RESUMEN
More than 30 proteins (Pex proteins) are known to participate in the biogenesis of peroxisomes-ubiquitous oxidative organelles involved in lipid and ROS metabolism. The Pex11 family of homologous proteins is responsible for division and proliferation of peroxisomes. We show that yeast Pex11 is a pore-forming protein sharing sequence similarity with TRPM cation-selective channels. The Pex11 channel with a conductance of Λ=4.1 nS in 1.0M KCl is moderately cation-selective (PK(+)/PCl(-)=1.85) and resistant to voltage-dependent closing. The estimated size of the channel's pore (r~0.6 nm) supports the notion that Pex11 conducts solutes with molecular mass below 300-400 Da. We localized the channel's selectivity determining sequence. Overexpression of Pex11 resulted in acceleration of fatty acids ß-oxidation in intact cells but not in the corresponding lysates. The ß-oxidation was affected in cells by expression of the Pex11 protein carrying point mutations in the selectivity determining sequence. These data suggest that the Pex11-dependent transmembrane traffic of metabolites may be a rate-limiting step in the ß-oxidation of fatty acids. This conclusion was corroborated by analysis of the rate of ß-oxidation in yeast strains expressing Pex11 with mutations mimicking constitutively phosphorylated (S165D, S167D) or unphosphorylated (S165A, S167A) protein. The results suggest that phosphorylation of Pex11 is a mechanism that can control the peroxisomal ß-oxidation rate. Our results disclose an unexpected function of Pex11 as a non-selective channel responsible for transfer of metabolites across peroxisomal membrane. The data indicate that peroxins may be involved in peroxisomal metabolic processes in addition to their role in peroxisome biogenesis.
Asunto(s)
Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo , Porinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Western Blotting , Dicroismo Circular , Ácidos Grasos/metabolismo , Espectrometría de Masas , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Oxidación-Reducción , Peroxinas , Fosforilación , Porinas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismoRESUMEN
The human MPV17-related mitochondrial DNA depletion syndrome is an inherited autosomal recessive disease caused by mutations in the inner mitochondrial membrane protein MPV17. Although more than 30 MPV17 gene mutations were shown to be associated with mitochondrial DNA depletion syndrome, the function of MPV17 is still unknown. Mice deficient in Mpv17 show signs of premature aging. In the present study, we used electrophysiological measurements with recombinant MPV17 to reveal that this protein forms a non-selective channel with a pore diameter of 1.8 nm and located the channel's selectivity filter. The channel was weakly cation-selective and showed several subconductance states. Voltage-dependent gating of the channel was regulated by redox conditions and pH and was affected also in mutants mimicking a phosphorylated state. Likewise, the mitochondrial membrane potential (Δψm) and the cellular production of reactive oxygen species were higher in embryonic fibroblasts from Mpv17(-/-) mice. However, despite the elevated Δψm, the Mpv17-deficient mitochondria showed signs of accelerated fission. Together, these observations uncover the role of MPV17 as a Δψm-modulating channel that apparently contributes to mitochondrial homeostasis under different conditions.
Asunto(s)
ADN Mitocondrial/genética , Potencial de la Membrana Mitocondrial , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Secuencia de Aminoácidos , Animales , Autofagia , Dicroismo Circular , Daño del ADN , Fibroblastos/metabolismo , Fluoresceínas/química , Genotipo , Homeostasis , Humanos , Concentración de Iones de Hidrógeno , Espectrometría de Masas , Ratones , Ratones Transgénicos , Membranas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Fosforilación , Filogenia , Pichia/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
To understand the functional role of the peroxisomal membrane channel Pxmp2, mice with a targeted disruption of the Pxmp2 gene were generated. These mice were viable, grew and bred normally. However, Pxmp2(-/-) female mice were unable to nurse their pups. Lactating mammary gland epithelium displayed secretory lipid droplets and milk proteins, but the size of the ductal system was greatly reduced. Examination of mammary gland development revealed that retarded mammary ductal outgrowth was due to reduced proliferation of epithelial cells during puberty. Transplantation experiments established the Pxmp2(-/-) mammary stroma as a tissue responsible for suppression of epithelial growth. Morphological and biochemical examination confirmed the presence of peroxisomes in the mammary fat pad adipocytes, and functional Pxmp2 was detected in the stroma of wild-type mammary glands. Deletion of Pxmp2 led to an elevation in the expression of peroxisomal proteins in the mammary fat pad but not in liver or kidney of transgenic mice. Lipidomics of Pxmp2(-/-)mammary fat pad showed a decrease in the content of myristic acid (C14), a principal substrate for protein myristoylation and a potential peroxisomal ß-oxidation product. Analysis of complex lipids revealed a reduced concentration of a variety of diacylglycerols and phospholipids containing mostly polyunsaturated fatty acids that may be caused by activation of lipid peroxidation. However, an antioxidant-containing diet did not stimulate mammary epithelial proliferation in Pxmp2(-/-) mice. The results point to disturbances of lipid metabolism in the mammary fat pad that in turn may result in abnormal epithelial growth. The work reveals impaired mammary gland development as a new category of peroxisomal disorders.
