RESUMEN
Ghrelin is a 28-amino-acid peptide secreted during starvation by gastric cells. Ghrelin physiologically induces food intake and seems to alter lipid and glucid metabolism in several tissues such as adipose tissue and liver. Liver has a key position in lipid metabolism as it allows the metabolic orientation of fatty acids between oxidation and esterification. We investigated the effects of peripheral ghrelin administration on 2 crucial parameters of fatty acid oxidation: the levocarnitine (L-carnitine)-dependent entry of the fatty acids in the mitochondria and the mitochondrial fatty acid oxidation. Ghrelin was either given to rats prior to the hepatocyte preparation and culture or used to treat hepatocytes prepared from control animals. Direct incubation of ghrelin to raw hepatocytes did not induce any change in the studied parameters. In hepatocytes prepared from 3 nmol ghrelin-treated rats, a 44% reduction of the mitochondrial fatty acid oxidation while no alteration of the L-carnitine-related parameters were observed. These results suggested (a) that ghrelin has no direct effect on liver, and (b) that when administrated to a whole organism, ghrelin may alter the lipid metabolism and the energy balance through a marked decrease in liver fatty acid oxidation.
Asunto(s)
Ácidos Grasos/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Hormonas Peptídicas/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Carnitina/farmacología , Células Cultivadas , Metabolismo Energético/efectos de los fármacos , Ghrelina , Mitocondrias Hepáticas/metabolismo , Oxidación-Reducción/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
The carnitine system plays a key role in beta-oxidation of long-chain fatty acids by permitting their transport into the mitochondrial matrix. The effects of hypothyroidism and hyperthyroidism were studied on gamma-butyrobetaine hydroxylase (BBH), the enzyme responsible for carnitine biosynthesis in the rat. In rat liver, BBH activity was decreased in the hypothyroid state and increased in hyperthyroid animals. The modifications in BBH activity correlated with changes in the enzyme Vmax values. These changes were shown to be related to hepatic BBH mRNA abundance. Thyroid hormones are known to interact with lipid metabolism, in particular by increasing long-chain fatty acid oxidation through activation of carnitine-dependent fatty acid import into mitochondria. Our study showed that thyroid hormones also increased carnitine bioavailability.
Asunto(s)
Carnitina/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Oxigenasas de Función Mixta/metabolismo , Hormonas Tiroideas/farmacología , Animales , Cinética , Hígado/enzimología , Masculino , Oxigenasas de Función Mixta/biosíntesis , Oxigenasas de Función Mixta/genética , Especificidad de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/metabolismo , gamma-Butirobetaína DioxigenasaRESUMEN
Carnitine is involved in the transfer of fatty acids across mitochondrial membranes. Carnitine is found in dairy and meat products, but is also biosynthesized from lysine and methionine via a process that, in rat, takes place essentially in the liver. After intestinal absorption or hepatic biosynthesis, carnitine is transferred to organs whose metabolism is dependent on fatty acid oxidation, such as heart and skeletal muscle. In skeletal muscle, carnitine concentration was found to be 50 times higher than in the plasma, implicating an active transport system for carnitine. In this study, we characterized this transport in isolated rat myotubes, established mouse C2C12 myoblastic cells, and rat myotube plasma membranes and found that it was Na(+)-dependent and partly inhibited by a Na(+)/K(+) ATPase inhibitor. L-carnitine analogues such as D-carnitine and gamma-butyrobetaine interfere with this system as does acyl carnitine. Among these inhibitors, the most potent was mildronate (3-(2,2,2-trimethylhydrazinium)propionate), known as a gamma-butyrobetaine hydroxylase inhibitor. It also induced a marked decrease in carnitine transport into muscle cells. Removal of carnitine or treatment with mildronate induced growth inhibition of cultured C2C12 myoblastic cells. These data suggest that myoblast growth and/or differentiation is dependent upon the presence of carnitine.
