RESUMEN
Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b(5) reductase and cytochrome b(5).
Asunto(s)
Transporte de Electrón/fisiología , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Animales , Fraccionamiento Celular , Centrifugación , Citocromos b5/metabolismo , Hígado/fisiología , Microscopía Electrónica , Microsomas/enzimología , Dilatación Mitocondrial/fisiología , NAD/metabolismo , NADH Deshidrogenasa/metabolismo , Oxidación-Reducción , Ratas , Sonicación , EstereoisomerismoRESUMEN
Microsomal membranes are postulated to contain either a homogeneous arrangement of individual enzymes or groupings of functionally related enzymes. In the present study we attempt to distinguish between these hypotheses in subfractions of rough microsomes from rat liver. After sonication, the individual vesicles that make up the rough-membrane fraction average less than 1/100 of their previous mass. The vesicles in the sonicated suspension are fractionated roughly according to size on a continuous sucrose gradient. Enzyme activity or concentration in fractions of the gradient is expressed on a phospholipid basis. Fractions containing primarily small vesicles differ from those containing larger vesicles in a manner suggesting a certain degree of separation of NADH-linked from NADPH-linked enzymes. NADH-ferricyanide reductase, NADH-cytochrome c reductase and cytochrome b(5) are most concentrated within the large vesicles in the lowest third of the gradient. In contrast, NADPH-cytochrome c reductase and cytochrome P-450 are found in highest concentration in the small vesicles that make up the upper third of the gradient. The results suggest a nonrandom distribution of these two enzyme groups in the membranes of the endoplasmic reticulum.
Asunto(s)
Citocromos , Hígado/enzimología , Microsomas/enzimología , Oxidorreductasas/metabolismo , Animales , Isótopos de Carbono , Centrifugación por Gradiente de Densidad , Glucosa-6-Fosfatasa/metabolismo , Glicerol/metabolismo , Leucina/metabolismo , Masculino , Membranas , Microscopía Electrónica , NAD/metabolismo , NADP/metabolismo , Ácido Orótico/metabolismo , Fosfolípidos/metabolismo , ARN/metabolismo , Ratas , Ribosomas/metabolismo , Tritio , UltrasonidoRESUMEN
We have examined the effects of adding glutathione and isolated cytosolic glutathione S-transferases A, B, and C to rat liver microsomes metabolizing benzo(a)pyrene. Addition of glutathione alone resulted in the conjugation of 15 to 20% of the total metabolites of benzo(a)pyrene, and this conjugation could be inhibited almost entirely by bromosulfophthalein (an inhibitor of glutathione S-transferases), indicating that it is catalyzed by the glutathione S-transferase present in microsomes. Addition of purified cytosolic glutathione S-transferases A, B, and C yielded about 30 to 40% conjugate formation. Analysis of metabolites by high-pressure liquid chromatography demonstrated that the formation of 4,5-diol of benzo(a)pyrene was decreased by at least 80% by conjugation and that the 7,8-diol was also decreased significantly (40 to 60%). In addition, it was found that glutathione S-transferase B is capable of conjugating benzo(a)pyrene 1,6- and 3,6-quinones.
Asunto(s)
Benzopirenos/metabolismo , Carcinógenos/metabolismo , Glutatión Transferasa/metabolismo , Microsomas Hepáticos/metabolismo , Animales , Benzo(a)pireno , Citosol/enzimología , Glutatión Transferasa/aislamiento & purificación , Cinética , Hígado/enzimología , Masculino , Ratas , Ratas EndogámicasRESUMEN
The epoxide hydrase assay developed by Oesch et al. (Biochim. Biophys. Acta, 227: 685-691, 1971) using [3H]styrene oxide as substrate was modified in three ways for use with rat lung microsomes: the substrate was purified before use, the volume of the incubation mixture was scaled down 4-fold, and the incubation time was extended to 45 min (activity was found to be linear for at least 60 min). These modifications increased the sensitivity of the assay procedure 75- to 150-fold. The procedure was found to be linear with lung microsomal protein up to at least 1.8 mg protein per incubation mixture. This modified assay for epoxide hydrase was used to characterize the enzyme in rat lung. Its apparent vmax is 0.5 nmole of styrene glycol formed per min per mg microsomal protein, and its apparent Km was 0.11 to 0.25 mM. The pH optimum is around 9.7. Upon subcellular fractionation of lung tissue, expoxide hydrase distributes in the same manner as a marker for the endoplasmic reticulum (reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase) and in a different way from markers for the nuclei, mitochondria, concentric lamellar organelles, lysosomes, Golgi membranes, plasma membrane and soluble cytoplasm. The specific activity of epoxide hydrase in rough and smooth lung microsomes is aobut the same. Treatment i.p. of rats with methylcholanthrene (3 injections of 20 mg/kg), phenobarbital (5 daily injections of 80 mg/kg) or styrene oxide (5 daily injections of 40 mg/kg), did not induce lung microsomal epoxide hydrase activity. 1,1,1-Trichloropropene 2,3-oxide was shown to be an uncompetitive inhibitor, and cyclohexene oxide was a noncompetitive inhibitor of this enzyme. Ethanol and butanol activate the epoxide hydrase of lung microsomes at low concentrations and inhibit it at higher concentrations.
