RESUMEN
In this work, the quasi-analog to discrete transition occurring in the current-voltage characteristic of oxygen engineered yttrium oxide-based resistive random-access memory (RRAM) devices is investigated in detail. In particular, the focus of our research is not on the absolute conductance values of this characteristic but on the magnitude of its conductance changes occurring during the reset process of the device. It is found that the detected changes correspond to conductance values predominantly of the order of the quantum unit of conductance G0 = 2e2/h, where e is the electron charge and h the Planck constant. This feature is observed even at conductance levels far above G0, i.e. where electron transport is seemingly diffusive. It is also observed that such behavior is reproducible across devices comprising yttrium oxide layers with different oxygen concentrations and measured under different voltage sweep rates. While the oxygen deficiency affects the total number of quantized conductance states, the magnitude of the changes in conductance, close to 1 G0, is invariant to the oxygen content of the functional layer.
RESUMEN
The lipolysis stimulated lipoprotein receptor (LSR) is an apolipoprotein (Apo) B and ApoE receptor that participates in the removal of triglyceride-rich lipoproteins during the postprandial phase. LSR gene is located upstream of APOE, an important risk factor for cardiovascular disease (CVD). Since the APOE common polymorphism significantly affects the variability of lipid metabolism, this study aimed to determine the potential impact of a functional SNP rs916147 in LSR gene on lipid traits in healthy subjects and to investigate potential epistatic interaction between LSR and APOE. Unrelated healthy adults (N = 432) and children (N = 328, <18 years old) from the STANISLAS Family Study were used. Age-specific epistasis was observed between APOE and LSR, reversing the protective effect of APOE ε2 allele on cholesterol, ApoE and low-density lipoprotein levels (ß: .114, P: .777 × 10-8 , ß: .125, P: .639 × 10-3 , ß: .059, P: .531 × 10-3 , respectively). This interaction was verified in an independent adult population (n = 1744). These results highlight the importance of the LSR polymorphism and reveal the existence of complex molecular links between LSR and ApoE for the regulation of lipid levels, revealing potential new pathways of interest in type III hyperlipidemia and its involvement in CVD pathology.
Asunto(s)
Apolipoproteínas E/genética , Enfermedades Cardiovasculares/genética , Lípidos/genética , Receptores de Lipoproteína/genética , Adolescente , Alelos , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/patología , Niño , Preescolar , Colesterol , Epistasis Genética , Femenino , Genotipo , Humanos , Lípidos/sangre , Masculino , Polimorfismo Genético , Factores de TranscripciónRESUMEN
The aim of this study was to evaluate the fatty acids, tocopherols, squalene, sterols and phenolic antioxidants in three types of argan oil (Moroccan food, Moroccan aesthetic and a French commercial variety) along with a basic comparison with extra virgin olive and sunflower oil. The fatty acid profiles in the argan oils were very similar, with oleic acid (43%) and linoleic acid (36%) and their respective monoacylglycerols predominating. The major vitamer identified was -tocopherol with a mean of 483+/-11 mg/kg, in contrast to -tocopherol, which is the major vitamer in olive (190+/-1 mg/kg) and sunflower oil (532+/-6 mg/kg). The squalene content of the argan oils was very similar with a mean of 313+/-4 mg/100 g, which is lower than that of the olive oil (499 mg/100 g) but significantly higher than in the sunflower oil (6 mg/100 g). In contrast to olive and sunflower oils in which -sitosterol is predominant, the major sterols detected in the argan oils were schottenol (mean 147+/-10 mg/kg) and spinasterol (mean 122+/-10 mg/kg). The only phenolic compounds other than the tocopherol vitamers which could be readily detected and quantitated were vanillic, syringic and ferulic (probably conjugated to glucose) acids along with tyrosol. In contrast to the extra virgin olive oil (793 mg/kg), the concentration of total phenolic compounds is extremely low (<5.0 mg/kg). Nevertheless, argan oil with its high content of the vitamer -tocopherol, squalene and oleic acid is likely to enhance the cancer prevention effects of the Moroccan diet.