Asunto(s)
Carnitina/metabolismo , Metilhidrazinas/farmacología , Músculo Esquelético/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , División Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Técnicas In Vitro , Masculino , Ratones , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Carnitine biosynthesis from lysine and methionine involves five enzymatic reactions. gamma-butyrobetaine hydroxylase (BBH; EC 1.14. 11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of gamma-butyrobetaine to carnitine. The cDNA encoding this enzyme has been isolated and characterized. The cDNA contained an open reading frame of 1161 bp encoding a protein of 387 amino acids with a deduced molecular weight of 44.5 kDa. The sequence of the cDNA showed an important homology with the human cDNA recently isolated. Northern analysis showed gamma-butyrobetaine hydroxylase expression in the liver and in some extend in the testis and the epididymis. During this study, it also appeared that BBH mRNA expression was undetectable by Northern analysis during the perinatal period. During the development of the rat, the amount of BBH mRNA appeared after the weaning of the young rat and reached a maximal expression at the adult stage.
Asunto(s)
Hígado/enzimología , Oxigenasas de Función Mixta/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Regulación del Desarrollo de la Expresión Génica , Masculino , Oxigenasas de Función Mixta/química , Datos de Secuencia Molecular , Ratas , Ratas Wistar , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , gamma-Butirobetaína DioxigenasaRESUMEN
The biosynthesis of carnitine from lysine and methionine involves five enzymatic reactions. Gamma-butyrobetaine hydroxylase (BBH; EC 1.14.11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of gamma-butyrobetaine to carnitine. This enzyme had never been purified to homogeneity from rat tissue. This paper describes the purification and characterization of the rat liver BBH. This protein has been purified some 413 fold by ion exchange, affinity and gel-filtration chromatographies and appears as a dimere of 43,000 Daltons subunits by PAGE. The affinity chromatography column used in the purification process utilizes 3-(2,2,2-trimethylhydrazinium)propionate (THP), a BBH inhibitor, as the ligand. Polyclonal antibodies were raised against the liver enzyme. They were able to precipitate BBH activity in either a crude liver extract or a purified fraction of the enzyme. Furthermore, it crossreacts with a 43 kDa protein in the liver. No evidence for extra hepatic enzyme was found.
Asunto(s)
Carnitina , Hígado/enzimología , Oxigenasas de Función Mixta/aislamiento & purificación , Animales , Ácido Ascórbico/farmacología , Betaína/análogos & derivados , Betaína/metabolismo , Catalasa/metabolismo , Catálisis , Cromatografía de Afinidad , Inhibidores Enzimáticos/metabolismo , Compuestos Ferrosos/farmacología , Hidroxilación , Ácidos Cetoglutáricos/metabolismo , Cinética , Ligandos , Masculino , Metilhidrazinas/metabolismo , Peso Molecular , Ratas , Ratas Wistar , gamma-Butirobetaína DioxigenasaRESUMEN
The five-fold higher carnitine content in the liver of fenofibrate-treated rats addresses the question about the possible role of this enhancement in the hypolipidaemic effect of the drug and the underlying mechanisms. When fenofibrate was administered with mildronate (a gamma-butyrobetaine hydroxylase inhibitor) in suitable amount, the content in carnitine was found to be normalized in liver. However, triglyceride contents of liver and serum were then at least as low as in rats treated by fenofibrate only. When carnitine concentration was lowered by mildronate to the third of the normal value, a marked increase in triglycerides occurred both in liver and serum, while the five-fold increase in carnitine due to fenofibrate enhanced blood ketone body concentration with no effect on liver and serum triglycerides. Data suggest that the normal carnitine concentration is largely sufficient to meet the usual requirement for carnitine palmitoyltransferase I activity (CPT I). In rat liver, increase in mitochondrial CPT I activity and in peroxisomal fatty acid oxidation may constitute part of the hypolipidaemic effect of fenofibrate.