Asunto(s)
Epóxido Hidrolasas/metabolismo , Hidroliasas/metabolismo , Pulmón/enzimología , Alcoholes/farmacología , Animales , Benzopireno Hidroxilasa/metabolismo , Ciclohexanos/farmacología , Ciclohexenos , Retículo Endoplásmico/enzimología , Inducción Enzimática/efectos de los fármacos , Epóxido Hidrolasas/análisis , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/biosíntesis , Éteres Cíclicos/farmacología , Hidrocarburos Clorados/farmacología , Técnicas In Vitro , Cinética , Hígado/enzimología , Masculino , Métodos , Metilcolantreno/farmacología , Microsomas/enzimología , NADPH-Ferrihemoproteína Reductasa/metabolismo , Fenobarbital/farmacología , Compuestos Policíclicos/metabolismo , Ratas , Estirenos/metabolismoRESUMEN
This presentation is a brief review of current knowledge concerning some biochemical, physiological and medical aspects of the function of ubiquinone (coenzyme Q) in mammalian organisms. In addition to its well-established function as a component of the mitochondrial respiratory chain, ubiquinone has in recent years acquired increasing attention with regard to its function in the reduced form (ubiquinol) as an antioxidant. Ubiquinone, partly in the reduced form, occurs in all cellular membranes as well as in blood serum and in serum lipoproteins. Ubiquinol efficiently protects membrane phospholipids and serum low-density lipoprotein from lipid peroxidation, and, as recent data indicate, also mitochondrial membrane proteins and DNA from free-radical induced oxidative damage. These effects of ubiquinol are independent of those of exogenous antioxidants, such as vitamin E, although ubiquinol can also potentiate the effect of vitamin E by regenerating it from its oxidized form. Tissue ubiquinone levels are regulated through the mevalonate pathway, increasing upon various forms of oxidative stress, and decreasing during aging. Drugs inhibiting cholesterol biosynthesis via the mevalonate pathway may inhibit or stimulate ubiquinone biosynthesis, depending on their site of action. Administration of ubiquinone as a dietary supplement seems to lead primarily to increased serum levels, which may account for most of the reported beneficial effects of ubiquinone intake in various instances of experimental and clinical medicine.
