RESUMEN
Molecular mechanisms determining the turn-over rate and specificity of catechol O-methylation were studied by combining enzyme kinetic measurements, computational modeling of substrate properties and fitting ligands in a 3D model of the active site of the enzyme. Enzyme kinetic measurements were carried out for 46 compounds, including most clinically used catechol drugs, by using recombinant human soluble catechol O-methyltransferase (COMT). The most important mechanism decreasing the turnover rate and increasing affinity was the electron withdrawing effect of substituents. Several other mechanisms by which substituents affected reactivity and affinity were identified. Highest turnover rates were determined for unsubstituted catechol and pyrogallol. Pyrogallol derivatives generally seemed to be more specific substrates than catechols. Catecholestrogens were the most specific endogenous substrates, whereas catecholamines were rather poor substrates. Among the catechol drugs used in the L-DOPA treatment of Parkinson's disease, the COMT inhibitors entacapone and tolcapone were not methylated, whereas the DOPA decarboxylase inhibitor benserazide was 15 times more specific substrate than L-DOPA, the target of COMT inhibition. The structure-activity relationships found allow the prediction of reactivity, affinity, and specificity with useful accuracy for catechols with a wide range of structures and properties. The knowledge can be used in the evaluation of metabolic interactions of endogenous catechols, drugs and dietary catechols, and in the designing of drugs with the catechol pharmacophore.
Asunto(s)
Catecol O-Metiltransferasa/metabolismo , Catecoles/metabolismo , Sitios de Unión , Unión Competitiva , Catecol O-Metiltransferasa/química , Inhibidores de Catecol O-Metiltransferasa , Catecoles/química , Humanos , Cinética , Metilación , Modelos Moleculares , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
A single nucleotide polymorphism at the nucleotide 1947 in the catechol-O-methyltransferase (COMT) gene encodes the high and low activity forms of the enzyme. We investigated COMT genotypes of 73 Korean patients with Parkinson's disease (PD), 29 with multiple system atrophy (MSA), and 49 controls, and analyzed the response to levodopa challenge in the PD patients. We found no significant difference in the distribution of the COMT genotypes among the three groups. The frequencies of the G- and A-alleles in the total population were 75 and 25%, respectively. The levodopa response was determined by a single oral levodopa challenge test with Sinemet (25/250 mg) in the patients with PD. The motor response evaluated by the time to peak response, the duration and magnitude of the response in the motor part of the Unified Parkinson's Disease Rating Scale; tapping or walking times showed no significant difference between the genotypes. Thus, pharmacokinetic or pharmacodynamic factors other than the investigated genetic variant of the COMT enzyme seem to determine the response to levodopa in PD.
Asunto(s)
Antiparkinsonianos/administración & dosificación , Catecol O-Metiltransferasa/genética , Levodopa/administración & dosificación , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Adulto , Anciano , Femenino , Genotipo , Humanos , Corea (Geográfico) , Masculino , Persona de Mediana Edad , Atrofia de Múltiples Sistemas/tratamiento farmacológico , Atrofia de Múltiples Sistemas/genética , Polimorfismo de Nucleótido SimpleRESUMEN
Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), also known as autoimmune polyglandular syndrome Type I (APS1), is an autosomal recessive autoimmune disease caused by mutations in a gene designated as AIRE (autoimmune regulator). Here we have studied the expression of Aire in transfected cell lines and in adult mouse tissues. Our results show that Aire has a dual subcellular location and that it is expressed in multiple immunologically relevant tissues such as the thymus, spleen, lymph nodes, and bone marrow. In addition, Aire expression was detected in various other tissues such as kidney, testis, adrenal glands, liver, and ovary. These findings suggest that APECED protein might also have a function(s) outside the immune system.(J Histochem Cytochem 49:197-208, 2001)
Asunto(s)
Poliendocrinopatías Autoinmunes/metabolismo , Fracciones Subcelulares/metabolismo , Factores de Transcripción/metabolismo , Animales , Western Blotting , Clonación Molecular , Cricetinae , ADN Complementario/genética , Expresión Génica , Haplorrinos , Humanos , Inmunohistoquímica , Hibridación in Situ , Ratones , Mutación , Especificidad de Órganos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Transfección , Proteína AIRERESUMEN
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is an autoimmune disease with autosomal recessive inheritance. APECED is characterized by the breakdown of tolerance to several organ-specific selfantigens. The symptoms of APECED fall into three main categories: autoimmune polyendocrinopathies, chronic mucocutaneous candidiasis, and ectodermal dystrophies. The gene defective in APECED, AIRE, has been cloned and numerous mutations in this gene have been found in patients with APECED. AIRE is predicted to encode a 545-amino-acid protein containing structural domains characteristic for transcription regulators. The protein has been shown to act as a transcriptional activator in vitro. The AIRE protein is mainly localized to the nucleus, where it can be detected as speckles resembling nuclear bodies. In humans, the expression of AIRE has been observed predominantly in immunologically relevant tissues, especially the thymus. Recently, we have shown in the mouse that Aire is also expressed in various tissues and cell types outside the immune system.
