RESUMEN
Up-regulation of detoxifying enzymes in insecticide-resistant strains of the house fly is a common mechanism for metabolic resistance. However, the molecular basis of this increased insecticide metabolism is not well understood. In the multiresistant Rutgers strain, several cytochromes P450 and glutathione S-transferases are constitutively overexpressed at the transcriptional level. Overexpression is the result of trans-regulation, and a regulatory gene has been located on chromosome 2. A Gly137 to Asp point mutation in alphaE7 esterase gene, leading to the loss of carboxylesterase activity, has been associated with organophosphate resistance in the house fly and the sheep blowfly. We show here that purified recombinant CYP6A1 is able to detoxify diazinon with a high efficiency. We also show that either the Gly137 to Asp point mutation in alphaE7 esterase gene or a deletion at this locus confer resistance and overproduction of the CYP6A1 protein. Based on these findings, we propose it is the absence of the wild-type Gly137 allele of the alphaE7 gene that releases the transcriptional repression of genes coding for detoxification enzymes such as CYP6A1, thereby leading to metabolic resistance to diazinon.
Asunto(s)
Hidrolasas de Éster Carboxílico/genética , Sistema Enzimático del Citocromo P-450/biosíntesis , Diazinón/metabolismo , Moscas Domésticas/enzimología , Alelos , Animales , Carboxilesterasa , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Diazinón/química , Diazinón/farmacología , Femenino , Genes de Insecto , Ligamiento Genético , Glicina/genética , Moscas Domésticas/efectos de los fármacos , Moscas Domésticas/genética , Resistencia a los Insecticidas/genética , Masculino , Mutación PuntualRESUMEN
Recombinant house fly (Musca domestica) cytochrome P450 reductase has been purified by anion exchange and affinity chromatography. Steady-state kinetics of cytochrome c reductase activity revealed a random Bi-Bi mechanism with formation of a ternary P450 reductase-NADPH-electron acceptor complex as catalytic intermediate. NADP(H) binding is essential for fast hydride ion transfer to FAD, as well as for electron transfer from FMN to cytochrome c. Reduced cytochrome c had no effect on the enzyme activity, while NADP+ and 2'-AMP inhibited P450 reductase competitively with respect to NADPH and noncompetitively with respect to cytochrome c. The affinity of the P450 reductase to NADPH is 10 times higher than to NADP+ (Kd of 0.31 and 3.3 microM, respectively). Such an affinity change during catalysis could account for a +30 mV shift of the redox potential of FAD. Cys560 was substituted for Tyr by site-directed mutagenesis. This mutation decreased enzyme affinity to NADPH 35-fold by decreasing the bimolecular rate constant of nucleotide binding with no detectable effect on the kinetic mechanism. The affinity of the C560Y mutant enzyme to NADP+ decreased 9-fold compared to the wild-type enzyme, while the affinity to 2'-AMP was not significantly affected, suggesting that Cys560 is located in the nicotinamide binding site of the active, full-size enzyme in solution.
