RESUMEN
Analysis of a mutant strain of Drosophila subobscura revealed that most (80%) mitochondrial genomes have undergone a large scale deletion (5 kb) in the coding region. Compared with the wild-type strain, complex I and III activities are, respectively, reduced by 50% and 30% in the mutant. However, the ATP synthesis capacities remain unchanged. In order to elucidate how the ATP synthesis is maintained at a normal level, despite a significant decrease in complex I and III activities, we progressively inhibited respiratory chain complex activities, respiration rate and ATP synthesis. Complex I, III and IV activities were inhibited by rotenone, antimycin and KCN, respectively. Threshold curves were thus determined for each complex. Our results demonstrated that in the mutant strain, both mitochondrial respiration and ATP synthesis had decreased when complex I activity was inhibited by more than 20%, whereas 70% inhibition is required to induce similar changes in the wild-type. The complex I inhibition pattern of the wild-type was restored by a backcross (mutant female/wild-type male). The complex III activity threshold is below 20% in both strains, and we observed some difference in antimycin sensitivity, suggesting a modification of the complex enzymatic properties in the mutant. In contrast, threshold values of 70% were measured for complex IV inhibition. Our data suggest that the difference in the complex I threshold curves between the wild-type and mutant strains could partially account for the absence of pathological phenotype in the mutant.
Asunto(s)
Antimicina A/análogos & derivados , Drosophila/genética , Mitocondrias/enzimología , Adenosina Trifosfato/biosíntesis , Animales , Antimicina A/farmacología , Citrato (si)-Sintasa/antagonistas & inhibidores , Citrato (si)-Sintasa/metabolismo , Relación Dosis-Respuesta a Droga , Drosophila/metabolismo , Transporte de Electrón/fisiología , Complejo I de Transporte de Electrón/metabolismo , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Glicerofosfatos/metabolismo , Cinética , Masculino , Mitocondrias/efectos de los fármacos , Complejos Multienzimáticos/metabolismo , Mutación , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/metabolismo , Oxidación-Reducción/efectos de los fármacos , Consumo de Oxígeno , Cianuro de Potasio/farmacología , Rotenona/farmacologíaRESUMEN
KC167 Drosophila cells were incubated with low concentrations of ethidium bromide (200 ng/ml), causing changes in mitochondrial DNA (mtDNA) content (2-184% of that of controls). SSCP (single strand conformational polymorphism) analysis of mtDNA indicated that the incubation with ethidium bromide also generated mutations. Compared with controls, there were marked reductions in the activities of respiratory complexes III and IV measured in these cells, and in respiration and ATP synthesis capacities measured in isolated mitochondria. These reductions matched that in mtDNA content. In contrast, no link could be demonstrated between mtDNA content and steady-state concentrations of the transcripts of genes COIII and Cyt b.
Asunto(s)
ADN Mitocondrial/efectos de los fármacos , Drosophila , Complejo III de Transporte de Electrones/metabolismo , Etidio , Adenosina Trifosfato/biosíntesis , Animales , Línea Celular , Complejo III de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Oxidación-Reducción , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero , Especificidad por SustratoRESUMEN
Eighty percent of DNA molecules are deleted in the mitochondrial population of an adult mutant strain of D. subobscura. Both intact and deleted genomes are autonomous monomers. The heteroplasmy level, which is lower in germ tissue, increases from the oocytes (60%) to the third larval instar (83%), and is then maintained throughout the life of the fly. The mtDNA/nuclear DNA ratio is on average two-times greater in the heteroplasmic strain than in the wild-type strain, irrespective of the stage, but the cellular content of mitochondria is elevated only in the embryos and pupae of the mutant strain. The steady state concentrations (SSCs) of the transcripts affected by the deletion are greatly reduced at the larval and adult stages, and less so at the pupal stage of the mutant strain compared with the wild-type. The SSCs of these transcripts are identical in the two strains at the embryonic stage. The fusion transcript, indicating that the deleted genome is expressed, was detected at all stages. The mechanisms involved in the changes in the heteroplasmy level during the course of development and in its maintenance from the third larval instar onwards are discussed.
Asunto(s)
ADN Mitocondrial/genética , ADN/genética , Drosophila/crecimiento & desarrollo , Drosophila/genética , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Transcripción Genética , Animales , Núcleo Celular/metabolismo , Citosol/metabolismo , Drosophila/embriología , Embrión no Mamífero/fisiologíaRESUMEN
In the studied mutant strain of Drosophila subobscura, 78% of the mitochondrial genomes lost >30% of the coding region by deletion. The mutations was genetically stable. Despite this massive loss of mitochondrial genes, the mutant did not seem to be affected. Distribution of the two genome types, cell levels of mitochondrial DNA, steady-state concentrations of the mitochondrial gene transcripts, mitochondrial enzymatic activities, and ATP synthesis capacities were measured in the head, thorax, and abdomen fractions of the mutant strain in comparison with a wild type strain. Results indicate that the deleted genomes are detected in all fractions but to a lesser extent in the male and female abdomen. In all fractions, there is a 50% increase in cellular mitochondrial DNA content. Although there is a decrease in steady-state concentrations of mitochondrial transcripts of genes affected by deletion, this is smaller than expected. The variations in mitochondrial biochemical activities in the different fractions of the wild strain are upheld in the mutant strain. Activity of complex I (involved in mutation) nevertheless shows a decrease in all fractions; activity of complex III (likewise involved) shows little or no change; finally, mitochondrial ATP synthesis capacity is identical to that observed in the wild strain. This latter finding possibly accounts for the lack of phenotype. This mutant is a good model for studying mitochondrial genome alterations and the role of the nuclear genome in these phenomena.