Asunto(s)
Poliendocrinopatías Autoinmunes/etiología , Animales , Femenino , Expresión Génica , Genes Recesivos , Humanos , Masculino , Ratones , Mutación , Poliendocrinopatías Autoinmunes/genética , Poliendocrinopatías Autoinmunes/metabolismo , Distribución Tisular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína AIRERESUMEN
Human UDP-glucuronosyltransferases (UGTs) 1A6 and 1A9 were expressed using Semliki Forest virus (SFV) vectors. Infection of chinese hamster lung fibroblast V79 cells with recombinant SFV-UGT viruses resulted in efficient protein expression as detected by metabolic labeling, Western blot analyses and immunofluorescence microscopy. The expression of UGT 1A6 and UGT1A9 in the SFV-infected cells was approximately two fold higher than in a stable V79 cell line. No UGT signal was detected in noninfected cells. In addition, SFV-UGT viruses also efficiently infected other mammalian cells, such as baby hamster kidney (BHK), chinese hamster ovary (CHO) and human lung (WI-26 VA4) cells leading to high production of recombinant enzyme. The measurement of enzyme activities and kinetic parameters using p-nitrophenol and nitrocatechol (entacapone) as substrates for UGT1A6 and UGT1A9, respectively, showed that the overall kinetic properties of the enzymes produced by the two systems were similar. We conclude that the SFV expression system represents an efficient, fast and versatile method for production of metabolic enzymes for in vitro assays.
Asunto(s)
Expresión Génica , Glucuronosiltransferasa/biosíntesis , Glucuronosiltransferasa/genética , Virus de los Bosques Semliki/genética , Animales , Células CHO/enzimología , Células CHO/virología , Catecoles/metabolismo , Células Cultivadas , Cricetinae , Cartilla de ADN/química , Fibroblastos/enzimología , Fibroblastos/virología , Vectores Genéticos , Humanos , Riñón/enzimología , Riñón/virología , Pulmón/enzimología , Pulmón/virología , Nitrilos , Nitrofenoles/metabolismo , ARN Mensajero/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus de los Bosques Semliki/enzimología , Especificidad por Sustrato , Transfección , UDP Glucuronosiltransferasa 1A9RESUMEN
Catechol-O-methyltransferase (COMT) catalyses the O-methylation of neurotransmitters, catechol hormones and drugs such as levodopa and methyldopa. Ethnic differences in COMT activity have been observed in several populations. Previous studies suggest that the g1947G>A low activity allele is less common in individuals of African origin. COMT genotyping was performed using a mini-sequencing method in 195 healthy Ghanaians with a frequency of the homozygous g1947G>A of 6%. This study provides confirmation that the low activity COMT allele is less common in individuals of African origin. This finding may be important clinically with regards to the treatment of many neuropsychiatric disorders and in the pathophysiology of various human disorders including estrogen-induced cancers, Parkinson's disease, depression and hypertension.