Asunto(s)
Metabolismo de los Lípidos , Glándulas Mamarias Animales/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Peroxisomas/metabolismo , Tejido Adiposo/metabolismo , Animales , Ácidos y Sales Biliares/química , Células Epiteliales/citología , Ácidos Grasos/química , Femenino , Homeostasis , Lactancia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Poliaminas/química , Fracciones Subcelulares , Factores de TiempoRESUMEN
Peroxisomes perform a large variety of metabolic functions that require a constant flow of metabolites across the membranes of these organelles. Over the last few years it has become clear that the transport machinery of the peroxisomal membrane is a unique biological entity since it includes nonselective channels conducting small solutes side by side with transporters for 'bulky' solutes such as ATP. Electrophysiological experiments revealed several channel-forming activities in preparations of plant, mammalian, and yeast peroxisomes and in glycosomes of Trypanosoma brucei. The properties of the first discovered peroxisomal membrane channel - mammalian Pxmp2 protein - have also been characterized. The channels are apparently involved in the formation of peroxisomal shuttle systems and in the transmembrane transfer of various water-soluble metabolites including products of peroxisomal ß-oxidation. These products are processed by a large set of peroxisomal enzymes including carnitine acyltransferases, enzymes involved in the synthesis of ketone bodies, thioesterases, and others. This review discusses recent data pertaining to solute permeability and metabolite transport systems in peroxisomal membranes and also addresses mechanisms responsible for the transfer of ATP and cofactors such as an ATP transporter and nudix hydrolases.
Asunto(s)
Membranas Intracelulares/metabolismo , Peroxisomas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Ácidos y Sales Biliares/metabolismo , Transporte Biológico , Coenzimas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cuerpos Cetónicos/metabolismo , Metabolismo de los Lípidos , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/fisiología , Oxidación-Reducción , Permeabilidad , Peroxisomas/enzimologíaRESUMEN
The review is dedicated to recent progress in the study of peroxisomal membrane permeability to solutes which has been a matter of debate for more than 40 years. Apparently, the mammalian peroxisomal membrane is freely permeable to small solute molecules owing to the presence of pore-forming channels. However, the membrane forms a permeability barrier for 'bulky' solutes including cofactors (NAD/H, NADP/H, CoA, and acetyl/acyl-CoA esters) and ATP. Therefore, peroxisomes need specific protein transporters to transfer these compounds across the membrane. Recent electrophysiological studies have revealed channel-forming activities in the mammalian peroxisomal membrane. The possible involvement of the channels in the transfer of small metabolites and in the formation of peroxisomal shuttle systems is described.
Asunto(s)
Membranas Intracelulares/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Modelos Biológicos , Peroxisomas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico , Coenzima A/metabolismo , NAD/metabolismo , NADP/metabolismo , Permeabilidad , Pirofosfatasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Hidrolasas NudixRESUMEN
Fatty acid degradation in most organisms occurs primarily via the beta-oxidation cycle. In mammals, beta-oxidation occurs in both mitochondria and peroxisomes, whereas plants and most fungi harbor the beta-oxidation cycle only in the peroxisomes. Although several of the enzymes participating in this pathway in both organelles are similar, some distinct physiological roles have been uncovered. Recent advances in the structural elucidation of numerous mammalian and yeast enzymes involved in beta-oxidation have shed light on the basis of the substrate specificity for several of them. Of particular interest is the structural organization and function of the type 1 and 2 multifunctional enzyme (MFE-1 and MFE-2), two enzymes evolutionarily distant yet catalyzing the same overall enzymatic reactions but via opposite stereochemistry. New data on the physiological roles of the various enzymes participating in beta-oxidation have been gathered through the analysis of knockout mutants in plants, yeast and animals, as well as by the use of polyhydroxyalkanoate synthesis from beta-oxidation intermediates as a tool to study carbon flux through the pathway. In plants, both forward and reverse genetics performed on the model plant Arabidopsis thaliana have revealed novel roles for beta-oxidation in the germination process that is independent of the generation of carbohydrates for growth, as well as in embryo and flower development, and the generation of the phytohormone indole-3-acetic acid and the signal molecule jasmonic acid.