Asunto(s)
Carnitina/metabolismo , Fenofibrato/farmacología , Hipolipemiantes/farmacología , Hígado/metabolismo , Metilhidrazinas/farmacología , Animales , Carnitina/sangre , Colesterol/sangre , Colesterol/metabolismo , Cuerpos Cetónicos/sangre , Cuerpos Cetónicos/metabolismo , Masculino , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Fosfolípidos/sangre , Fosfolípidos/metabolismo , Ratas , Ratas Wistar , Triglicéridos/metabolismoRESUMEN
This study was designed to determine which enzyme activities were first impaired in mitochondria exposed to 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), a known radical initiator. EPR spin-trapping revealed generation of reactive oxygen species although malondialdehyde formation remained very low. With increasing AAPH concentrations, State-3 respiration was progressively depressed with unaltered ADP/O ratios. A top-down approach demonstrated that alterations were located at the phosphorylation level. As shown by inhibitor titrations, ATP/ADP translocase activity was unaffected in the range of AAPH concentrations used. In contrast, AAPH appeared to exert a deleterious effect at the level of F1F0-ATPase, comparable with dicyclohexylcarbodi-imide, which alters Fo proton channel. A comparison of ATP hydrolase activity in uncoupled and broken mitochondria reinforced this finding. In spite of its pro-oxidant properties, AAPH was shown to act as a dose-dependent inhibitor of cyclosporin-sensitive permeability transition initiated by Ca2+, probably as a consequence of its effect on F1F0-ATPase. Resveratrol, a potent antiperoxidant, completely failed to prevent the decrease in State-3 respiration caused by AAPH. The data suggest that AAPH, when used under mild conditions, acted as a radical initiator and was capable of damaging F1F0-ATPase, thereby slowing respiratory chain activity and reducing mitochondrial antioxidant defences.
Asunto(s)
Amidinas/farmacología , Membranas Intracelulares/fisiología , Mitocondrias Hepáticas/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , ATPasas de Translocación de Protón/metabolismo , Animales , Atractilósido/análogos & derivados , Atractilósido/farmacología , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Óxidos N-Cíclicos , Diciclohexilcarbodiimida/farmacología , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Radicales Libres/metabolismo , Membranas Intracelulares/efectos de los fármacos , Cinética , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/análisis , Potenciales de la Membrana/fisiología , Mitocondrias Hepáticas/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , ATPasas de Translocación de Protón/efectos de los fármacos , Ratas , Ratas Wistar , Marcadores de Spin , Succinatos/metabolismoRESUMEN
This study was designed to examine whether short- and long-term treatments by a low level of dietary L-carnitine are capable of altering enzyme activities related to fatty acid oxidation in normal Wistar rats. Under controlled feeding, ten days of treatment changed neither body weights nor liver and gastrocnemius weights, but succeeded in reducing the weight of peri-epididymal adipose tissues. Triacylglycerol contents were lowered in liver and ketone body concentrations were found slightly more elevated in blood. In the liver, mitochondrial carnitine palmitoyltransferase I (CPT I) exhibited a slightly higher specific activity and a lower sensitivity to malonyl-CoA inhibition, while peroxisomal fatty acid oxidizing system (PFAOS) was found to be less active. Carnitine supplied for one month reduced the mass of the periepididymal fat tissue, but not those of the other studied organs, and produced a slight but non-significant gain in body weight after ten days of treatment. In the liver, CPTI characteristics were comparable in control and treated groups, while PFAOS activity was less in rats receiving carnitine. Data show that L-carnitine at a low level in the diet exerted two paradoxical effects before and after ten days of treatment. Results are discussed in regard to fatty acid oxidation in mitochondria and peroxisomes, and to the possible altered acyl-CoA/acylcarnitine ratio with increased concentrations of L-carnitine in the liver.
