RESUMEN
This report, the first in a series on diet-dependent changes in the serum metabolome (metabolic serotype), describes validation of the use of high performance liquid chromatography (HPLC) separations coupled with Coulometric array detectors to characterize changes in the metabolome. The long-term aim of these studies is to improve understanding of the effects of significant variation in nutritive status on physiology and on disease processes. Initial studies focus on identifying the effects of dietary (or caloric) restriction on the redox-active components of rat serum. Identification of compounds of interest is being carried out using HPLC separations coupled with coulometric array analysis, an approach allowing simultaneous examination of nearly 1200 serum compounds. The technical and practical issues discussed in this report are related to both analytical validity (HPLC running conditions, computer-automated peak identification, mathematical compensation for chromatographic drift, etc.) and biological variability (individual variability, cohort-cohort variability, outliers). Attention to these issues suggests approximately 250 compounds in serum are sufficiently reliable, both analytically and biologically, for potential use in building mathematical models of serotype.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Dieta , Privación de Alimentos/fisiología , Estado Nutricional/fisiología , Animales , Estudios de Cohortes , Interpretación Estadística de Datos , Femenino , Masculino , Modelos Teóricos , Oxidación-Reducción , Ratas , Ratas Endogámicas F344 , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , SerotipificaciónRESUMEN
Induction of the mitochondrial permeability transition (PT) has been proposed to contribute to neuronal cell death. Nearly all studies of the biochemistry of PT induction, however, have been conducted in isolated liver mitochondria. To better understand PT induction in brain mitochondria, we used Ficoll gradients to purify nonsynaptosomal mitochondria from the forebrains of male Fischer 344 rats. Incubation of these mitochondria with Ca(2+) was associated with a loss of absorbance. Inorganic phosphate enhanced this loss of absorbance, and the PT inhibitor cyclosporin A reduced it, especially in conjunction with ADP. These findings suggest that Ca(2+)-mediated loss of absorbance resulted from PT induction. Na(+), which enhances mitochondrial Ca(2+) efflux, but stimulates mitochondrial free radical production, had no effect on PT induction. These data confirm the existence of tissue-specific differences in the nature of PT induction.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Membranas Intracelulares/metabolismo , Mitocondrias/metabolismo , Prosencéfalo/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Animales , Calcio/metabolismo , Calcio/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ciclosporina/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Membranas Intracelulares/efectos de los fármacos , Malatos/metabolismo , Malatos/farmacología , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Fosfatos/metabolismo , Fosfatos/farmacología , Povidona/farmacología , Ratas , Ratas Endogámicas F344 , Dióxido de Silicio/farmacología , Ácido Succínico/metabolismo , Ácido Succínico/farmacologíaRESUMEN
Recent studies have demonstrated the need for complementing cellular genomic information with specific information on expressed proteins, or proteomics, since the correlation between the two is poor. Typically, proteomic information is gathered by analyzing samples on two-dimensional gels with the subsequent identification of specific proteins of interest by using trypsin digestion and mass spectrometry in a process termed peptide mass fingerprinting. These procedures have, as a rule, been labor-intensive and manual, and therefore of low throughput. The development of automated proteomic technology for processing large numbers of samples simultaneously has made the concept of profiling entire proteomes feasible at last. In this study, we report the initiation of the (eventual) complete profile of the rat mitochondrial proteome by using high-throughput automated equipment in combination with a novel fractionation technique using minispin affinity columns. Using these technologies, approximately one hundred proteins could be identified in several days. In addition, separate profiles of calcium binding proteins, glycoproteins, and hydrophobic or membrane proteins could be generated. Because mitochondrial dysfunction has been implicated in numerous diseases, such as cancer, Alzheimer's disease and diabetes, it is probable that the identification of the majority of mitochondrial proteins will be a beneficial tool for developing drug and diagnostic targets for associated diseases.
Asunto(s)
Proteínas/aislamiento & purificación , Proteoma , Animales , Automatización , Electroforesis en Gel Bidimensional , Masculino , Mitocondrias Hepáticas/metabolismo , Ratas , Ratas Endogámicas LewRESUMEN
Intracellular free Zn(2+) is elevated in a variety of pathological conditions, including ischemia-reperfusion injury and Alzheimer's disease. Impairment of mitochondrial respiration is also associated with these pathological conditions. To test whether elevated Zn(2+) and impaired respiration might be linked, respiration of isolated rat liver mitochondria was measured after addition of Zn(2+). Zn(2+) inhibition (K(i)(app) = approximately 1 micrometer) was observed for respiration stimulated by alpha-ketoglutarate at concentrations well within the range of intracellular Zn(2+) reported for cultured hepatocytes. The bc(1) complex is inhibited by Zn(2+) (Link, T. A., and von Jagow, G. (1995) J. Biol. Chem. 270, 25001-25006). However, respiration stimulated by succinate (K(i)(app) = approximately 6 micrometer) was less sensitive to Zn(2+), indicating the existence of a mitochondrial target for Zn(2+) upstream from bc(1) complex. Purified pig heart alpha-ketoglutarate dehydrogenase complex was strongly inhibited by Zn(2+) (K(i)(app) = 0.37 +/- 0.05 micrometer). Glutamate dehydrogenase was more resistant (K(i)(app) = 6 micrometer), malate dehydrogenase was unaffected, and succinate dehydrogenase was stimulated by Zn(2+). Zn(2+) inhibition of alpha-ketoglutarate dehydrogenase complex required enzyme cycling and was reversed by EDTA. Reversibility was inversely related to the duration of exposure and the concentration of Zn(2+). Physiological free Zn(2+) may modulate hepatic mitochondrial respiration by reversible inhibition of the alpha-ketoglutarate dehydrogenase complex. In contrast, extreme or chronic elevation of intracellular Zn(2+) could contribute to persistent reductions in mitochondrial respiration that have been observed in Zn(2+)-rich diseased tissues.