Asunto(s)
Redes y Vías Metabólicas , Peroxisomas/fisiología , Acetil-CoA C-Aciltransferasa/metabolismo , Acil-CoA Oxidasa/metabolismo , Animales , Arabidopsis/genética , Arabidopsis/fisiología , Complejos Multienzimáticos/metabolismo , Oxidación-Reducción , Especificidad por SustratoRESUMEN
The liver isoform of fatty-acid-binding protein (L-FABP) facilitates the cellular uptake, transport and metabolism of fatty acids and is also involved in the regulation of gene expressions and cell differentiation. Consistent with these functions, L-FABP is predominantly present in the cytoplasm and to a lesser extent in the nucleus; however, a significant portion of this protein has also been detected in fractions containing different organelles. More recent observations, notably on L-FABP-deficient mice, indicated a possible direct involvement of L-FABP in the peroxisomal oxidation of long-chain fatty acids. In order to clarify the links between L-FABP and peroxisomal lipid metabolism, we reinvestigated the subcellular distribution of the protein. Analytical subcellular fractionation by a method preserving the intactness of isolated peroxisomes, two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with MS analysis, and immunoelectron microscopy of liver sections demonstrate the presence of L-FABP in the matrix of peroxisomes as a soluble protein. Peroxisomal L-FABP was highly inducible by clofibrate. The induction of L-FABP was accompanied by a marked increase in the binding capacity of peroxisomal matrix proteins for oleic acid and cis-parinaric acid. The peroxisomal beta-oxidation of palmitoyl-CoA and acyl-CoA thioesterase activity were stimulated by L-FABP, indicating that the protein modulates the function of peroxisomal lipid-metabolizing enzymes. The possible role of intraperoxisomal L-FABP in lipid metabolism is discussed.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/metabolismo , Hígado/citología , Hígado/metabolismo , Peroxisomas/metabolismo , Animales , Clofibrato/farmacología , Ácidos Grasos/metabolismo , Hígado/efectos de los fármacos , Masculino , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Ratas , Ratas Sprague-DawleyRESUMEN
Mulibrey nanism (MUL) is a rare autosomal recessive multi-organ disorder characterized by severe prenatal-onset growth failure, infertility, cardiopathy, risk for tumors, fatty liver, and type 2 diabetes. MUL is caused by loss-of-function mutations in TRIM37, which encodes an E3 ubiquitin ligase belonging to the tripartite motif (TRIM) protein family and having both peroxisomal and nuclear localization. We describe a congenic Trim37 knock-out mouse (Trim37(-/-)) model for MUL. Trim37(-/-) mice were viable and had normal weight development until approximately 12â months of age, after which they started to manifest increasing problems in wellbeing and weight loss. Assessment of skeletal parameters with computer tomography revealed significantly smaller skull size, but no difference in the lengths of long bones in Trim37(-/-) mice as compared with wild-type. Both male and female Trim37(-/-) mice were infertile, the gonads showing germ cell aplasia, hilus and Leydig cell hyperplasia and accumulation of lipids in and around Leydig cells. Male Trim37(-/-) mice had elevated levels of follicle-stimulating and luteinizing hormones, but maintained normal levels of testosterone. Six-month-old Trim37(-/-) mice had elevated fasting blood glucose and low fasting serum insulin levels. At 1.5â years Trim37(-/-) mice showed non-compaction cardiomyopathy, hepatomegaly, fatty liver and various tumors. The amount and morphology of liver peroxisomes seemed normal in Trim37(-/-) mice. The most consistently seen phenotypes in Trim37(-/-) mice were infertility and the associated hormonal findings, whereas there was more variability in the other phenotypes observed. Trim37(-/-) mice recapitulate several features of the human MUL disease and thus provide a good model to study disease pathogenesis related to TRIM37 deficiency, including infertility, non-alcoholic fatty liver disease, cardiomyopathy and tumorigenesis.
RESUMEN
BACKGROUND: Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. METHODS/PRINCIPAL FINDINGS: We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T. brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70-80 pA, 20-25 pA, and 8-11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20-25 pA is anion-selective (P(K+)/P(Cl-)â¼0.31), while the other two types of channels are slightly selective for cations (P(K+)/P(Cl-) ratios â¼1.15 and â¼1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel's pore. CONCLUSIONS/SIGNIFICANCE: These results indicate that the membrane of glycosomes apparently contains several types of pore-forming channels connecting the glycosomal lumen and the cytosol.