Asunto(s)
Enfermedad , Ubiquinona/fisiología , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Daño del ADN , Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Humanos , Peroxidación de Lípido/efectos de los fármacos , Lipoproteínas LDL/sangre , Mamíferos , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Especificidad de Órganos , Oxidantes/metabolismo , Oxidantes/farmacología , Oxidación-Reducción , Fosfolípidos/metabolismo , Valores de Referencia , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Ubiquinona/farmacologíaRESUMEN
(1) Certain metal chelates of 4,7-diphenyl-1,10-phenanthroline (bathophenanthroline, BPh) are potent inhibitors of soluble mitochondrial F1-ATPase. (2) The BPh-metal chelate inhibition of soluble mitochondrial F1-ATPase is relieved by uncouplers of oxidative phosphorylation. (3) The uncouplers appear to interact directly with the inhibitory chelates, forming stoichiometric adducts. (4) A complex between F1 and bPh3Fe2+, containing 3 mol BPh3Fe2+/mol F1, has been isolated. The enzymically inactive F1-BPh3Fe2+ complex binds uncouplers, yielding an enzymically active F1-BPh3Fe2+-uncoupler complex.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Quelantes/farmacología , Hierro/farmacología , Magnesio/farmacología , Mitocondrias Cardíacas/metabolismo , Mitocondrias/metabolismo , Factores de Acoplamiento de la Fosforilación Oxidativa/farmacología , Fenantrolinas/farmacología , Partículas Submitocóndricas/metabolismo , Desacopladores/farmacología , Animales , Bovinos , Cinética , Mitocondrias Cardíacas/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , ATPasas de Translocación de Protón , Espectrofotometría , Relación Estructura-Actividad , Partículas Submitocóndricas/efectos de los fármacosRESUMEN
(1) Trisbathophenanthroline-Fe2+ (BPh3Fe2+)alters the hyperbolic relationship between concentration of ATP and reaction velocity of F1-ATPase to sigmoidal, with a simultaneous decrease in maximal velocity. (2) BPh3Fe2+ binds to the beta-subunit of F1 and competes with the binding of aurovertin. The reversal of this effect uncouplers in enhanced by ADP and diminished by ATP. BPh3Fe2+ also changes the hyperbolic concentration dependence of aurovertin binding to sigmoidal. (3) BPh3Fe2+ stabilizes F1 against the cold inactivation and cold dissociation in an uncoupler-reversible manner. (4) BPh3Fe2+ efficiently protects F1 against the light-induced inactivation occurring in the presence of Rose Bengal, and the effect is reversed by uncouplers. (5) The results are discussed in relation to the reaction mechanism of F1-ATPase and other enzymes catalyzing the reversible hydrolysis of pyrophosphate bonds.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Mitocondrias Cardíacas/enzimología , Factores de Acoplamiento de la Fosforilación Oxidativa/metabolismo , Fenantrolinas/farmacología , Desacopladores/farmacología , Animales , Aurovertinas/farmacología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Bovinos , Hierro/farmacología , Cinética , ATPasas de Translocación de Protón , Rosa Bengala/farmacologíaRESUMEN
H2O2 production and accumulation during incubation of isolated rat-brain mitochondria with substrates of monoamine oxidase A and B were investigated. All substrates gave rise to an accumulation of H2O2 which was inhibited by malate + pyruvate or isocitrate, consistent with a need for mitochondrial NADPH to maintain glutathione in the reduced state. However, in the absence of these additions the level of reduced glutathione decreased only by about 30%, indicating that only a fraction of the mitochondrial glutathione pool was accessible to the glutathione peroxidase and glutathione reductase activities responsible for the continuous removal of H2O2 generated by monoamine oxidase. The H2O2 accumulation was also inhibited by externally added reduced glutathione or NADPH but not NADH. External NADPH was oxidized by added oxidized glutathione but not alpha-ketoglutarate + NH4+. These results suggest that the removal of H2O2 generated by monoamine oxidase proceeds by way of special fractions of glutathione peroxidase and glutathione reductase that are located in the intermembrane space of mitochondria in such a way that they can react with both intra- and extra-mitochondrial glutathione and NADPH, possibly at the contact sites between the inner and outer mitochondrial membranes. Evidence is also presented that H2O2 generated by monoamine oxidase enhances Ca2+ release from mitochondria and may thus function as a regulator of mitochondrial Ca2+ efflux.