Asunto(s)
Alelos , Catecol O-Metiltransferasa/genética , Catecol O-Metiltransferasa/metabolismo , Frecuencia de los Genes/efectos de los fármacos , Adolescente , Adulto , Asia Occidental/epidemiología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Femenino , Ghana/epidemiología , Humanos , Kenia/epidemiología , Levodopa/farmacología , Masculino , Metildopa/farmacología , Persona de Mediana EdadRESUMEN
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autosomal disease with recessive inheritance. It is characterized by multiple autoimmune endocrinopathies, chronic mucocutaneous candidiasis, and ectodermal dystrophies. The defective gene responsible for this disease was recently isolated, and several different mutations in the novel gene, AIRE, have been identified, by us and by others, in patients with APECED. We have shown that the APECED protein is mainly localized, both in vitro and in vivo, to the cell nucleus, where it forms distinct speckles. This accords with the predicted structural features of the protein, which suggest involvement of AIRE in the regulation of gene transcription. Here, we report the results of mutational analyses of a series of 112 patients with APECED who were from various ethnic backgrounds. A total of 16 different mutations, covering 91% of disease alleles, were observed; of these, 8 were novel. The mutations are spread throughout the coding region of AIRE, yet four evident mutational hotspots were observed. In vitro expression of four different naturally occurring nonsense and missense mutations revealed a dramatically altered subcellular location of the protein in cultured cells. Interestingly, the wild-type APECED protein tethered to the Gal4 DNA-binding domain acted as a strong transcriptional activator of reporter genes in mammalian cells, whereas most of the analyzed mutant polypeptides had lost this capacity.
Asunto(s)
Mutación/genética , Poliendocrinopatías Autoinmunes/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Alelos , Animales , Transporte Biológico , Línea Celular , Codón sin Sentido/genética , Citoplasma/metabolismo , Etnicidad/genética , Exones/genética , Femenino , Genes Reporteros/genética , Haplotipos/genética , Humanos , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Mutación Missense/genética , Poliendocrinopatías Autoinmunes/metabolismo , Polimorfismo Conformacional Retorcido-Simple , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/química , Proteína AIRERESUMEN
The levels of 26 kDa-soluble (S) and 30 kDa-membrane-bound (MB) catechol-O-methyltransferase (COMT) polypeptides were determined in paired samples from normal and neoplastic breast tissue of 32 patients with breast cancer. Immunohistochemical staining showed that the COMT reaction in normal mammary tissue was restricted to the epithelial cells in the ducti and lobuli, whereas in the tumors a strong reaction was also seen in the malignant cells. The amounts of COMT proteins in tumors could not be correlated with various clinical or pathological parameters. Quantitative immunoblotting analysis revealed that the total amount of COMT proteins in tumors was more than 50% higher than in respective normal samples in 26 out of 32 patients. Five cases showed less than a 50% difference and in one case less COMT was detected in the tumor. In most cases the amount of both S- and MB-COMT forms was increased. The average amount of total COMT was 178 +/- 57 pg/microg total protein in normal tissue and 566 +/- 94 pg/microg total protein in tumor. Respective values for S-COMT were 137 +/- 52 pg/microg total protein in normal tissue and 369 +/- 62 pg/microg total protein in tumor and for MB-COMT 41 +/- 10 and 197 +/- 41 pg/microg total protein, respectively. Analysis of COMT-specific transcripts suggested that the COMT enzyme level in tumors is determined in some cases by transcriptional and in some cases by post-transcriptional mechanisms.