Asunto(s)
Neoplasias/prevención & control , Extractos Vegetales/análisis , Sapotaceae/química , Dieta , Ácidos Grasos/análisis , Ácidos Grasos/química , Ácidos Grasos/farmacología , Humanos , Marruecos , Fenoles/análisis , Fenoles/química , Fenoles/farmacología , Extractos Vegetales/farmacología , Aceites de Plantas , Escualeno/análisis , Escualeno/química , Escualeno/farmacología , Esteroles/análisis , Esteroles/química , Esteroles/farmacología , Tocoferoles/análisis , Tocoferoles/química , Tocoferoles/farmacologíaRESUMEN
Polycyclic aromatic hydrocarbons are xenobiotics whose elevated toxicity for living organisms requires to efficiently monitor air pollution, either by evaluating their levels in the environment, or by assessing their biological impacts on sentinel organisms. We investigated the effects of naphthalene exposure on some xenobiotic-metabolising enzyme activities in three organs of Helix aspersa. Particular activities depending on cytochrome P450 (ethoxyresorufin O-deethylase, EROD; ethoxycoumarin O-deethylase, ECOD; pentoxyresorufin O-dealkylase, PROD) and associated with glutathione (glutathione S-transferase, GST; glutathione peroxidase, GPX; glutathione reductase, GR) were assessed. In control animals, the P450-dependent specific activities were distributed according to the range kidney > digestive gland > mantle cavity forming tissues (MCFT). Neither ECOD nor PROD activities could be detected in MCFT. In the two other organs, the major phase I activities were due to ECOD, the level of PROD being very low or null. The glutathione-associated activities showed comparable levels in the three organs, except GPX activity that was higher in the digestive gland. Naphthalene (NAP) exposure did not affect any activity in MCFT, but it significantly decreased EROD and ECOD activities in the kidney as opposed to their increase in the digestive gland, whereas PROD activities were not influenced by the treatment. Glutathione-dependent activities were not significantly affected by NAP exposure, except for GPX which activity diminished in the digestive gland. This study demonstrates that complex detoxification pathways should exist in Helix aspersa as in mammals and that they could be used as potential biomarkers of NAP exposure.
Asunto(s)
Sistema Enzimático del Citocromo P-450/farmacología , Exposición a Riesgos Ambientales , Contaminantes Ambientales/efectos adversos , Caracoles Helix/fisiología , Naftalenos/efectos adversos , Xenobióticos/metabolismo , Animales , Biomarcadores/análisis , Sistema Enzimático del Citocromo P-450/análisisRESUMEN
PURPOSE: The glutathione detoxification pathway includes glutathione S-transferase (GST) and peroxidase (GPX) isoenzymes as well as glutathione reductase (GSSR). Though well established from cultured cancer cell lines, its involvement in resistance is still unclear in the tumours. This study aimed to describe the parameters that influence the glutathione contents and associated activities in breast cancer. METHODS: The components of the glutathione pathway were measured in the tumours from 41 women with primary breast cancer in comparison with those in the matched tumour-free samples. Appropriate statistical studies (regression analysis, Wilcoxon signed rank test) explored the influence of clinical and prognostic factors. RESULTS: Reduced and total glutathione contents were largely increased (P < 0.0001) and all related activities were significantly enhanced in the tumours. Interindividual variations were described, probably due to various parameters (age, menopause, axillary lymph node status, S and G2 + M cell fractions, ER, cathepsin-D and c-ErbB-2 expressions) that influence particular components of the glutathione pathway, especially the glutathione levels. CONCLUSIONS: The breast tumours improved their redox status and detoxification capacities depending on various parameters of significance for cell proliferation and aggressiveness, which supports the involvement of the glutathione pathway in malignant cell resistance to oxidative stress and apoptosis.
Asunto(s)
Neoplasias de la Mama/enzimología , Glutatión/metabolismo , Adulto , Anciano , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Catepsina D/análisis , Resistencia a Antineoplásicos , Femenino , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Inactivación Metabólica , Menopausia , Persona de Mediana Edad , Receptor ErbB-2/análisis , Receptores de Estrógenos/análisisRESUMEN
Glutathione and the associated enzymes, glutathione S-transferases, peroxidases, and reductase, have been implicated in cancer chemoresistance. This pathway was investigated in paired cancerous and peritumoral breast samples from 41 women. The tumours exhibited a higher redox status as deduced from increased transferase, peroxidase, and reductase activities and from higher total and reduced glutathione contents. Several components were strongly correlated in peritumoral tissues, suggesting a highly co-ordinated glutathione pathway that appeared disrupted in breast tumours with only a few correlations left. Therefore, resistance could spontaneously result from deregulated variations in the glutathione pathway, which might be relevant to the malignant disease progression.