Asunto(s)
Moscas Domésticas/enzimología , NADPH-Ferrihemoproteína Reductasa/metabolismo , Animales , Cisteína/genética , Grupo Citocromo c/metabolismo , Electrones , Cinética , Mutagénesis Sitio-Dirigida , NADP/metabolismo , NADPH-Ferrihemoproteína Reductasa/genética , NADPH-Ferrihemoproteína Reductasa/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Tirosina/genéticaRESUMEN
P450c21 catalyzes an important step in steroid synthesis. Its deficiency leads to symptoms of steroid imbalance. To obtain enough P450c21 for structure and function studies, we developed a method to express P450c21 in Escherichia coli. The 5'-region of the human P450c21 cDNA was modified to ensure efficient translation and the C terminus of the protein was extended with four His residues for easy purification. Mutant proteins with substitutions at residues 172 and 281 exhibited decreased enzymatic activities similar to those found in mammalian cells. One new mutation changing Glu-380 to Asp (D380) caused 3-fold reduction in enzymatic activity. The amount of apoprotein production detected by immunoblotting and the affinity of the mutant protein towards substrate as measured by Km were normal. The defect lies in the decreased ability of the apoprotein to bind heme, which was measured by CO difference and substrate-binding spectra. The D380 mutant protein had 3-fold reduction in peak heights in both spectra. This reduced heme binding resulted in 3-fold lower enzymatic activity.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Escherichia coli/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Escherichia coli/genética , Humanos , Cinética , Mutación , Plásmidos , Alineación de Secuencia , Esteroide 21-HidroxilasaRESUMEN
Eukaryotic P450 proteins are membrane proteins found predominantly in the endoplasmic reticulum. In vertebrates, several biosynthetic P450s are found in mitochondria as well. We cloned three putative insect mitochondrial P450s from larval house fly cDNA. These P450s are members of a new P450 family, CYP12. The CYP12 proteins are most closely related to the mammalian mitochondrial P450 of the CYP11, CYP24, and CYP27 families. The most abundant cDNA, CYP12A1, was expressed in Escherichia coli and purified. NADPH-dependent reduction of CYP12A1 was rapid and efficient with the bovine mitochondrial proteins adrenodoxin reductase and adrenodoxin as electron transfer partners. In contrast, house fly microsomal NADPH cytochrome P450 reductase reduced CYP12A1 only poorly. In a reconstituted system with the bovine mitochondrial electron donors, CYP12A1 metabolized a variety of insecticides and other xenobiotics, but did not metabolize ecdysteroids, juvenoids, or fatty acids. Subcellular localization of CYP12A1 by immunogold histochemistry established the mitochondrial nature of this protein. CYP12A1 mRNA levels are constitutively higher in an insecticide-resistant strain than in a susceptible strain, and this trait maps to chromosome II in the house fly, where the constitutive overexpression of the pesticide-metabolizing microsomal CYP6A1 also maps. Multiple mitochondrial P450s have evolved in insects and may play a role in the metabolism of xenobiotics in addition to their possibly ancestral functions in steroidogenesis.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Mitocondrias/enzimología , Secuencia de Aminoácidos , Animales , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Activación Enzimática , Moscas Domésticas , Resistencia a los Insecticidas/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Especificidad por SustratoRESUMEN
The cytochrome P450 gene Cyp6a2 from Drosophila melanogaster is located on the right arm of chromosome 2 at position 43A1-2 and comprises two exons separated by a 69-bp intron. Phenobarbital treatment of flies leads to a rapid increase in the level of CYP6A2 mRNA and to an increased production of the CYP6A2 protein. DNA from the Cyp6a2 promoter region was functional when linked to a luciferase reporter gene and transfected into D. melanogaster Schneider cells. Moreover, a dose-dependent induction of luciferase activity by phenobarbital indicated that elements necessary for phenobarbital induction are located within 428 bp of the translation start site. Heterologous expression of the CYP6A2 protein in lepidopteran cells infected with a Cyp6a2-recombinant baculovirus was observed by Western blotting of cell lysates and by spectral characterization of the reduced-CO complex of the P450. The CYP6A2 protein produced in this system metabolized aldrin and heptachlor to their epoxides and metabolized the insecticide diazinon by desulfuration to diazoxon and by oxidative ester cleavage to 2-isopropyl-4-methyl-6-hydroxypyrimidine. Metabolism in lysates of cells infected with recombinant baculovirus was greatly enhanced by the addition of purified housefly NADPH cytochrome P450 reductase and cytochrome b5. These results show that CYP6A2 catalyzes the metabolism of organophosphorus insecticides and they implicate Cyp6a2 overexpression in metabolic resistance. The Cyp6a2 gene appears to be a suitable model for a genetic analysis of the phenobarbital induction process.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Drosophila melanogaster/enzimología , Expresión Génica , Fenobarbital/farmacología , Aldrín/farmacología , Secuencia de Aminoácidos , Animales , Baculoviridae , Secuencia de Bases , Catálisis , Sistema Enzimático del Citocromo P-450/biosíntesis , Familia 6 del Citocromo P450 , ADN/química , Proteínas de Drosophila , Drosophila melanogaster/genética , Inducción Enzimática , Vectores Genéticos , Heptacloro/farmacología , Moscas Domésticas/enzimología , Moscas Domésticas/genética , Insecticidas/farmacología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Regiones Promotoras GenéticasRESUMEN