Asunto(s)
Neoplasias de la Mama/enzimología , Mama/enzimología , Catecol O-Metiltransferasa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/patología , Catecol O-Metiltransferasa/genética , Femenino , Humanos , Immunoblotting , Inmunohistoquímica , Persona de Mediana Edad , ARN Mensajero/análisisRESUMEN
Gelsolin, an actin-modulating protein, derived from a single gene exists in intracellular and secreted forms. A point mutation at position 187 of both forms of gelsolin causes familial amyloidosis of the Finnish type (FAF). Here, we expressed both isoforms of the wild-type and FAF mutant gelsolin in mouse embryonic gelsolin-null fibroblasts. We demonstrate that the FAF mutation does not interfere with the normal actin-modulating function of intracellular gelsolin, and that aberrant processing of secreted FAF gelsolin to FAF amyloid precursor takes place in the gelsolin-negative background. These results suggest that, in patients with FAF, symptoms are caused by the accumulation in their tissues of amyloid derived from plasma gelsolin and are not due to functional differences in cytoplasmic gelsolin.
Asunto(s)
Actinas/metabolismo , Amiloidosis/metabolismo , Fibroblastos/metabolismo , Gelsolina/genética , Gelsolina/metabolismo , Amiloidosis/genética , Animales , Células Cultivadas , Ratones , Ratones Noqueados , MutaciónRESUMEN
Autoimmune-polyendocrinopathy-candidiasis-ecto-dermaldystrophy (APECED) is the only systemic autoimmune disease with a monogenic background known so far revealing no association with the major histocompatibility complex region. We have recently isolated the gene defective in this syndrome and characterized several different mutations in individuals with the disorder. The novel gene, AIRE, contains a putative bipartite nuclear targeting signal predicting a nuclear location of the corresponding protein. The presence of two PHD-type zinc finger domains as well as the newly described putative DNA-binding domain, SAND, in the amino acid sequence of the APECED protein implies that it may be involved in the regulation of gene expression. Using transient expression of AIRE cDNA in mammalian cells we demonstrate here the nuclear location of the APECED protein. Immunohistochemical staining of transfected cells revealed that most of the recombinant 58 kDa APECED protein is present in the form of nuclear dots. By double immuno-fluorescence labelling we further show that these APECED-containing structures and the previously described PML nuclear bodies are largely non-overlapping. The AIRE protein was also visualized in multiple human tissues: a subset of the cells in thymus, in spleen and in lymph node showed nuclear staining with APECED antiserum. Immunofluorescence labelling of peripheral blood mononuclear leukocytes also revealed a nuclear body-like staining pattern in a fraction of these cells. These data from both in vitro and ex vivo systems, together with the predicted structural features of the APECED protein, suggest that this protein is most probably involved in the regulation of gene expression.
Asunto(s)
Núcleo Celular/química , Factores de Transcripción/análisis , Animales , Células CHO , Línea Celular , Cricetinae , ADN Complementario/genética , Expresión Génica , Células HeLa , Humanos , Inmunohistoquímica , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , Proteínas Recombinantes de Fusión/genética , Distribución Tisular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Proteínas Supresoras de Tumor , Proteína AIRERESUMEN
A new chromatographic catechol O-methyltransferase (COMT) assay based on S-adenosyl-L-[methyl-14C]methionine and on-line radioactivity detection was developed. With minor modifications in the mobile phase composition the methylation velocities for 30 structurally diverse compounds including simple catechols, neurotransmitters, catecholestrogens and catecholic drugs could be measured using human and rat recombinant soluble COMT. The enzymes showed very similar substrate selectivities. The radiochemical method was validated using 3,4-dihydroxybenzoic acid as a model substrate and it was shown that accurate and reproducible methylation velocity values could be achieved for both of the catecholic hydroxyls. The method proved to be suited for determining the enzyme kinetic parameters and can probably be further used for gathering enzyme kinetic data on differentially substituted catechols in order to construct proper structure-activity relationships for COMT.