Asunto(s)
Neoplasias de la Mama/metabolismo , Mama/metabolismo , Glutatión/metabolismo , Inactivación Metabólica/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Mama/enzimología , Neoplasias de la Mama/enzimología , Femenino , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Persona de Mediana EdadRESUMEN
To maintain the success of recommended methods and to allow comparison among various methods of enzyme analysis, enzyme reference materials are required, having catalytic properties as close as possible to those of the corresponding human enzymes. Though human sources are preferable, ethical reasons require the extraction and purification from animal tissues. By providing theoretically unlimited amounts of material, gene transfer technologies and mass culture can overcome the need of human or mammalian tissues. We have used these technologies to produce human gamma-glutamyltransferase (GGT) and pancreatic lipase (PL) in various types of host cells. Different strategies were tested, especially for GGT, depending on the inherent properties and requirements of the human enzyme. Expression and purification protocols were optimized, yielding good amounts of recombinant enzymes which share many physico-chemical and catalytic features with their natural counterparts. Kinetic constants and catalytic behavior were very similar, demonstrating the usefulness of these products as reference materials. We assume recombinant DNA technologies could be successfully applied to most enzymes or proteins assayed in clinical chemistry laboratories.
Asunto(s)
Enzimas/biosíntesis , Enzimas/genética , Técnicas de Transferencia de Gen , Biosíntesis de Proteínas , Proteínas/genética , Animales , Butiratos/farmacología , Ácido Butírico , Células CHO , Carcinoma Hepatocelular , Cricetinae , Enzimas/normas , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Lipasa/biosíntesis , Lipasa/genética , Páncreas/enzimología , Proteínas/normas , Ratas , Proteínas Recombinantes/biosíntesis , Estándares de Referencia , Saccharomyces cerevisiae/genética , Transfección , Transformación Bacteriana , Células Tumorales Cultivadas , gamma-Glutamiltransferasa/biosíntesis , gamma-Glutamiltransferasa/genética , gamma-Glutamiltransferasa/normasRESUMEN
During the last decade, the unbound glutathione content of cultured adherent cells has become a very important biological marker for many pharmacological and toxicological in vitro studies with regard to the protective role of the tripeptide in its reduced form (GSH). However, the literature does not provide extensive information on the influence of sample preparation on cellular GSH and thiol analyses. Using the fibroblast-like V79 cell line as model, we undertook a comparative study of the efficiency of different procedures reported in the literature with respect to GSH recovery. Depending on the preanalytical step, up to 10-fold discrepancies could be observed in the recovery of intracellular GSH. Different parameters that must be controlled in order to maximize GSH recovery are discussed. The optimal strategy consisted in rapid perchloric acid deproteinization performed directly in the dish, which was extremely valuable for preparing GSH samples from adherent cells, and especially from cells expressing elevated gamma-glutamyl transferase activity.
Asunto(s)
Glutatión/análisis , Animales , Adhesión Celular , Línea Celular , Separación Celular , Cricetinae , Cricetulus , Técnicas Citológicas , Estudios de Evaluación como Asunto , Glutatión/aislamiento & purificación , Glutatión/metabolismo , HumanosRESUMEN
We previously described the establishment of a transfected cell line (V79HGGT) that stably produces the highest recombinant human gamma-glutamyltransferase (GGT) activity. We now report the utilization of V79HGGT as a model system for studying human GGT. The papain-solubilized recombinant enzyme has been highly purified from cultured cells by a new procedure. Studies on the purified enzyme, either by N-terminal sequencing or by characterization of its enzymic activities, confirmed that recombinant GGT shares structural and catalytic identity with native human enzymes. The circular dichroism analysis indicated an alpha-helical content of 19%. Based on these data, we have undertaken a study on the functional consequences of elevated GGT activity on the reduced glutathione (GSH) content. GSH status was followed in V79 and V79HGGT cells throughout growth. A particular pattern was observed for each cell line, depending on, but differentially affected by, alteration of the culture medium. Elevated GGT activity was associated with a 2.5-fold reduced GSH content, clearly suggesting a negative influence of the highly expressed enzyme on the GSH level under normal growth conditions. Possible mechanisms involved are proposed. Our findings pointed out that, among the GSH-related enzymes, GGT could constitute an important factor determining the steady-state content of GSH.
