterized following their addition to the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenyl-nitrone (PBN). Carbon-centered radical adducts were obtained during nitrosamine photolysis and nitrogen-centered radical adducts during nitrosamide photolysis. Since both the nitrosamines and nitrosamides initially form nitrogen-centered radicals on photolysis, a secondary reaction or rearrangement must occur after initial N-nitroso bond cleavage in the nitrosamines. Mechanisms are proposed to account for these results.
Asunto(s)
Nitrosaminas/efectos de la radiación , Compuestos Nitrosos/efectos de la radiación , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Concentración de Iones de Hidrógeno , Fotólisis , Marcadores de SpinRESUMEN
Two hydrazine spin labels, 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carbonyl ethyl hydrazine and 1-oxyl-2,2,6,6-tetramethylpiperidino-4-hydrazine, were synthesized as probes of the FAD binding site of monoamine oxidase. The reporter nitroxide moiety showed an ESR spectrum classified as partially immobilized which is indicative of FAD near the surface of the enzyme. Attempts to pick up flavin semiquinone or free radical intermediates during substrate oxidation with the spin traps 5,5-dimethyl-1-pyrroline-1-oxidase and phenyl-t-butylnitrone were not successful.
Asunto(s)
Flavina-Adenina Dinucleótido , Hígado/enzimología , Monoaminooxidasa , Animales , Bovinos , Óxidos N-Cíclicos , Espectroscopía de Resonancia por Spin del Electrón , Marcadores de SpinRESUMEN
Rat thymocytes, spleen lymphocytes and isolated nuclei were incubated with fatty acids and then labelled with 5-doxylstearic acid and 12-doxylstearic acid. The ESR spectra only in the case of 5-doxylstearic acid showed changes which were demonstrable only under those conditions which resulted in cytolysis. Thymocytes in medium with 10% serum showed the effect at 10 microM, splenic lymphocytes at 100 microM. The effect was maximal at 2 min and was not enhanced by higher concentrations. The uptake of fatty acid by spleen cells required to cause this change was determined using 14C-oleic acid, to be 0.6 mumol/g tissue. This quantity is less than that required (label:lipid ratio less than 1:10) to produce major perturbations in membranes. Free fatty acids of C-8 to C-18 produced the effect, but not esters or amides. It was concluded that free fatty acids induce changes proximal to the polar region of membrane lipids which, if not progressive and essential to the ultimate process of lysis, are at least indicative of impending cell death at an early time.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Ácidos Grasos/toxicidad , Animales , Femenino , Humanos , Ácido Oléico , Ácidos Oléicos/metabolismo , Ratas , Marcadores de Spin , Bazo/citología , Factores de Tiempo , Esclerosis Tuberosa/genéticaRESUMEN
An imidazole spin-label was used to study the role of tyrosyl residues in the reassociation process for the nucleosome core particle. The nucleosome core particle, containing 145 base pairs of DNA and a histone core (two each of the four histones H2S, H2B, H3, and H4), was isolated from chicken erythrocytes. Native particles were first dissociated in 2 M NaCl and labeled with varying concentrations of imidazole spin-label. The labeled histone core and endogenous DNA were then reassociated back by salt step dialysis. Reconstituted spin-labeled complexes, purified by an isokinetic sucrose gradient, were found to have physical properties identical with those of unlabeled native particles. Spin-labeling the surface tyrosines of the histone core did not interfere with proper reassociation of the nucleosome core complex. ESR spectra of the reconstituted nucleosomes core complex are not the strongly anisotropic type, suggesting that labeled surface tyrosines in the histone core are not involved in specific DNA-histone interaction nor does wrapping of DNA on the histone core involve very close contact with the label. When labeling was carried out under denaturing conditions following exposure of the histone core to urea, additional histone tyrosine residues were spin-labeled. The resulting histone-DNA complexes that formed after reassociation had physical properties different from those of the native nucleosomes core. This result suggested that some of the "buried" tyrosines are essential for specific histone-histone interactions that lead to stable histone core structures. Spin-labeling the buried tyrosines prevented to compact supercoiling of DNA into nucleosome core particle.
Asunto(s)
Nucleosomas/metabolismo , Animales , Sitios de Unión , Pollos , ADN/metabolismo , Histonas/metabolismo , Hidroxilamina , Hidroxilaminas , Técnicas In Vitro , Conformación de Ácido Nucleico , Conformación Proteica , Marcadores de Spin , TirosinaAsunto(s)
Mononucleótido de Flavina/farmacología , Flavina-Adenina Dinucleótido/farmacología , Microsomas Hepáticos/enzimología , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxígeno/metabolismo , Superóxidos/metabolismo , Animales , Espectroscopía de Resonancia por Spin del Electrón , Cinética , Masculino , NADPH-Ferrihemoproteína Reductasa/aislamiento & purificación , Ratas , Ratas EndogámicasRESUMEN
A series of nitroxide spin-labeled reagents have been employed to explore the environment of the cysteine residues in bovine plasma amine oxidase. When the enzyme was reduced by substrate or phenylhydrazine, 1 essential sulfhydryl residue/subunit was liberated. This cysteine residue was reacted then with the spin label 3-(maleimido-methyl)-2,2,5,5-tetramethyl-1-pyrrolinyloxyl. The ESR spectra of the labeled enzyme derivatives suggested that this essential sulfhydryl residue is located in a pocket, whereas the nonessential sulfhydryl residues are probably located near the surface. By varying the length of the nitroxide spin-labeled N-ethylmaleimide derivatives, it was determined that the liberated essential cysteine residues are in a restricted environment. The ESR spectral data also suggested that the nitroxide radical and the essential copper in the enzyme do not interact with one another. The effect of ionic strength, pH, and urea denaturation on the environment of the essential sulfhydryl residue were also investigated.