Asunto(s)
Encéfalo/metabolismo , Calcio/metabolismo , Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Monoaminooxidasa/metabolismo , Animales , Encéfalo/enzimología , Glutatión/farmacología , Cinética , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , NADP/metabolismo , RatasRESUMEN
This paper is a study of factors influencing the rate of lipid peroxidation in beef heart submitochondrial particles induced by NAD(P)H via the NADH-ubiquinone oxidoreductase (Complex I) of the respiratory chain. In accordance with earlier observations, both NADH and NADPH initiated lipid peroxidation in the presence of ADP-Fe3+. The rate of the reaction, measured as oxygen consumption and formation of thiobarbituric acid reactive substances, was biphasic as a function of NADH concentration, reaching a maximum at low NADH concentrations and then declining. In contrast, the NADPH-initiated lipid peroxidation showed a monophasic concentration profile of hyperbolic character. Rotenone did not eliminate the biphasicity of the NADH-induced reaction, indicating that this was not due to an antioxidant effect of reduced ubiquinone at high NADH concentrations. This conclusion was further supported by the demonstration that extraction of ubiquinone from the particles did not relieve the inhibition of lipid peroxidation by high NADH concentrations. However rhein, another inhibitor of Complex I, eliminated the biphasicity, and even caused a substantial stimulation of the NADH-induced lipid peroxidation in the particles upon extraction of ubiquinone by pentane. No similar effect occurred in the case of NADPH-induced lipid peroxidation. Furthermore, rhein facilitated both NADH- and NADPH-induced lipid peroxidation even in the absence of added ADP-Fe3+, in a fashion similar to that earlier reported with succinate in the presence of theonyltrifluoroacetone. Based on these findings and measurements of the redox states of ubiquinone and cytochromes in the presence of KCN and NADH or NADPH, it is concluded that Complex I may distinguish between electron input from NADH and NADPH by differences in the site(s) of substrate binding and in the pathways and rates of NADH and NADPH oxidation.
Asunto(s)
Peroxidación de Lípido , Mitocondrias Cardíacas/metabolismo , NADP/metabolismo , NAD/metabolismo , Animales , Antraquinonas/farmacología , Antioxidantes/metabolismo , Bovinos , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Complejos Multienzimáticos/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/antagonistas & inhibidores , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas/metabolismo , Oxidantes/metabolismo , Oxidación-Reducción , Consumo de Oxígeno/efectos de los fármacos , Rotenona/farmacología , Partículas Submitocóndricas/efectos de los fármacos , Partículas Submitocóndricas/metabolismo , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Ubiquinona/metabolismo , Desacopladores/farmacologíaRESUMEN
The antibiotic funiculosin mimics the action of antimycin in several ways. It inhibits the oxidation of NADH and succinate, but not TMPD+ascorbate. The titer for maximal inhibition in Mg2+-ATP particles (0.4-0.6 nmol/mg protein) is close to the concentrations of cytochromes b and cc1. Funiculosin also induces the oxidation of cytochromes cc1 and an extra reduction of cytochrome b in the aerobic steady state, and it inhibits duroquinol-cytochrome c reductase activity in isolated Complex III. The location of the funiculosin binding site is clearly similar to that of antimycin. In addition, funiculosin, like antimycin, prevents electron transport from duroquinol to cytochrome b in isolated Complex III if the complex is pre-reduced with ascorbate. Funiculosin and antimycin differ, however, in the manner in which they modulate the reduction of cytochrome b by ascorbate+TMPD.
Asunto(s)
Antraquinonas/farmacología , Antifúngicos/farmacología , Mitocondrias Musculares/metabolismo , Aerobiosis , Animales , Bovinos , Citocromos/metabolismo , Citocromos c1/metabolismo , Ditionita/farmacología , Cinética , Mitocondrias Musculares/efectos de los fármacos , Miocardio , NADH NADPH Oxidorreductasas/metabolismo , Oxidación-Reducción , Espectrofotometría , Succinato Deshidrogenasa/metabolismoRESUMEN
1. Oxidation of NADPH by various acceptors catalyzed by submitochondrial particles and a partially purified NADH dehydrogenase from beef heart was investigated. Submitochondrial particles devoid of nicotinamide nucleotide transhydrogenase activity catalyze an oxidation of NADPH by oxygen. The partially purified NADH dehydrogenase prepared from these particles catalyzes an oxidation of NADPH by acetylpyridine-NAD. In both cases the rates of oxidation are about two orders of magnitude lower than those obtained with NADH as electron donor. 2. The kinetic characteristics of the NADPH oxidase reaction and reduction of acetylpyridine-NAD by NADPH are similar with regard to pH dependences and affinities for NADPH, indicating that both reactions involve the same binding site for NADPH. The binding of NADPH to this site appears to be rate limiting for the overall reactions. 3. At redox equilibrium NADPH and NADH reduce FMN and iron-sulphur center 1 of NADH dehydrogenase to the same extents. The rate of reduction of FMN by NADPH is at least two orders of magnitude lower than with NADH. 4. It is concluded that NADPH is a substrate of NADH dehydrogenase and that the nicotinamide nucleotide is oxidized by submitochondrial particles via the NADH--binding site of the enzyme.