Asunto(s)
Glutatión/metabolismo , gamma-Glutamiltransferasa/metabolismo , Secuencia de Aminoácidos , Boratos/farmacología , Catálisis , División Celular , Línea Celular , Dicroismo Circular , Humanos , Isoxazoles/farmacología , Leucotrieno C4/metabolismo , Leucotrieno D4/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Serina/farmacología , gamma-Glutamiltransferasa/antagonistas & inhibidores , gamma-Glutamiltransferasa/química , gamma-Glutamiltransferasa/aislamiento & purificaciónRESUMEN
gamma-Glutamyltransferase (GGT) is a glutathione-metabolizing enzyme whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse and induction by drugs. To elucidate the implication of GGT in various metabolic pathways, we established a stable transfected V79 cell line highly producing the human GGT. A full length cDNA, encoding the human hepatoma Hep G2 GGT, was subcloned into an expression vector under the control of the SV40 early promoter and was used to transfect V79 cells. A cell line was selected, exhibiting a GGT activity of 2 units per mg of protein, one of the highest levels reported to date. The recombinant GGT purified from this cell line showed the expected heterodimeric structure, with two subunits existing as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. Catalytic features were found to be identical to those of human serum and Hep G2 GGTs. Thus, the newly engineered cell line should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.
Asunto(s)
Línea Celular , gamma-Glutamiltransferasa/genética , Animales , Cricetinae , Cricetulus , ADN Complementario , Humanos , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transfección , Células Tumorales Cultivadas , gamma-Glutamiltransferasa/biosíntesisRESUMEN
Protein reference materials are traditionally prepared by purification from mammalian or human tissues. The supply of these tissues is limited; consequently, there is a growing need for applied molecular and cellular biology technologies for the production of human recombinant proteins. This is especially true when only small amounts of the proteins are available in the tissues. We review the current knowledge necessary for high-level production of such proteins in different heterologous expression systems, using our data on gamma-glutamyltransferase (EC 2.3.2.2) as an example. We describe the steps required to achieve the expression of enzymes and other proteins in Escherichia coli, yeast, or mammalian cells. We list many of the problems investigators may face in preparing recombinant proteins, and provide information on selecting the most appropriate system as well as the most favorable experimental conditions. Depending on the expression system, recombinant proteins can potentially be obtained for most, if not all, enzymes of interest in clinical chemistry, and such proteins should possess characteristics very similar to those of the corresponding human native proteins. Studies suggest that these products can be used as reference materials in clinical chemistry laboratories.
Asunto(s)
Transfección , gamma-Glutamiltransferasa/genética , Animales , Secuencia de Bases , Línea Celular , Escherichia coli/genética , Expresión Génica , Humanos , Proteínas Recombinantes/biosíntesis , Estándares de Referencia , Saccharomyces cerevisiae/genética , gamma-Glutamiltransferasa/biosíntesisRESUMEN
Sodium butyrate was used to enhance biosynthesis rates of recombinant human gamma-glutamyl transferase (GGT) expressed under the control of the SV40 or the cytomegalovirus immediate early promoter, respectively, in transfected V79 and CHO Chinese hamster cell lines. Maximal induction of GGT specific activity in butyrate-treated cells ranged from 3 to 5-fold and resulted from a strong increase in the GGT mRNA ratio. We also observed that maximal transcription level in V79 cells occurred within 12 hr of treatment, whilst the cell proliferation was transiently arrested. Despite its processing requirements, induced GGT exhibited unchanged catalytic and physico-chemical features relative to human serum or hepatoma enzyme, thus appearing as an excellent model for further studies on human GGT.