Asunto(s)
Membranas Intracelulares/metabolismo , Canales Iónicos/metabolismo , Proteínas de la Membrana/metabolismo , Microcuerpos/metabolismo , Trypanosoma brucei brucei/metabolismo , Animales , Transporte Biológico , Citosol/metabolismo , Glucólisis , Membrana Dobles de Lípidos/metabolismo , Orgánulos/metabolismo , Ratas , Ratas WistarRESUMEN
Peroxisomes are multifunctional organelles with an important role in the generation and decomposition of reactive oxygen species (ROS). In this review, the ROS-producing enzymes, as well as the antioxidative defense system in mammalian peroxisomes, are described. In addition, various conditions leading to disturbances in peroxisomal ROS metabolism, such as abnormal peroxisomal biogenesis, hypocatalasemia, and proliferation of peroxisomes are discussed. We also review the role of mammalian peroxisomes in some physiological and pathological processes involving ROS that lead to mitochondrial abnormalities, defects in cell proliferation, and alterations in the central nervous system, alcoholic cardiomyopathy, and aging. Antioxid.
Asunto(s)
Peroxisomas/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Animales , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Modelos Biológicos , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Peroxisomas/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The separate localization of glyoxylate cycle enzymes in the peroxisomes and the cytosol of the yeast Saccharomyces cerevisiae indicates that the peroxisomal membrane must permit the flow of metabolites between the two compartments. The transfer of these metabolites may require peroxisomal membrane channel(s). We used an electrophysiological approach (reconstitution assay in lipid bilayers) to assess the ability of peroxisomal membrane channels to conduct different solutes including metabolites of the glyoxylate cycle. At least two distinct channel-forming activities were detected in peroxisomal preparations. One of these activities was highly inducible by dithiothreitol and showed large-amplitude current increments when 1M KCl was used as a bath solution. Single-channel analysis revealed that the inducible channel is anion-selective (P(Cl(-)) / P(K(+)) = 2.6; P(citrate)/P(K(+)) = 1.6) and displays flickering at holding potentials over + or - 30mV directed upward or downward relative to the main open state of the channel. The channel inducible by DTT facilitates the transfer of solutes with a molecular mass up to 400Da, sufficient to allow the transmembrane trafficking of glyoxylate cycle intermediates between the peroxisomal lumen and the cytoplasm.
Asunto(s)
Glioxilatos/metabolismo , Canales Iónicos/metabolismo , Peroxisomas/enzimología , Saccharomyces cerevisiae/enzimología , Transporte Biológico Activo , Fraccionamiento Celular , Ditiotreitol/farmacología , Técnicas In Vitro , Membrana Dobles de Lípidos , Potenciales de la Membrana/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Peroxisomas/efectos de los fármacosRESUMEN
Highly-purified peroxisomes from the yeast Saccharomyces cerevisiae grown on oleic acid were investigated for the presence of channel (pore)-forming proteins in the membrane of these organelles. Solubilized membrane proteins were reconstituted in planar lipid bilayers and their pore-forming activity was studied by means of multiple-channel monitoring or single-channel analysis. Two abundant pore-forming activities were detected with an average conductance of 0.2 and 0.6 nS in 1.0 m KCl, respectively. The high-conductance pore (0.6 nS in 1.0 m KCl) is slightly selective to cations (P(K+)/P(Cl-) approximately 1.3) and showed an unusual flickering at elevated (> +/-40 mV) holding potentials directed upward relative to the open state of the channel. The data obtained for the properties of the low-conductance pore (0.2 nS in 1.0 m KCl) support the notion that the high-conductance channel represents a cluster of two low-conductance pores. The results lead to conclusion that the yeast peroxisomes contain membrane pore-forming proteins that may aid the transfer of small solutes between the peroxisomal lumen and cytoplasm.
Asunto(s)
Canales Iónicos/metabolismo , Peroxisomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Electroforesis en Gel de Poliacrilamida , Membrana Dobles de Lípidos , Microscopía Electrónica de Transmisión , Peroxisomas/enzimologíaRESUMEN
BACKGROUND: Peroxisomal metabolic machinery requires a continuous flow of organic and inorganic solutes across peroxisomal membrane. Concerning small solutes, the molecular nature of their traffic has remained an enigma. METHODS/PRINCIPAL FINDINGS: In this study, we show that disruption in mice of the Pxmp2 gene encoding Pxmp2, which belongs to a family of integral membrane proteins with unknown function, leads to partial restriction of peroxisomal membrane permeability to solutes in vitro and in vivo. Multiple-channel recording of liver peroxisomal preparations reveals that the channel-forming components with a conductance of 1.3 nS in 1.0 M KCl were lost in Pxmp2(-/-) mice. The channel-forming properties of Pxmp2 were confirmed with recombinant protein expressed in insect cells and with native Pxmp2 purified from mouse liver. The Pxmp2 channel, with an estimated diameter of 1.4 nm, shows weak cation selectivity and no voltage dependence. The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane. CONCLUSIONS/SIGNIFICANCE: Pxmp2 is the first peroxisomal channel identified, and its existence leads to prediction that the mammalian peroxisomal membrane is permeable to small solutes while transfer of "bulky" metabolites, e.g., cofactors (NAD/H, NADP/H, and CoA) and ATP, requires specific transporters.