Active human cytochrome P-450c21 was expressed in Escherichia coli and purified to homogeneity. To increase expression, cDNA encoding for the N-terminal fragment of cytochrome P-450c21 was modified. Four histidine codons were added to cDNA encoding for the C-terminus of the protein; thus, recombinant protein could have been rapidly and effectively purified by metal-affinity chromatography. Modified human cytochrome P-450c21 was expressed (40-50 nmoles/l of culture according to spectrophotometry) which was able to bind to bacterial membrane. Modifications of N- and C-terminal regions of cytochrome P-450c21 did not change Km and Vmax for hydroxylation of progesterone and 17 alpha-hydroxyprogesterone in reconstituted system. Recombinant cytochrome P-450c21 was purified to apparent homogeneity from Escherichia coli membrane extract by metal-affinity chromatography. Purified cytochrome P-450c21 migrates as a single 54 kD band on polyacrylamide gel and exhibits type I spectral changes during interaction with progesterone and 17 alpha-hydroxyprogesterone. Activity of purified cytochrome P-450c21 was reconstituted with mouse liver microsomal NADPH-cytochrome P-450-reductase and NADPH-regenerating system. Purified enzyme had Km 12.2 and 3.21 microM and Vmax 192.9 and 198 nmoles/min/nmole of P-450c21 for 17 alpha-hydroxyprogesterone and progesterone, respectively. According to titration spectra, dissociation constants for progesterone and 17 alpha-hydroxy-progesterone were 14.7 and 31.1 microM, respectively.
Asunto(s)
Cromatografía de Afinidad/métodos , Sistema Enzimático del Citocromo P-450/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/metabolismo , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Ratones , Datos de Secuencia Molecular , Plásmidos , Esteroide 21-Hidroxilasa , Especificidad por SustratoRESUMEN
A microsomal cytochrome b5 cDNA from the house fly, Musca domestica, was cloned and sequenced. The deduced amino acid sequence of the full-length house fly cytochrome b5 (134 residues) is 48% identical to that of rat microsomal cytochrome b5. The house fly cytochrome b5 protein was overexpressed in Escherichia coli, purified, and characterized. Absorption and EPR spectroscopy reveal properties very similar to cytochromes b5 from vertebrates. NMR spectra indicate that the orientation of the heme in the protein relative to its alpha,gamma meso axis is about 1:1. A redox potential of -26 mV versus standard hydrogen electrode was measured by cyclic voltammetry on a modified gold electrode in the presence of hexamminechromium(III) chloride. The cytochrome b5 is reduced by house fly cytochrome P450 reductase in a reconstituted system at a high rate (5.5 s-1), and it stimulates heptachlor epoxidation when reconstituted with house fly cytochrome P450 reductase, cytochrome P450 6A1, phospholipid, and detergent. Cytochrome b5 decreases the apparent Km for P450 reductase and increases the Vmax for heptachlor epoxidation at constant cytochrome P450 6A1 concentrations. The results indicate that cytochrome b5 stimulates a step following the first electron transfer during cytochrome P450 6A1 turnover.
Asunto(s)
Citocromos b5/genética , Dípteros/enzimología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Sistema Enzimático del Citocromo P-450/metabolismo , Citocromos b5/química , Citocromos b5/metabolismo , ADN Complementario , Escherichia coli/genética , Cinética , Datos de Secuencia Molecular , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Análisis EspectralRESUMEN
We have investigated the structure and function of P450c21 with regard to a conserved site around Ile-172 by site-directed mutagenesis making single amino acid substitutions of residues 169 173. Substitutions of Ile-171 and -172 resulted in production of mutant proteins with dramatic reductions in enzymatic activities, indicating the importance of these two residues in maintaining the structure and function of P450c21. The I171N protein was present at a slightly lower level, due to a decreased rate of protein synthesis. The I172N apoprotein was synthesized at the normal rate, but its heme-bound P450 form was present at a much lower level. This I172N protein was tightly integrated into the membrane of endoplasmic reticulum, similar to the wild type P450c21, as shown by immunofluorescence detection, alkaline extraction, and cellular fractionation. Kinetic studies indicated that I172N had a lower Vmax value. In addition, the I172N protein was more sensitive to proteinase K digestion, indicating a possible alteration of conformation. This conformational change may result in the lower yield of the I172N hemoprotein and the reduced catalytic activity.