Asunto(s)
Butiratos/farmacología , gamma-Glutamiltransferasa/biosíntesis , Animales , Ácido Butírico , Células CHO , Línea Celular , Cricetinae , Inducción Enzimática/efectos de los fármacos , Humanos , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Transfección , gamma-Glutamiltransferasa/genética , gamma-Glutamiltransferasa/metabolismoRESUMEN
A rapid, sensitive and selective method for the assay of glutathione in cultured cells has been developed using ion-pair reversed phase rapid high performance liquid chromatography. The use of a 4 micron particle, 5 cm long column (Superspher 100 RP 18 end-capped) allowed complete analysis of glutathione within 3 min. A postcolumn derivatization reaction with o-phthalaldehyde and fluorometric detection made the assay fully selective with regard to other endogenous thiols and sensitive (the detection limit was 0.5 ng of glutathione injected). The linearity range was between 0.1 and 2.0 microgram/mL with good repeatability (relative standard deviation less than 5% for the lowest concentration quantitated). Recoveries of GSH from cultured cell samples were above 98%. The rapid analysis enabled the processing of a large number of samples in a short time (up to 20 per hour). The method was applied to the measurement of the intracellular glutathione amount in V79 fibroblasts along cell growth in culture.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Glutatión/análisis , Animales , Células Cultivadas , Cricetinae , Cricetulus , Fibroblastos/química , Pulmón/química , Pulmón/citología , Reproducibilidad de los Resultados , Espectrometría de FluorescenciaRESUMEN
gamma-Glutamyltransferase (GGT, EC 2.3.2.2) is an enzyme involved in glutathione metabolism and drug and xenobiotic detoxification. Using human hepatoma Hep G2 GGT cDNA as probe, we isolated a cDNA from a human pancreatic cDNA library. Analysis of the nucleotide sequences revealed a 2244-bp insert that includes an open reading frame of 1710 bp, encoding a protein identical to the Hep G2 and human placenta GGTs. Similarly, the 5' untranslated region, though shorter, is highly homologous to that of Hep G2 cDNA. These data suggest strongly that the same gene encodes human GGT in the placenta, Hep G2 and the pancreas. We further studied the distribution of the corresponding mRNA, called type I mRNA, in different human tissues. Using a highly sensitive method associating reverse transcription with specific amplification by polymerase chain reaction, cDNA was synthesized from total RNA isolated from the tissues and GGT specific fragments were amplified. We observed the presence of a specific cDNA fragment corresponding to the type I mRNA in the human tissues and cells tested, providing the evidence for a ubiquitous expression of this GGT mRNA in human tissues.
Asunto(s)
Páncreas/enzimología , gamma-Glutamiltransferasa/química , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular/enzimología , ADN/biosíntesis , ADN/aislamiento & purificación , Sondas de ADN , Feto/enzimología , Expresión Génica , Humanos , Datos de Secuencia Molecular , Placenta/enzimología , Reacción en Cadena de la Polimerasa , ARN/análisisRESUMEN
Cytochrome P-450BM-3 is a catalytically self-sufficient fatty acid omega-hydroxylase with two domains. Functional and primary structure analyses of the hemo- and flavoprotein domains of cytochrome P-450BM-3 and the corresponding microsomal cytochrome P-450 system have shown that these proteins are highly homologous. Prior attempts to reconstitute the fatty acid hydroxylation function of cytochrome P-450BM-3, utilizing the two domains, obtained either by trypsinolysis or by recombinant methods, were unsuccessful. In this paper, we describe the reconstitution of the fatty acid hydroxylation activity of cytochrome P-450BM-3 utilizing the recombinantly produced flavoprotein domain (Oster, T., Boddupalli, S. S., and Peterson, J. A. (1991) J. Biol. Chem. 266, 22718-22725) and its hemoprotein counterpart. The rate of fatty acid-dependent oxygen consumption was shown to be linear when increasing concentrations of the hemoprotein domain are added to a fixed concentration of the flavoprotein domain and vice versa. The combination of the hemo- and flavoprotein domains in a ratio of 20:1 respectively, in the reaction mixture, results in the transfer of 80% of the reducing equivalents from NADPH for the hydroxylation of palmitate at 25 degrees C. The ratio of the regioisomeric products obtained for lauric, myristic, and palmitic acids was similar to that obtained with the holoenzyme form of cytochrome P-450BM-3. The reconstitution of the fatty acid omega-hydroxylase activity, using the soluble domains of cytochrome P-450BM-3, without added factors such as lipids, may be useful for structure/function comparisons to their eukaryotic counterparts.