Asunto(s)
Catecol O-Metiltransferasa/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Animales , Anticarcinógenos/metabolismo , Radioisótopos de Carbono , Humanos , Hidroxibenzoatos/metabolismo , Cinética , Metilación , Ratas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Especificidad por SustratoRESUMEN
Uridine diphosphate (UDP)-glucuronosyltransferases (UGTs), presently called UDP-glycosyltransferases, catalyse the detoxification of many toxic and carcinogenic compounds. Glucuronidation is also a major metabolic pathway for numerous drugs. The UGT1A6 gene (formerly known as UGT1*06 and UGT1A1) has been suggested to belong to the aryl hydrocarbon (Ah) gene battery, which consists of several genes encoding for drug-metabolising enzymes regulated by dioxin and other ligands of the Ah receptor. In this study, we analysed the localisation of UGT1A6 expression in rat liver by in situ hybridisation to mRNA. Two different RNA probes were used, one which was specific to UGT1A6 and the other against the C terminal sequence shared by all UGT1 genes. In this study, no UGT1A6 mRNA was detected in the control animals. However, other gene(s) of the UGT1 family were expressed in the perivenous region surrounding the central veins as detected by hybridisation with the probe against the common region of the UGT1 genes. Treatment with the lower dose (5 mg/kg) of 3-methylcholanthrene (3MC) induced expression of UGT1A6 perivenously. Treatment with the higher dose (25 mg/kg) of 3-Methylcholanthrene resulted in a more panacinar expression pattern. In contrast to the perivenous induction observed with 3-methylcholanthrene, treatment with 15 mg/kg of beta-naphthoflavone (BNF) resulted in strong induction in the periportal region. The results reveal an inducer-specific pattern of UGT1A6 expression similar to that demonstrated earlier for other Ah battery genes, namely CYP1A1, CYP1A2, GSTYalpha and ALDH3. The finding further supports the notion that common factors regulate the regional hepatic expression of Ah battery genes.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucuronosiltransferasa/genética , Hígado/efectos de los fármacos , Metilcolantreno/farmacología , Familia de Multigenes , beta-naftoflavona/farmacología , Animales , Hibridación in Situ , Hígado/enzimología , Masculino , ARN Mensajero/biosíntesis , Ratas , Ratas WistarRESUMEN
Catechol O-methyltransferase (COMT) inactivates neurotransmitters, hormones and drugs such as levodopa. COMT activity is inherited in an autosomal recessive manner and individuals with low activity have thermolabile COMT protein. A low activity allele has been demonstrated at codon 108/158 of the soluble and membrane bound COMT protein, respectively, whereby a G to A transition results in a valine to methionine substitution, rendering the protein more thermolabile. As ethnic differences in erythrocyte COMT activity have been previously demonstrated, the frequency of low activity alleles were investigated in 265 British Caucasian, 99 British South-west Asian and 102 Kenyan individuals. Genotyping of COMT codon 108/158 was performed using a minisequencing method. Erythrocyte COMT activity was measured in 60 British Caucasian individuals by radiochemical assay. The frequency of low activity alleles was 0.54 in Caucasians, 0.49 in South-west Asians, and 0.32 in Kenyans. There was a much lower frequency of individuals with homozygous low activity allele in the Kenyan population (9%) than in Caucasians (31%) or South-west Asians (27%). Erythrocyte COMT activity was lower and less thermostable in individuals with homozygous low activity alleles. The data provide molecular evidence that low COMT is less common in African individuals than the Caucasian population.
Asunto(s)
Población Negra/genética , Catecol O-Metiltransferasa/genética , Población Blanca/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Codón , Eritrocitos/enzimología , Femenino , Frecuencia de los Genes , Genotipo , Humanos , India/etnología , Kenia , Masculino , Persona de Mediana Edad , Pakistán/etnología , EscociaRESUMEN
The three-dimensional structure of calcium-loaded regulatory, i.e. N-terminal, domain (1-91) of human cardiac troponin C (cNTnC) was determined by NMR in water/trifluoroethanol (91:9 v/v) solution. The single-calcium-loaded cardiac regulatory domain is in a "closed" conformation with comparatively little exposed hydrophobic surface. Difference distance matrices computed from the families of Ca2+-cNTnC, the apo and two-calcium forms of the skeletal TnC (sNTnC) structures reveal similar relative orientations for the N, A, and D helices. The B and C helices are closer to the NAD framework in Ca2+-cNTnC and in apo-sNTnC than in 2.Ca2+-sNTnC. However, there is an indication of a conformational exchange based on broad 15N resonances for several amino acids measured at several temperatures. A majority of the amides in the alpha-helices and in the calcium binding loop exhibit very fast motions with comparatively small amplitudes according to the Lipari-Szabo model. A few residues at the N and C termini are flexible. Data were recorded from nonlabeled and 15N-labeled samples, and backbone dynamics was investigated by 15N T1, T2, and heteronuclear nuclear Overhauser effect as well as by relaxation interference measurements.