Asunto(s)
Carnitina/administración & dosificación , Ácidos Grasos/metabolismo , Hígado/metabolismo , Animales , Peso Corporal , Carnitina/farmacocinética , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/metabolismo , Dieta , Cuerpos Cetónicos/sangre , Cuerpos Cetónicos/metabolismo , Masculino , Microcuerpos/metabolismo , Mitocondrias/metabolismo , Tamaño de los Órganos , Oxidación-Reducción , Ratas , Ratas Wistar , Distribución Tisular , Triglicéridos/sangre , Triglicéridos/metabolismoRESUMEN
This study was designed to examine whether the depletion of L-carnitine may induce compensatory mechanisms allowing higher fatty acid oxidative activities in liver, particularly with regard to mitochondrial carnitine palmitoyltransferase I activity and peroxisomal fatty acid oxidation. Wistar rats received D-carnitine for 2 days and 3-(2,2,2,-trimethylhydrazinium)propionate (mildronate), a noncompetitive inhibitor of gamma-butyrobetaine hydroxylase, for 10 days. They were starved for 20 hr before being sacrificed. A dramatic reduction in carnitine concentration was observed in heart, skeletal muscles and kidneys, and to a lesser extent, in liver. Triacylglycerol content was found to be significantly more elevated on a gram liver and whole liver basis as well as per mL of blood (but to a lesser extent), while similar concentrations of ketone bodies were found in the blood of D-carnitine/mildronate-treated and control rats. In liver mitochondria, the specific activities of acyl-CoA synthetase and carnitine palmitoyltransferase I were enhanced by the treatment, while peroxisomal fatty acid oxidation was higher per gram of tissue. It is suggested that there may be an enhancement of cellular acyl-CoA concentration, a signal leading to increased liver fatty acid oxidation in acute carnitine deficiency.
Asunto(s)
Carnitina/deficiencia , Ácidos Grasos/metabolismo , Hígado/metabolismo , Oxigenasas de Función Mixta/antagonistas & inhibidores , Animales , Peso Corporal , Carnitina/antagonistas & inhibidores , Carnitina/biosíntesis , Masculino , Metilhidrazinas/farmacología , Tamaño de los Órganos , Oxidación-Reducción , Ratas , Ratas Wistar , gamma-Butirobetaína DioxigenasaRESUMEN
The urea cycle takes place in the hepatocyte of ureothelic animals. The conversion of ammonia into urea involves five reactions. The first 2 take place in the matrix of the mitochondria, the last 2 occur in the cytosol. Argininosuccinate synthetase (AS) is the third reaction of the urea cycle. It catalyses the condensation of citrulline and aspartate into argininosuccinate. We have previously reported that rat AS activity was present in the cytosol and the outer membrane of the mitochondria. We have shown that, at the activity level, the colocation of AS was changing during fetal and neonatal development and was under the control of corticosteroid and pancreatic hormones. However, an unresolved issue was whether both AS had the same specific activity and that their location was changing during ontogenesis or that the specific activities of mitochondrial and cytosolic enzymes were different and/or modified during this period. In the present report, we compared the compartmentalization of AS activity and protein level in the fetus, the new-born and the adult rat and the role of corticosteroid and pancreatic hormones. Specific activities of both AS remained unchanged during ontogenesis. Glucocorticoids induced an increase in mitochondrial AS while glucagon appeared to induce a concomitant decrease in the level of mitochondrial AS and an increase in cytosolic AS.
Asunto(s)
Corticoesteroides/fisiología , Argininosuccinato Sintasa/metabolismo , Hígado/embriología , Hormonas Pancreáticas/fisiología , Adrenalectomía , Animales , Animales Recién Nacidos , Células Cultivadas , Citoplasma/metabolismo , Dexametasona/farmacología , Diabetes Mellitus Experimental/metabolismo , Femenino , Glucagón/farmacología , Hidrocortisona/farmacología , Hipofisectomía , Hígado/citología , Hígado/metabolismo , Masculino , Mitocondrias Hepáticas/metabolismo , Embarazo , Ratas , Ratas WistarRESUMEN
Argininosuccinate synthetase (AS) is the third enzyme in ureogenesis, it catalyses the reaction of condensation of citrulline and aspartate into argininosuccinate. In the present report, we described the first characterization of AS within the outer membrane of rat liver mitochondria. Mitochondria-associated AS displayed the same kinetic characteristics as the cytoplasmic enzyme, but was found to be thermostable while cytoplasmic AS was not. The evolution of the co-location of AS was analyzed during ontogenesis. Total AS activity increased throughout rat fetal development. Simultaneously, the subcellular distribution of the enzyme has changed. AS activity was mainly mitochondrial in fetal and new-born liver liver and cytoplasmic in adult rat liver. The variation in subcellular distribution of AS may be due to the dramatic changes in hormonal levels that occur during this period. The role of corticosteroid and pancreatic hormones was studied. During fetal period, corticosteroid hormones induced an increase in mitochondria-associated AS activity. This was prevented by insulin. Glucagon did not modify total AS activity but reduced mitochondrial AS activity, meanwhile, a comparable increase in cytoplasmic AS activity was observed. One may hypothesize that glucagon may participate in the transfer of mitochondrial enzyme into the cytosol.