Asunto(s)
Mitocondrias Cardíacas/metabolismo , NADP/metabolismo , Animales , Bovinos , Concentración de Iones de Hidrógeno , Cinética , NAD/análogos & derivados , NAD/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Organoides/metabolismoRESUMEN
Three aspects of the induction of drug-metabolizing enzymes brought about by trans-stilbene oxide have been investigated. (1) The liver hypertrophy in rats treated with trans-stilbene oxide was found to result solely from an increase in the number of cells in this organ, without any increase in the size of each individual cell. (2) Administration of trans-stilbene oxide also produces a 27% increase in the phospholipid content of the hepatic endoplasmic reticulum, i.e., a limited proliferation of this organelle occurs. (3) Furthermore, induction causes changes in the lipid composition of the endoplasmic reticulum. The cholesterol content is decreased, the relative content of sphingo-myelin is also lowered, and a number of changes in the fatty-acid composition occur as well. All of these effects would tend to increase the fluidity of the phospholipid bilayer of the endoplasmic-reticulum membrane and may thus affect drug metabolism.
Asunto(s)
Retículo Endoplásmico/metabolismo , Inducción Enzimática/efectos de los fármacos , Hígado/efectos de los fármacos , Fosfolípidos/metabolismo , Estilbenos/farmacología , Animales , División Celular/efectos de los fármacos , Colesterol/metabolismo , Ácidos Grasos Insaturados/metabolismo , Hígado/citología , Hígado/enzimología , Masculino , Microsomas Hepáticos/metabolismo , RatasRESUMEN
The hydrolytic and phosphorylation activities of the ATPase complex of bovine heart mitochondria are regulated by the ATPase inhibitor of Pullman and Monroy [1]. The inhibiting action of the peptide on ATPase activity can be overcome by a proton-motive force. Submitochondrial particles that contain the inhibitor, either intrinsically or externally added, show a lag that precedes phosphorylation. Particles devoid of the inhibitor, of particles that are in an 'active' state fail to present the lag. Accordingly, the data indicate that, prior to the onset of phosphorylation, the ATPase complex undergoes a transition to an active state through a process that involves the inhibitor. The transition depends on the concentration of ATP, 50 microM ATP giving 50% inhibition of the proton-motive force-induced transition.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Mitocondrias Cardíacas/enzimología , Proteínas Musculares/fisiología , Animales , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Bovinos , Cinética , Malonatos/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Nigericina/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Fosforilación , Partículas Submitocóndricas/enzimología , Valinomicina/farmacologíaRESUMEN
The ATPase complex of submitochondrial particles exhibits activity transitions that are controlled by the natural ATPase inhibitor (Gómez-Puyou, A., Tuena de Gómez-Puyou, M. and Ernster, L. (1979) Biochim. Biophys. Acta 547, 252-257). The ATPase of intact heart mitochondria also shows reversible activity transitions; the activation reaction is induced by the establishment of electrochemical gradients, whilst the inactivation reaction is driven by collapse of the gradient. In addition it has been observed that the influx of Ca2+ into the mitochondria induces a rapid inactivation of the ATPase; this could be due to the transient collapse of the membrane potential in addition to a favorable effect of Ca2+-ATP on the association of the ATPase inhibitor peptide to F1-ATPase. This action of Ca2+ may explain why mitochondria utilize respiratory energy for the transport of Ca2+ in preference to phosphorylation. It is concluded that the mitochondrial ATPase inhibitor protein may exert a fundamental regulatory function in the utilization of electrochemical gradients.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Calcio/farmacología , Mitocondrias Cardíacas/enzimología , Animales , Antimicina A/farmacología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Metabolismo Energético/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Glutamatos/farmacología , ATPasas de Translocación de Protón , Ratas , Rotenona/farmacología , Succinatos/farmacologíaRESUMEN
Optimal conditions for the preparation of relatively pure microsomes and microsomal subfractions from rat lung have been determined. The most importnat of these conditions is homogenization of a 20% (w/v) suspension of lung tissue in 0.44 M sucrose/1% (w/v) bovine serum albumin with four up-and-down strokes at 440 rev./min in a Potter-Elvehjem homogenizer. The 10000 X g supernatant prepared from this homogenate can be centrifuged at 105000 X g to obtain total microsomes or subfractionated into rough and smooth microsomes on a Cs+-containing discontinuous sucrose gradient. The total, rough and smooth microsomes have been characterized in terms of their chemical composition, enzymatic activity, and morphology. These preparations should prove useful in studies of various enzymes in lung (e.g. benzpyrene monooxygenase, epoxide hydrase, enzymes of phospholipid and ascorbic acid synthesis) and in subfractionations designed to reveal heterogeneites in the lateral plane of the lung endoplasmic reticulum.