Asunto(s)
Miocardio/química , Troponina C/química , Secuencia de Aminoácidos , Calcio/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , SolucionesRESUMEN
The catechol-O-methyltransferase (COMT) gene occurs as two polymorphic alleles, which code for a high activity thermostable and low activity thermolabile form of the enzyme. We devised a fast solid-phase minisequencing assay for genotyping the COMT gene at nucleotide position 544 encoding amino acid residue 158. The method was applied to correlate the genotype of the COMT gene with the biological activity of the COMT enzyme. In red blood cells from individuals homozygous for G at nucleotide position 544 coding for Val-158, the activity of COMT ranged from 0.55-1.03 pmol min-1 mg-1 protein, and in individuals homozygous for A at position 544 coding for Met-158, the activity ranged from 0.21-0.43 pmol min-1 mg-1. Heterozygotes showed intermediate activities of 0.20-0.88 pmol min-1 mg-1. The thermostability (heated/unheated) at 48 degrees C of the high activity form was shown to be about two-fold compared to that of the low activity form of the enzyme. By analysing 76 individual samples and three pooled samples representing altogether 3140 individuals using the solid-phase minisequencing method, the two COMT alleles were shown to be equally distributed in the Finnish population. No statistically significant difference in the frequencies of the COMT alleles was found when comparing the normal population with a sample of 158 Finnish patients with Parkinson's disease.
Asunto(s)
Catecol O-Metiltransferasa/genética , Frecuencia de los Genes , Variación Genética , Enfermedad de Parkinson/genética , Polimorfismo Genético , Catecol O-Metiltransferasa/química , Catecol O-Metiltransferasa/metabolismo , ADN/sangre , Cartilla de ADN , Estabilidad de Enzimas , Eritrocitos/enzimología , Femenino , Finlandia , Genotipo , Calor , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Valores de ReferenciaRESUMEN
The intracellular localization of soluble and membrane-bound isoforms of rat and human catechol O-methyltransferase (COMT) was studied by expressing the recombinant COMT proteins either separately or together in mammalian cell lines (HeLa and COS-7 cells) and in rat primary neurons. The distribution of soluble and membrane-bound COMT enzyme was visualized by immunocytochemistry. For comparison, the localization of native COMT was studied in rat C6 glioma cells by immunoelectron microscopy. Staining of cells expressing membrane-bound COMT with a COMT-specific antiserum revealed an immunofluorescence signal in intracellular reticular structures and in the nuclear membrane. Double-staining of the cells with antisera against proteins specific for the rough endoplasmic reticulum indicated that they colocalized with membrane-bound COMT, suggesting that it resided in the endoplasmic reticulum. Notably, no COMT-specific fluorescence of plasma membranes was detected. The signal in the endoplasmic reticulum was also evident in the cells expressing both recombinant COMT forms. Intracellular native COMT reaction was detected by immunoelectron microscopy in rat C6 glioma cells and an intense cytoplasmic signal was seen in the primary neurons infected with the recombinant Semliki Forest virus. The cells expressing recombinant soluble COMT revealed intense nuclear staining together with diffuse cytoplasmic immunoreactivity, suggesting that a part of soluble COMT is transported to nuclei. Western blotting from rat liver and brain revealed soluble COMT in the nuclei. Enzyme activity measurements from liver cytoplasmic and nuclear fractions suggested that about 5% of the soluble COMT resided in nuclei. The intracellular localization of both COMT forms implies that COMT acts in the cytoplasm and possibly also in the nuclear compartment, and that the physiological substrates of COMT enzymes may have to be internalized before their methylation by COMT.