Asunto(s)
Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Isoleucina , Mutación Puntual , Conformación Proteica , Secuencia de Aminoácidos , Animales , Bovinos , Secuencia Conservada , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Cinética , Ratones , Microsomas/enzimología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , Esteroide 21-HidroxilasaRESUMEN
Steroid 21-hydroxylase (P450c21) deficiency is the major cause of a common genetic disease, congenital adrenal hyperplasia, with the symptoms of virilization due to steroid imbalance. We have devised a fast diagnostic method to detect common mutations in the c21B gene by a two-step gene amplification procedure coupled to restriction digestion. This procedure does not require isotopes and is suitable for routine use in a hospital setting. In addition, we have developed a procedure for the production of active P450c21 in E. coli. We tested many different vector and bacterial strain combinations to find out the best condition for P450c21 expression. The bacteria harboring the P450c21 expression plasmid were grown in a rich media supplemented with trace metals, heme biosynthesis precursor delta-levulinic acid, and induced with IPTG at 20 degrees C for 48 h. We found that low growth temperature and long induction time were important for abundant synthesis of P450c21 in E. coli.
Asunto(s)
Hiperplasia Suprarrenal Congénita/genética , Regulación Enzimológica de la Expresión Génica/fisiología , Esteroide 21-Hidroxilasa/fisiología , Secuencia de Bases , Escherichia coli , Humanos , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Esteroide 21-Hidroxilasa/genéticaRESUMEN
To elucidate the general principles of the structural organization and functioning among mitochondrial and microsomal steroidogenic cytochromes P-450, a comparative study of the molecular organization of microsomal cytochrome P-450c21 and mitochondrial cytochrome P-450scc from adrenal cortex has been performed using immunochemical approaches and limited proteolysis. An efficient method to isolate and purify cytochrome P-450c21 has developed and the antibodies against the hemoprotein raised and characterized. Limited trypsinolysis of cytochrome P-450c21 leads to the formation of two fragments tightly bound to the microsomal membrane. Proteolytic modification is accompanied by spectral changes and the decreases of the catalytic activity. Possible models of the molecular organization of cytochrome P-450c21 are discussed in comparison with mitochondrial cytochrome P-450scc and liver microsomal cytochrome P-450.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Isoenzimas/metabolismo , Corteza Suprarrenal/enzimología , Animales , Bovinos , Cromatografía en Gel , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Inmunodifusión , Membranas Intracelulares/metabolismo , Isoenzimas/aislamiento & purificación , Microsomas/enzimología , Mapeo Peptídico , Relación Estructura-ActividadRESUMEN
The possibility of functioning of microsomal steroid hydroxylases in the adrenal cortex in the presence of the electron-transporting proteins in mitochondrial monooxygenase systems has been studied. It was found that adrenodoxin reductase stimulates both the 21- and 17 alpha-steroid hydroxylation activities in microsomes of the adrenal cortex with the same efficiency as does NADPH-dependent cytochrome P-450 reductase. Using cytochrome b5 and its specific antibody, an attempt has been made to investigate possible mechanisms of the stimulating effect of adrenodoxin reductase.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Microsomas/metabolismo , Mitocondrias/metabolismo , Oxigenasas de Función Mixta/metabolismo , Esteroides/metabolismo , Animales , Bovinos , Citocromos b5/metabolismo , Transporte de Electrón , Hidroxilación , Microsomas/enzimología , Mitocondrias/enzimología , NADPH-Ferrihemoproteína Reductasa/metabolismo , Esteroide Hidroxilasas/metabolismo , Esteroides/biosíntesisRESUMEN
To overcome the adverse consequences of antibody immobilization (impairment of the antigen-binding parameters) the interaction of a number of antigens with both soluble and covalently immobilized antibodies in the presence of water-soluble polymers has been investigated. Incorporation of some water-soluble polymers (Ficoll, Dextran) into the assay mixture was shown to substantially increase the antigen-binding capacity of both soluble and immobilized antibodies. The use of dextran T70 enhanced the sensitivity of competitive radioimmunoassays for CEA and beta 2-microglobulin 3-4-fold. This procedure may be also applied to shorten the incubation period when performing an assay.