Asunto(s)
ADN Mitocondrial/genética , Drosophila/genética , Eliminación de Gen , Mutación , Abdomen , Adenosina Trifosfato/biosíntesis , Animales , Drosophila/metabolismo , Femenino , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
A mutant strain of D. Subobscura possesses two populations of mitochondrial genomes: a population identical to that of the wild strain (20%) and a dominant population (80%) which has lost more than 30% of its coding zone by deletion. Spectrophotometric determination of respiratory complex activities shows that: complex I (5 genes implicated in deletion) presents maximal activity reduced by 40%, whereas that of complex III (concerned by cytochrome b) is lowered by 30%. Nevertheless, polarographic determinations of substrate oxidation show activity of complex I to be reduced by 30%. In contrast, complex III activity is similar to that measured in the wild strain. The predominant use of one part of the respiratory chain may account for the fact that the mutant strain is apparently unaffected by mutation.
Asunto(s)
ADN Mitocondrial/fisiología , Drosophila/genética , Genes de Insecto , Consumo de Oxígeno/genética , Eliminación de Secuencia , Animales , ADN Mitocondrial/efectos de los fármacos , Complejo III de Transporte de Electrones/genética , Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Glutamatos/metabolismo , Glicerofosfatos/metabolismo , Malatos/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Oxidación-Reducción , Rotenona/farmacología , Succinatos/metabolismoRESUMEN
After osmotic shock with 50 mM Tricine buffer (pH 7.9), isolated mitochondria from D. Melanogaster embryos are treated with a low concentration of Triton X-100 (25 micrograms/mg of protein). The lysed mitochondria are still capable of RNA and protein synthesis. While incorporation of labeled precursor is often higher in lysed than in intact mitochondria, neosynthesized proteins exhibit similar electrophoretic patterns. Studies of labeled precursor incorporation in the presence of various effectors indicate a better accessibility to the translation machinery in lysed mitochondria than in intact mitochondria. Such a system has proven capable of translating an exogenous synthetic mRNA, i.e., poly (U).
Asunto(s)
Mitocondrias/metabolismo , Poli U/genética , Animales , Drosophila melanogaster , Cinética , Poli U/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/biosíntesisRESUMEN
Release of Ca++, Mg++ and K+ by the carboxylic ionophore X-14547 A was studied in the mitochondrial membrane. A comparison was made with A.23187 ( Calcimycin ) and X.537 A (Lasalocid A) under the same experimental conditions. It was shown that in this test system X.14547 A is primarily a K+ carrier comparable with X.537 A.
Asunto(s)
Antibacterianos/farmacología , Indenos/farmacología , Membranas Intracelulares/efectos de los fármacos , Ionóforos/farmacología , Mitocondrias/efectos de los fármacos , Animales , Calcimicina/farmacología , Calcio/metabolismo , Lasalocido/farmacología , Magnesio/metabolismo , Potasio/metabolismoRESUMEN
We have characterized the ribosomal proteins from Spinacia chloroplasts using two-dimensional gel electrophoresis. The 30S and 50S subunits contain 23-25 and 36 ribosomal proteins, respectively. In contrast to prokaryotic ribosomes, chloroplast ribosomes contain at least one (and possibly two) phosphorylated ribosomal proteins. Isolated chloroplasts synthesize in the presence of ((35)S) labeled methionine and cysteine at least seven 30S and thirteen 50S ribosomal proteins which are assembled into (pre)ribosomes. This suggests that about one third of the chloroplast ribosomal proteins is encoded by the chloroplast DNA itself. The identity of several labeled proteins in the two-dimensional gel electrophoretic patterns which did not comigrate with stained chloroplast ribosomal proteins is discussed.
RESUMEN
The alkali cations discrimination on a liquid membrane electrodes system, was determined for the carboxylic ionophores grisorixin, alborixin and two derivatives, dihydrogrisorixin and hexahydroalborixin. The two antibiotics exhibited a great perference for K+. Dihydrogrisorixin again showed the selectivity curve of a carboxylic ionophore, but with a discrimination power lowered compared with grisorixin. Hexahydroalborixin had lost all the complexing properties of the natural molecule. The selectivity scales measured for cations, were directly correlated with the K+ and glutamate effluxes measured in rat liver mitochondria. The chemical modifications of the natural structures of grisorixin and alborixin resulted in a drastic reduction of their ionophoric properties. The loss of K+-glutamate might occur in two steps, the efflux of K+ catalysed by the ionophores then causing a loss of negative charges in the form of glutamate.