Asunto(s)
Membranas Intracelulares/metabolismo , Canales Iónicos/metabolismo , Proteínas de la Membrana/fisiología , Peroxisomas/metabolismo , Animales , Secuencia de Bases , Permeabilidad de la Membrana Celular , Cartilla de ADN , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Mammalian UK114 belongs to a highly conserved family of proteins with unknown functions. Although it is believed that UK114 is a cytosolic or mitochondrial protein there is no detailed study of its intracellular localization. Using analytical subcellular fractionation, electron microscopic colloidal gold technique, and two-dimensional gel electrophoresis of peroxisomal matrix proteins combined with mass spectrometric analysis we show here that a large portion of UK114 is present in rat liver peroxisomes. The peroxisomal UK114 is a soluble matrix protein and it is not inducible by the peroxisomal proliferator clofibrate. The data predict involvement of UK114 in peroxisomal metabolism.
Asunto(s)
Hepatocitos/metabolismo , Hígado/metabolismo , Proteínas de Neoplasias/metabolismo , Peroxisomas/metabolismo , Animales , Bacterias/metabolismo , Células Cultivadas , Secuencia Conservada , Masculino , Mamíferos/metabolismo , Proteínas de Neoplasias/química , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
It has been known for a long time that mammalian peroxisomes are extremely fragile in vitro. Changes in the morphological appearance and leakage of proteins from purified particles demonstrate that peroxisomes are damaged during isolation. However, some properties of purified peroxisomes, e.g., the latency of catalase, imply that their membranes are not disrupted. In the current study, we tried to ascertain the mechanism of this unusual behavior of peroxisomes in vitro. Biochemical and morphological examination of isolated peroxisomes subjected to sonication or to freezing and thawing showed that the membrane of the particles seals after disruption, restoring permeability properties. Transient damage of the membrane leads to the formation of peroxisomal "ghosts" containing nucleoid but nearly devoid of matrix proteins. The rate of leakage of matrix proteins from broken particles depended inversely on their molecular size. The effect of polyethylene glycols on peroxisomal integrity indicated that these particles are osmotically sensitive. Peroxisomes suffered an osmotic lysis during isolation that was resistant to commonly used low-molecular-mass osmoprotectors, e.g., sucrose. Damage to peroxisomes was partially prevented by applying more "bulky" osmoprotectors, e.g., polyethylene glycol 1500. A method was developed for the isolation of highly purified and nearly intact peroxisomes from rat liver by using polyethylene glycol 1500 as an osmoprotector.
Asunto(s)
Peroxisomas/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Alcohol Deshidrogenasa/metabolismo , Animales , Proteínas Portadoras/metabolismo , Catalasa/metabolismo , Fraccionamiento Celular , Centrifugación , Hidroxibutirato Deshidrogenasa/metabolismo , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Hígado/metabolismo , Masculino , Mamíferos , Microscopía Electrónica , Presión Osmótica , Peroxisomas/ultraestructura , Povidona , Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Dióxido de SilicioRESUMEN
The functional role of the peroxisomal membrane as a permeability barrier to metabolites has been a matter of controversy for more than four decades. The initial conception, claiming free permeability of the membrane to small solute molecules, has recently been challenged by several observations suggesting that the peroxisomal membrane forms a closed compartment. We have characterized in vitro the permeability of rat liver peroxisomal membrane. Our results indicate that the membrane allows free access into peroxisomes for small hydrophilic molecules, such as substrates for peroxisomal enzymes (glycolate, urate), but not to more bulky cofactors (NAD/H, NADP/H, CoA). Although access for cofactors is not prevented completely by the membrane, the membrane barrier severely restricts their rate of entry into peroxisomes. The data lead to conclusion that, in vivo, peroxisomes may possess their own pool of cofactors, while they share a common pool of small metabolites with the cytoplasm. The results also indicate that molecular size plays an important role in in vivo distinction between cofactors and metabolic intermediates.