Asunto(s)
Proteínas Bacterianas , Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Grasos/metabolismo , Flavoproteínas/metabolismo , Hemoproteínas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Secuencia de Bases , Clonación Molecular , Sistema Enzimático del Citocromo P-450/química , Escherichia coli/metabolismo , Flavoproteínas/genética , Hemoproteínas/genética , Hidroxilación , Cinética , Microsomas/enzimología , Oxigenasas de Función Mixta/química , Datos de Secuencia Molecular , NADPH-Ferrihemoproteína Reductasa , Oxígeno/metabolismo , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Comparison of the amino acid sequences of several microsomal cytochrome P-450 reductases to the flavoprotein domain (BMR) of cytochrome P-450BM-3 has revealed that this class of flavoproteins contains evolutionarily conserved regions that are important for their interaction with nucleotide substrates and cofactors. In order to understand the properties of BMR, the region encoding this protein, beginning at residue Lys-472 of cytochrome P-450BM-3, was subcloned and expressed in Escherichia coli. The recombinant protein (more than 50% of host-soluble proteins) was purified to homogeneity using conventional purification procedures. BMR (Mr 66,000) showed typical flavoenzyme absorbance spectra, contained FAD and FMN in a stoichiometry of 1:1, and catalyzed reduction of several artificial electron acceptors with rates comparable to those of the microsomal NADPH-cytochrome P-450 oxidoreductase. Limited trypsinolysis of BMR, under non-denaturing conditions, revealed that the protease removed the NH2-terminal 122 residues. This region was postulated to contain amino acids that are important for FMN binding (Porter, T. D. (1991) Trends Biochem. Sci. 16, 154-158). Consistent with this hypothesis, the major tryptic product of BMR (BMR-52, Mr 52,000) contained only FAD, in an equimolar ratio to the protein. Also, like the FMN-depleted microsomal NADPH-cytochrome P-450 oxidoreductase (Kurzban, G. P., Howarth, J., Palmer, G., and Strobel, H. W. (1990) J. Biol. Chem. 265, 12272-12279), BMR-52 was active for only catalyzing ferricyanide reduction. These data provide strong experimental evidence for a discrete multidomain structure of BMR, as proposed for the membrane-bound reductases, with an amino-terminal FMN binding region and carboxyl-terminal FAD- and NADPH binding regions. Thus, BMR strongly resembles the microsomal cytochrome P-450 reductase and offers an opportunity to better understand the structure-function relationships of this class of flavoproteins.
Asunto(s)
Bacillus megaterium/genética , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Mononucleótido de Flavina/metabolismo , Flavoproteínas/genética , Flavoproteínas/metabolismo , Secuencia de Aminoácidos , Bacillus megaterium/enzimología , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Reductasas del Citocromo/genética , Reductasas del Citocromo/aislamiento & purificación , Reductasas del Citocromo/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Flavoproteínas/aislamiento & purificación , Cinética , Microsomas/enzimología , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Espectrofotometría , TripsinaRESUMEN
gamma-Glutamyltransferase [GGT; (5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2] is a glutathione-metabolizing enzyme, whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse, and induction by xenobiotics. To elucidate the implication of GGT in various metabolic pathways, we established a stable transgenic V79 cell line, highly producing the human GGT. A full-length cDNA, encoding the human hepatoma HepG2 GGT, was subcloned in an expression vector under the control of the simian virus 40 early promoter and was used to transfect V79 cells. We selected a cell line exhibiting a GGT activity of 2 units per mg of protein, the highest GGT expression level reported to date. As described for the human kidney and liver enzymes, the recombinant GGT purified from this cell line showed a heterodimeric structure. Its two subunits existed as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. However, catalytic features were found to be identical to those of human serum and HepG2 GGTs. The newly engineered cell line thus should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.