Asunto(s)
Corticoesteroides/fisiología , Argininosuccinato Sintasa/metabolismo , Membranas Intracelulares/enzimología , Mitocondrias Hepáticas/enzimología , Hormonas Pancreáticas/fisiología , Animales , Animales Recién Nacidos , Fraccionamiento Químico , Citosol/metabolismo , Desarrollo Embrionario y Fetal/fisiología , Estabilidad de Enzimas , Femenino , Cinética , Masculino , Mitocondrias Hepáticas/ultraestructura , Ratas , Ratas Wistar , SolubilidadRESUMEN
Murine macrophages respond to endotoxins by inducing a vast array of genes that play a major role in the host's response to infection and tumor growth. We have isolated and characterized a 1.8-kb cDNA, designated IRG2, from a cDNA library prepared from RNA isolated from the murine cell line, RAW 264.7, after bacterial LPS stimulation. The cDNA encodes a protein of 47 kDa that is the murine homologue of a small family of proteins described from IFN-induced human cells. The IRG2 message does not appear until 3 h after LPS exposure and its induction is dependent on new protein synthesis. IRG2 induction by LPS is slightly inhibited by the anti-inflammatory steroid, dexamethasone. Increasing cytosolic cAMP with either forskolin, dibutyryl cAMP, or 8-(4-chlorophenylthio)-cAMP caused marked inhibition of the LPS induction of IRG2. In contrast, activation of PKC with phorbol ester potentiated the LPS response. Removing extracellular Ca2+ with EGTA inhibited IRG2 induction; increasing intracellular calcium with the calcium ionophore A23187 led to enhanced levels of the IRG2 transcript. These data suggest that the induction of IRG2 occurs via a PKC pathway.
Asunto(s)
ADN Complementario/genética , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cartilla de ADN/genética , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Proteínas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Sistemas de Mensajero Secundario/efectos de los fármacos , Sistemas de Mensajero Secundario/genética , Homología de Secuencia de AminoácidoRESUMEN
Citrullinemia is a recessive genetic disease caused by a deficiency in argininosuccinate synthetase (AS). Retroviruses were used to transduce the human AS gene into cultured human cells. Using amphotropic viruses with high titer (> 10(6) cfu/ml), we were able to correct the defect in cultured fibroblasts from citrullinemic patients. Retroviral transduction of the human AS gene into human bone marrow cells was also studied. Co-cultivation was used to infect the cells and up to 80% of progenitor cells were found to be carrying and expressing the AS retrovirus after infection. When the infected cells were kept in culture, integration and expression of the retrovirus was observed. Retroviral sequences were present and expressed in the cultured bone marrow-derived cells for up to 10 weeks.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/terapia , Argininosuccinato Sintasa/genética , Médula Ósea/enzimología , Citrulina/sangre , Terapia Genética , Retroviridae/genética , Células de la Médula Ósea , Trasplante de Médula Ósea , Células Cultivadas , Fibroblastos , Humanos , TransfecciónRESUMEN
Amphotropic and ecotropic packaging cell lines were used to obtain high titers (greater than 10(6) colony forming units/ml) of retroviruses encoding human argininosuccinate synthetase, and these viruses were used to transduce murine bone marrow cells using cocultivation in vitro. The bone marrow cells were transplanted into lethally irradiated recipient mice, and argininosuccinate synthetase activity was measured in peripheral blood. Transduction with amphotropic retrovirus resulted in short-term expression for a period of 1-8 weeks, and no animals expressed the human gene after 25 weeks. Over 60% of the animals transplanted with cells transduced with ecotropic retrovirus expressed the human gene 44 weeks post-transplant, although the level of expression varied over a wide range. Analysis of the DNA from transplanted animals demonstrated the presence of the human sequence in expressing animals, and S1 nuclease analysis of RNA confirmed the presence of the human RNA transcripts. Analysis of granulocyte/macrophage (GM) colonies derived from the bone marrow of transplanted, expressing animals revealed a correlation between the level of expression of the transduced gene with the percentage of GM colonies carrying the human gene sequence. These data demonstrate the feasibility of obtaining long-term expression of genes introduced into bone marrow cells using retroviral vectors and the feasibility of obtaining expression of a gene not normally expressed in bone marrow.