Asunto(s)
Antibacterianos/farmacología , Glutamatos/metabolismo , Ionóforos/farmacología , Mitocondrias Hepáticas/metabolismo , Nigericina/farmacología , Potasio/metabolismo , Animales , Fenómenos Químicos , Química , Química Física , Técnicas In Vitro , Iones , Nigericina/análogos & derivados , Piranos/farmacología , RatasAsunto(s)
Calcio/metabolismo , Glutamatos/metabolismo , Mitocondrias Hepáticas/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico Activo/efectos de los fármacos , Calcio/farmacología , Etilmaleimida/farmacología , Glutamatos/farmacología , Cinética , Mitocondrias Hepáticas/efectos de los fármacos , Dilatación Mitocondrial/efectos de los fármacos , Ratas , Valinomicina/farmacologíaAsunto(s)
Glutamatos/metabolismo , Mitocondrias Cardíacas/metabolismo , Piruvatos/farmacología , Ácido Aminooxiacético/farmacología , Animales , Arsénico/farmacología , Transporte Biológico/efectos de los fármacos , Calcio/farmacología , Cobayas , Cinética , Mitocondrias Cardíacas/efectos de los fármacos , Palmitoil Coenzima A/metabolismo , Rotenona/farmacología , Relación Estructura-ActividadAsunto(s)
Antibacterianos/farmacología , Glutamatos/metabolismo , Ionóforos , Mitocondrias Hepáticas/metabolismo , Nigericina/farmacología , Animales , Transporte Biológico , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Etilmaleimida/farmacología , Concentración de Iones de Hidrógeno , Malatos/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Dilatación Mitocondrial/efectos de los fármacos , Modelos Biológicos , Nigericina/análogos & derivados , Fosfatos/metabolismo , Potasio/metabolismo , Ratas , Rotenona/farmacología , Valinomicina/farmacologíaRESUMEN
Our data clearly demonstrate that protective effect of phosphate and protective effect of mersalyl against NEM-inhibition of phosphate transport act at the level of two kinds of proteins. (1)Two major components are phosphate and nigericin NEM sensitive. According to our previous data [13] it has been also demonstrated that these two proteins components are valinomycin NEM sensitive (results not shown here) suggesting a relationship between these proteins and the energy linked proton translocation process. Relationships between these proteins and the phosphate translocation process are not evident and are under further investigations. (2) Two other insoluble major components localised at the level of the subparticular fraction are mersalyl NEM sensitive. We can suggest that these proteins are implicated in the translocation of phosphate in pig heart mitochondria.
Asunto(s)
Mitocondrias Musculares/metabolismo , Proteínas Musculares/metabolismo , Fosfatos/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Etilmaleimida/farmacología , Mersalil/farmacología , Mitocondrias Musculares/análisis , Proteínas Musculares/análisis , Miocardio/ultraestructura , Nigericina/farmacología , Fosfatos/farmacología , Relación Estructura-Actividad , PorcinosRESUMEN
Phosphate transport in mitochondria was investigated with respect to its inhibition by NEM. The reactivity of the Pi carrier SH groups was influenced by phosphate or ionophores during preincubation before the addition of NEM. Furthermore in order to obtain some mitochondrial protein fractions where the typical effects of phosphate and ionophores on [14C]-NEM fixations were observed, mitochondria were submitted to hypotonic treatment and sonication. The following results were obtained: 1. -- Phosphate and grisorixin (a new ionophore of the nigericin group) decreased the inhibition of phosphate transport by NEM. The same effect was observed for [14C]-NEM incorporation. 2. -- Valinomycin increased [14C]-NEM incorporation. The valinomycin effect was abolished by phosphate. ClCCP alone affected [14C]-NEM incorporation slightly. Valinomycin plus ClCCP decreased NEM inhibition of phosphate transport and [14C]-NEM incorporation like grisorixin. 3. -- The variability of SH group reactivity can be interpreted by a control of SH group accessibility by transmembrane delta pH as previously suggested. 4. -- Typical effects of phosphate or ionophores were observed in whole pig heart and rat liver mitochondria. These effects were enhanced in the same supernatant protein fraction resulting from sonication in pig heart mitochondria : phosphate decreased [14C]-NEM incorporation by 1,50 nmoles/mg protein, grisorixin by 0.95 nmoles, whereas valinomycin increased it by 0.75 nmoles. For rat liver mitochondria the phosphate effect and the valinomycin increased it by 0.75 nmoles. For rat liver mitochondria the phosphate effect valinomycin effect on [14C]-NEM incorporation were observed in the subparticular fraction obtained after sonification.