Asunto(s)
Catecol O-Metiltransferasa/metabolismo , Animales , Células CHO , Células COS , Catecol O-Metiltransferasa/genética , Compartimento Celular , Núcleo Celular/enzimología , Cricetinae , Citoplasma/enzimología , Células HeLa , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Mutagénesis Sitio-Dirigida , Ratas , Ratas Wistar , Proteínas Recombinantes/metabolismoRESUMEN
The protein purification strategies used for obtaining homogeneous rat and human soluble catechol-O-methyltransferase (S-COMT) polypeptides are reviewed. Expression and purification of recombinant rat and human S-COMT in Escherichia coli and for human S-COMT in baculevirus-infected insect cells made it possible to elucidate the S-COMT polypeptides in more detail. The application of these purification methods has allowed the crystallization of the rat S-COMT protein and the analysis of the kinetic properties of the enzyme in great detail. The availability of the pure S-COMT protein together with the structural data has also greatly enhanced the development of more potent COMT inhibitors.
Asunto(s)
Catecol O-Metiltransferasa/aislamiento & purificación , Expresión Génica/genética , Secuencia de Aminoácidos , Animales , Western Blotting , Catecol O-Metiltransferasa/análisis , Catecol O-Metiltransferasa/química , Catecol O-Metiltransferasa/genética , Cromatografía Liquida/métodos , Electroforesis en Gel de Poliacrilamida , Humanos , Hígado/enzimología , Datos de Secuencia Molecular , Placenta/enzimología , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The localization of catechol-O-methyltransferase immunoreactivity in rat dorsal root ganglia and in the spinal cord and its co-existence with substance P, calcitonin gene-related peptide and fluoride-resistant acid phosphatase in dorsal root ganglion cells was examined with immunohistochemical and histochemical double-staining methods. Analysis of dorsal of dorsal root ganglia at both cervical and lumbar levels revealed catechol-O-methyltransferase immunoreactivity in numerous dorsal root ganglion cells. Double-staining studies showed that catechol-O-methyltransferase and substance P immunoreactivities were located in different cells with a few exceptions, whereas both catechol-O-methyltransferase and calcitonin gene-related peptide immunoreactivities were detected in about 10% of all labeled cells positive for one of the two markers at both levels studied. The great majority of fluoride-resistant alkaline phosphatase-positive cells were also immunoreactive for catechol-O-methyltransferase. Again, no difference was found between cervical and lumbar levels. Catechol-O-methyltransferase immunoreactivity was also found in the neuropil of the dorsal horn of the spinal cord. The staining was most intense in the superficial laminae (I-III) and overlapped partly with substance P and calcitonin gene-related peptide immunoreactivity. Western blotting analysis revealed that soluble catechol-O-methyltransferase was the clearly dominating form of the enzyme in dorsal root ganglia. The distribution pattern of catechol-O-methyltransferase in dorsal horn and sensory neurons suggests that the enzyme may modulate sensory neurotransmission.
Asunto(s)
Catecol O-Metiltransferasa/metabolismo , Ganglios Sensoriales/enzimología , Ganglios Espinales/enzimología , Fosfatasa Ácida/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Western Blotting , Péptido Relacionado con Gen de Calcitonina/metabolismo , Ganglios Sensoriales/ultraestructura , Ganglios Espinales/ultraestructura , Inmunohistoquímica , Masculino , Microscopía Inmunoelectrónica , Ratas , Ratas Wistar , Sustancia P/metabolismoRESUMEN
Previous biochemical and histochemical studies have suggested that catechol-O-methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers, COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18-day-old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double-staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, flat cells displaying type-1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type-2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane-bound 28-kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.