Asunto(s)
Transfección , gamma-Glutamiltransferasa/genética , Animales , Línea Celular , Clonación Molecular , Cricetinae , Cricetulus , Expresión Génica , Humanos , Immunoblotting , Cinética , Sustancias Macromoleculares , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , gamma-Glutamiltransferasa/aislamiento & purificación , gamma-Glutamiltransferasa/metabolismoRESUMEN
In this paper, we report the characterization of 4 isolated, constitutive cytochrome P-450 fractions from pig liver microsomes. The two predominant forms, A2 and A3, exhibit several similarities: a Mr of 54 kDa, a lambda max CO-Fe++ at 448 nm, a relatively high ratio of the high-spin form and an immunological cross-reaction with polyclonal antibodies against rat liver P-450 IIB1. It is shown that these forms and the minor form Ba, which are active as benzphetamine N-demethylase, play an important metabolic role in ochratoxin A oxidation. This mycotoxin was oxidized by at least 3 different pig liver cytochrome P-450 fractions, each producing different metabolites, namely (4R)-, (4S)-hydroxyochratoxin A, and a new lipophilic metabolite. Since the pig is particularly susceptible to ochratoxin A toxicity, it represents a good animal model for in vitro studies of the metabolism of such a xenobiotic.
Asunto(s)
Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Isoenzimas/aislamiento & purificación , Microsomas Hepáticos/enzimología , Ocratoxinas/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/metabolismo , Isoenzimas/metabolismo , Microsomas Hepáticos/metabolismo , PorcinosRESUMEN
To prepare a reference material for gamma-glutamyltransferase (GGT; EC 2.3.2.2) measurements in clinical chemistry, we constructed different vectors containing either the rat kidney or the human hepatoma Hep G2 GGT cDNA downstream from an inducible promoter for expression in Escherichia coli and Saccharomyces cerevisiae. Transformed bacterial and yeast cells were tested for GGT production by use of Western blot analysis and enzymatic activity measurements. Both rat renal and Hep G2 GGT cDNAs were expressed in E. coli, producing active and nonglycosylated enzymes localized in the periplasmic space. Recombinant Hep G2 GGT was synthesized as a single-chain protein, unlike rat renal GGT, which presented two polypeptides of 62 and 30 kDa, identified as the precursor and a GGT heavy-subunit-like peptide, respectively. Rat renal GGT was produced in S. cerevisiae as two polypeptides, 55 and 30 kDa, detected by antisera against rat renal GGT. These results suggest maturation mechanisms such as glycosylation and cleavage steps, enhancing the interest of S. cerevisiae as a useful expression system for producing active mammalian proteins as reference materials.
Asunto(s)
ADN/genética , Escherichia coli/genética , Saccharomyces cerevisiae/genética , gamma-Glutamiltransferasa/biosíntesis , Animales , Western Blotting , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Vectores Genéticos , Glicosilación , Humanos , Riñón/enzimología , Ratas , Proteínas Recombinantes/biosíntesis , Células Tumorales Cultivadas/enzimología , gamma-Glutamiltransferasa/genéticaRESUMEN
After screening different human hepatoma cell lines, we observed that both HepG2 and PLC/PRF/5 naturally produced large amounts of gamma-glutamyltransferase. We optimized HepG2 cell culture conditions and observed that higher cell densities were obtained when cells were cultured on microcarriers, particularly when Cytodex 3 was used and that cell growth was optimal when DMEM, the basic medium, was supplemented with 5% fetal calf serum and 6 mmol/l glutamine. These culture conditions allowed us to produce the highest amounts of GGT after about 150 h of culture. The GGT obtained from HepG2 cells was partially purified and some of its physico-chemical properties characterized. Successive Con A gel chromatography separated the activity into two peaks, suggesting that GGT from HepG2 is not uniformly glycosylated. Papain-treated HepG2 GGT showed a Mr of about 120 kDa and migrated as a single-chain protein in SDS-PAGE. Immunological and kinetic properties of the GGT were similar to other human GGTs (liver, kidney and serum). It appears that HepG2 GGT could be a source for the preparation of a human enzyme reference material.