Asunto(s)
Complejo Cetoglutarato Deshidrogenasa/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mitocondrias Hepáticas/metabolismo , Zinc/metabolismo , Animales , Transporte de Electrón/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Ácidos Cetoglutáricos/farmacología , Ratas , Zinc/farmacologíaRESUMEN
The aeration-dependent changes in content of various quinones in Escherichia coli were found to be unaffected by a prokaryotic translation inhibitor chloramphenicol. In addition, this process was shown to be completely intact in cells with mutated fnr, arc and appY loci. It is assumed that E. coli possesses a special system of oxygen-dependent post-transcriptional regulation of the quinone biosynthetic pathways.
Asunto(s)
Proteínas del Complejo de Cadena de Transporte de Electrón , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Quinonas/metabolismo , Vitamina K 2/análogos & derivados , Aerobiosis , Anaerobiosis , Cloranfenicol/farmacología , Grupo Citocromo b , Citocromos/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Genotipo , Oxidorreductasas/metabolismo , Quinonas/aislamiento & purificación , Especificidad de la Especie , Ubiquinona/metabolismo , Vitamina K/análogos & derivados , Vitamina K/metabolismoRESUMEN
Regulation of synthesis of cytochrome d in Escherichia coli has been studied using mutants with cytochrome-d--beta-galactosidase gene fusions. It was shown that various protonophorous uncouplers, when added to the growth medium, cause induction of the cytochrome d synthesis. The cytochrome-d-inducing activity of uncouplers correlates with their ability to inhibit such a delta mu (H+)-driven function as motility of the E. coli cells. An increase in the Na+ concentration in the growth medium from 1.5 mM to 25 mM results in induction of the cytochrome d synthesis. The cytochrome-d-inducing effect of uncouplers is much more pronounced when the Na+ concentration is high than when it is low. These data are in agreement with the assumption that cytochrome d is involved in the Na+ energetics substituting for the H+ energetics when the latter appears to be inefficient. Mutations in arcA or arcB genes (but not in fnr gene) completely prevent the increase in the cytochrome d level induced by uncouplers but are without effect on that induced by Na+. It is assumed that in the control of the cytochrome d synthesis, the Arc system is involved in the delta mu H+ sensing whereas sensing of delta mu Na+ (or of the Na+ concentration) is mediated by some other receptor system.
Asunto(s)
Grupo Citocromo d/biosíntesis , Escherichia coli/enzimología , beta-Galactosidasa/biosíntesis , Grupo Citocromo d/genética , Inducción Enzimática , Hidrógeno/farmacología , Iones , Mutación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Sodio/farmacología , beta-Galactosidasa/genéticaRESUMEN
The motility of Escherichia coli and of alkalo- and halotolerant Bacillus FTU has been studied. It is found that Bac. FTU motility (i) requires Na+, (ii) is resistant to the protonophorous uncoupler pentachlorophenol (PCP) if cells grow at high pH, and is sensitive to the uncouplers at neutral pH, (iii) is sensitized to the uncouplers with the addition of monensin, (iv) sensitive to amiloride and (v) can be supported by an artificially imposed Na+ gradient in the presence of uncoupler, cyanide and arsenate. On the other hand, E. coli motility (a) does not require Na+, (b) is always uncoupler-sensitive, (c) is amiloride-resistant, and (d) can be supported by an artificially-imposed gradient of H+, not Na+. It is concluded that the motilities of Bac. FTU and E. coli are due to the operation of the Na+ and the H+ motors, respectively. In Bac. FTU growing at alkaline pH, the Na+ motors are assumed to be energized by delta mu Na+ produced by the Na(+)-motive respiratory chain, and therefore delta mu H+ is not involved in the motility process. As to Bac. FTU growing in a neutral medium, delta mu Na+ is produced secondarily, via the Na+/H(+)-antiporter, i.e., at the expense of delta mu H+ formed by the H(+)-motive respiratory chain.