Asunto(s)
Fraccionamiento Celular/métodos , Pulmón/ultraestructura , Metilcolantreno/farmacología , Microsomas , Animales , Benzopireno Hidroxilasa/metabolismo , Cesio/farmacología , Masculino , Microscopía Electrónica , Microsomas/análisis , Microsomas/enzimología , Microsomas/ultraestructura , NADPH-Ferrihemoproteína Reductasa/metabolismo , Fosfolípidos/análisis , Proteínas/análisis , ARN/análisis , Ratas , Fracciones Subcelulares/enzimologíaRESUMEN
Bovine heart submitochondrial particles depleted of F1 by treatment with urea ("F1-depleted particles') were incubated with soluble F1-ATPase. The binding of F1 to the particles and the concomitant conferral of oligomycin sensitivity on the ATPase activity required the presence of cations in the incubation medium. NH4+, K+, Rb+, Na+ and Li+ promoted reconstitution maximally at 40-74 mM, guanidinium+ and Tris+ at 20-30 mM, and Ca2+ and Mg2+ at 3-5 mM. The particles exhibited a negative zeta-potential, as determined by microelectrophoresis, and this was neutralized by mono- and divalent cations in the same concentration range as that needed to promote F1 binding and reconstitution of oligomycin-sensitive ATPase. It is concluded that the cations act by neutralizing negative charges on the membrane surface, mainly negatively charged phospholipids. These results are discussed in relation to earlier findings reported in the literature with F1-depleted thylakoid membranes and with submitochondrial particles depleted of both F1 and the coupling proteins F6 and oligomycin sensitivity-conferring protein.
Asunto(s)
Proteínas Portadoras , Mitocondrias Cardíacas/enzimología , Oligomicinas/farmacología , ATPasas de Translocación de Protón/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Cationes Bivalentes/farmacología , Bovinos , Proteínas de la Membrana/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales , Partículas Submitocóndricas/enzimología , Urea/farmacologíaRESUMEN
trans-Stilbene oxide has been found to be a new type of inducer of drug-metabolizing systems. In order to identify the true inducer and to determine the structural requirements for induction, rats were treated with metabolites and structural analogues of stilbene. Subsequently, hepatic levels of cytochrome P-450, microsomal epoxide hydrolase, and cytoplasmic glutathione S-transferase were assayed. All three enzymes were induced by cis- and trans-stilbene and cis- and trans-stilbene oxide. In addition, epoxide hydrolase and glutathione S-transferase activities were induced by benzoin and benzil. In contrast, the diols and benzoic acid had little, if any, effect. The main conclusions drawn from these findings are that: (1) trans-stilbene oxide itself seems to be the inducer of drug-metabolizing enzymes; and (2) benzil is more selective as an inducer of epoxide hydrolase than is trans-stilbene oxide. Attempts to induce epoxide hydrolase with other structural analogues of stilbene led to the following conclusions: (1) two phenyl rings are required for induction; (2) the induction is not as great if the rings are substituted or one of the ring carbon atoms is replaced by a nitrogen; (3) a carbon bridge between the phenyl groups generally results in a greater induction, especially if the bridge contains an epoxy group or one or two keto groups.