Asunto(s)
Inmunoensayo/métodos , Animales , Reacciones Antígeno-Anticuerpo , Antígeno Carcinoembrionario/inmunología , Técnicas de Inmunoadsorción , Técnicas In Vitro , Polímeros , Conejos , Radioinmunoensayo/métodos , Solubilidad , Agua , Microglobulina beta-2/inmunologíaRESUMEN
Chemical modification of different amino acid residues and the carbohydrate moiety of antibodies to carcino-embryonic antigen (CEA) was used to elucidate their role in the interaction with CEA and to evaluate the effect of antibody modification and steric factor in immunosorbent synthesis. The distribution of the modified groups among the structural fragments of IgG and the levels of changes in the antigen-binding properties of modified antibodies were investigated. The Fc-fragment of IgG was shown to contain carboxylic groups, tyrosine and histidine residues, whose modification influences the antigen-binding properties as a result of generalized conformational changes in the IgG molecule. A comparison of these results with earlier obtained data on the functional properties, of immobilized antibodies revealed that the decrease of antigen-binding characteristics of anti-CEA after IgG immobilization via NH2- and COOH-groups, carbohydrate moiety, tyrosine and histidine residues is due to the direct effect of antibody modification, whereas the changes in parameters of antibody interaction with antigen after IgG immobilization via SH-groups, methionine and histidine residues is due to steric hindrances.
Asunto(s)
Anticuerpos/análisis , Antígeno Carcinoembrionario/inmunología , Inmunoglobulina G/análisis , Aminoácidos/análisis , Animales , Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo , Sitios de Unión de Anticuerpos , Carbohidratos/análisis , Fenómenos Químicos , Química , Fragmentos Fab de Inmunoglobulinas/análisis , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fc de Inmunoglobulinas/análisis , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , ConejosRESUMEN
To obtain highly efficient immunosorbents for solid-phase immunoassay and affinity chromatography, methods for immobilization of antibodies against the carcino-embryonic antigen (CEA) on insoluble matrices were optimized. The immunosorbents obtained were characterized by equilibrium parameters of the reaction between immobilized anti-CEA and CEA calculated from rather a simple kinetic model. This model describes the interaction of the monovalent antigen with two independent types of binding sites. The role of some amino acid residues of anti-CEA in the interaction with CEA was investigated. The effects of immobilization density and the spacer arm length on the functional properties of the immobilized antibodies were studied. The optimal immunosorbent was used to purify 125I-CEA by immunoaffinity chromatography.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Reacciones Antígeno-Anticuerpo , Antígeno Carcinoembrionario/inmunología , Cromatografía de Afinidad , Histidina/análisis , Humanos , Inmunoglobulina G/aislamiento & purificación , Técnicas de Inmunoadsorción , Compuestos de Sulfhidrilo/análisis , Tirosina/análisisRESUMEN
Methods for immobilization of anti-immunoglobulins on insoluble supports were optimized, and the interaction of immunoadsorbents obtained with [125I]-labeled rabbit IgG was investigated. It was shown that this interaction can be adequately described by a rather simple equilibrium model which reflects the interaction of a monovalent antigen with two independent types of binding sites. Within the framework of this model the association constants as well as the concentrations of high affinity binding sites which influence the capacity and efficiency of the separation system were determined. Optimization of the immobilization methods implicated a study on the role of certain functional groups of the antibody involved in the formation of covalent bonds, on the effect of the spacer arm length on the properties of immobilized antibody as well as on the role of the degree of immobilization. It was found that immunoadsorbents obtained after antibody immobilization via lysine or tyrosine residues on matrices with a specific spacer group are the optimal ones.