Asunto(s)
Argininosuccinato Sintasa/genética , Células Madre Hematopoyéticas/metabolismo , Retroviridae/genética , Transducción Genética , Animales , Argininosuccinato Sintasa/sangre , Secuencia de Bases , Médula Ósea/metabolismo , Células de la Médula Ósea , Trasplante de Médula Ósea , Línea Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , ADN Viral/sangre , Femenino , Expresión Génica , Vectores Genéticos , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , ARN Viral/sangre , Factores de TiempoRESUMEN
The development and hormonal regulation of thioredoxin and of the thioredoxin-reductase system were investigated during the perinatal period in rat liver. An immunological procedure was developed in order to quantify thioredoxin in fetal and neonatal hepatocytes. Both immunoreactive thioredoxin and thioredoxin-reductase activity appeared on day 16.5 of pregnancy. The level of immunoreactive thioredoxin increased during the late fetal period, and its level was the same 24 h after birth. Moreover, its development was not subjected to hormonal regulation by corticosteroids and glucagon. In contrast, thioredoxin-reductase activity increased 3 times during the late fetal period and presented a marked increase 24 h after birth. In the absence of glucocorticoids there was no increase in the level of thioredoxin reductase, while administration of hydrocortisone acetate and glucagon to fetuses prematurely evoked its activity. This study suggests that if thioredoxin acts physiologically, this activity is related to the state of reduction of the molecule rather than to the total concentration in the liver.
Asunto(s)
Glucagón/farmacología , Hígado/metabolismo , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Tiorredoxinas/metabolismo , Animales , Argininosuccinato Sintasa/metabolismo , Carbamoil-Fosfato Sintasa (Amoniaco)/metabolismo , Activación Enzimática/efectos de los fármacos , Femenino , Feto , Hidrocortisona/análogos & derivados , Hidrocortisona/farmacología , Hígado/efectos de los fármacos , Hígado/embriología , Embarazo , Ratas , Ratas EndogámicasRESUMEN
Argininosuccinate synthetase (ASS, EC 6.3.4.5), the third enzyme of urea-cycle, was studied in desactivated extracts of rat liver. The enzyme is activated, in vitro, by Mg2+ ions (5 mM) and dithiothreitol (DTT: 10 mM). After reduction by DTT, thioredoxins isolated from rat liver were able to activate ASS by 370%.
Asunto(s)
Argininosuccinato Sintasa/metabolismo , Proteínas Bacterianas/farmacología , Ditiotreitol/farmacología , Ligasas/metabolismo , Hígado/enzimología , Tiorredoxinas/farmacología , Animales , Cinética , Magnesio/farmacología , Masculino , Ratas , Ratas EndogámicasRESUMEN
The five urea cycle enzymes were studied in desactivated extracts of rat liver. After reduction by dithiothreitol (DTT) and in presence of Mg2+ ions, thioredoxines isolated from rat liver were able to activate carbamyl phosphate synthetase-I (CPS-I) and argininosuccinate synthetase (ASS) respectively by 468% and by 370%. Thioredoxines were purified from adult rat liver and an antiserum was raised to these proteins. After immunologic quantitation, their level in adult rat was 0.103 mg/g liver.