Asunto(s)
Inducción Enzimática/efectos de los fármacos , Estilbenos/farmacología , Animales , Fenómenos Químicos , Química , Sistema Enzimático del Citocromo P-450/biosíntesis , Epóxido Hidrolasas/biosíntesis , Glucosafosfato Deshidrogenasa/biosíntesis , Glutatión Transferasa/biosíntesis , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Preparaciones Farmacéuticas/metabolismo , Ratas , Estilbenos/metabolismo , Relación Estructura-Actividad , Uridina Difosfato Glucosa Deshidrogenasa/biosíntesisRESUMEN
The interaction of soluble mitochondrial ATPase from beef heart with the natural ATPase inhibitor was studied. It was found that the phosphorylation of small amounts of ADP by phosphoenolpyruvate and pyruvate kinase, and an ensuing catalytic cycle supports the binding of the inhibitor to the enzyme. The association of the inhibitor with F1-ATPase does not increase the content of ATP in the F1-ATPase-inhibitor complex. The inhibitor of catalytic activity bathophenanthroline-Fe2+ chelate prevents the interaction, while the association of the inhibitor with F1-ATPase is delayed if the reaction is carried out in 2H2O. The date indicate that a transient state involved in the catalytic cycle is the form of the enzyme that interacts with the inhibitor. The proton-motive force-induced dissociation of the inhibitor from particulate ATPase is prevented by bathophenanthroline-Fe2+ chelate and nitrobenzofurazan chloride, which indicates that a functional catalytic (beta) subunit is required for the proton-motive force-induced release of the inhibitor. The data suggest a direct involvement of catalytic (beta) subunit in the mechanism by which the F1-ATPase senses the proton-motive force.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Mitocondrias Cardíacas/enzimología , 4-Cloro-7-nitrobenzofurazano/farmacología , Adenosina Difosfato/metabolismo , Animales , Bovinos , Metabolismo Energético , Quelantes del Hierro/farmacología , NAD/metabolismo , Oxidación-Reducción , Fenantrolinas/farmacología , Fosfoenolpiruvato/metabolismo , Conformación Proteica , ATPasas de Translocación de Protón , Piruvato Quinasa/metabolismoRESUMEN
Respiratory activity of isolated rat brain mitochondria was measured following in vitro exposure to oxygen radicals. The radicals were generated by hypoxanthine and xanthine oxidase in the presence of a suitable iron chelate and caused a severe inhibition of respiration stimulated by phosphate plus ADP (with malate + glutamate as substrate). The damage could be prevented by catalase or high concentrations of mannitol, but not by superoxide dismutase. A similar effect was observed when hypoxanthine and xanthine oxidase were replaced by glucose and glucose oxidase or by hydrogen peroxide. Most of the findings indicate that the hydroxyl radical is the damaging agent. It is concluded that brain mitochondria exposed to oxygen radicals in vitro show an inhibition of respiratory activity similar to that reported by other investigators as occurring in mitochondria in vivo following transient cerebral ischemia. Therefore, oxygen radicals may contribute to this type of cell damage.
Asunto(s)
Encéfalo/ultraestructura , Mitocondrias/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Oxígeno/farmacología , Animales , Catalasa/farmacología , Radicales Libres , Técnicas In Vitro , Masculino , Métodos , Mitocondrias/patología , Ratas , Ratas EndogámicasRESUMEN
Respiratory activity and the ADP/O ratio of isolated rat brain mitochondria were measured following incubation with varying concentrations of lactic acid in reaction media buffered either with bicarbonate and CO2 or with phosphate alone, at a pH of 7.1. Increasing lactic acid levels caused a progressive decrease in substrate-, phosphate-, and ADP-stimulated (State 3) respiration and ADP/O ratios. Fifteen millimolar lactic acid, pH 6.4, caused approximately 50% inhibition of State 3 respiration (with malate + glutamate as substrate). At lower pH values (5.3-6.1), addition of ADP caused little or no increase in O2 consumption; i.e., ATP formation ceased. Addition of lactic acid at constant pH moderately affected respiratory control ratios but did not change State 3 respiration or ADP/O ratios. Thus, the effect of lactic acid was related to the pH change. Increasing CO2 concentrations in the reaction medium had similar effects on mitochondrial respiration, indicating that changes in extramitochondrial pH rather than in transmembrane H+ gradients determined the respiratory alterations. Following a 5-min incubation of mitochondria with lactic acid, pH 6.1, there was an incomplete recovery of State 3 respiration and respiratory control ratios. It is concluded that mitochondrial respiration is inhibited by a decrease in pH which, if excessive, may lead to a permanent suppression of ATP production. These results may, at least partly, explain the deleterious effects of enhanced lactic acidosis in brain ischemia.