RESUMO
Acyl-CoA hydrolase could provide a mechanism via its potency to modulate cellular concentrations of acyl-CoAs for the regulation of various cellular events including fatty acid metabolism and gene expression. However, only limited evidence of this is available. To better understand the physiological role of this enzyme, we characterized a mouse brain acyl-CoA hydrolase, mBACH. The cloned cDNA for mBACH encoded a 338-amino-acid polypeptide with >95% identity to the human and rat homologs, indicating that the BACH gene is highly conserved among species. This was supported by the similarity in genomic organization of the BACH gene between humans and mice. Bacterially expressed mBACH was highly active against long-chain acyl-CoAs with a relatively broad specificity for chain length. While palmitoyl-CoA hydrolase activity was widely distributed in mouse tissues, it was marked in the brain, consistent with mBACH being almost exclusively distributed in this tissue, where >80% of the enzyme activity was explained by mBACH present in the cytosol. Immunohistochemistry demonstrated a neuronal localization of mBACH in both the central and peripheral nervous systems. In neurons, mBACH was distributed throughout the cell body and neurites. Although four isoforms except mBACH itself, that may be generated by the alternative use of exons of a single mBACH gene, were cloned, their mRNA levels in the brain were estimated to be negligible. However, a 50-kDa polypeptide besides the major one of 43-kDa seemed to be translated from the mBACH mRNA with differential in-frame ATG triplets used as the initiation codon. These findings will contribute to the functional analysis of the BACH gene using mice including genetic studies.
Assuntos
Córtex Cerebral/enzimologia , Camundongos/genética , Proteínas do Tecido Nervoso/genética , Palmitoil-CoA Hidrolase/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Córtex Cerebral/citologia , Clonagem Molecular , Códon de Iniciação/genética , Citosol/enzimologia , DNA Complementar/genética , Éxons/genética , Feminino , Gônadas/enzimologia , Isoenzimas/genética , Masculino , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Peso Molecular , Miocárdio/enzimologia , Proteínas do Tecido Nervoso/fisiologia , Neuritos/enzimologia , Neuroblastoma/patologia , Especificidade de Órgãos , Palmitoil-CoA Hidrolase/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Células Tumorais Cultivadas , Vísceras/enzimologiaRESUMO
Acyl-CoA hydrolases cleave acyl-CoA thioesters to free fatty acids and coenzyme A. The potency of these enzymes may serve to modulate cellular levels of acyl-CoAs to affect various cellular functions, including lipid metabolism. In this study, we investigated the tissue distribution of this multigene family of enzymes, focusing on cytosolic (CTE-I) and mitochondrial acyl-CoA thioesterases (MTE-I) in adult rats, using an anti-CTE-I antibody which recognizes both the isoforms. Western blotting detected them mainly in organs closely related to fatty acid oxidation, of which kidney contained the highest levels of both enzymes. Immunohistochemistry localized the enzymes primarily in the proximal tubules, where a large energy demand is expected and fatty acids represent a major fuel, correlating well with the intrarenal distribution of peroxisomal beta-oxidation. In situ hybridization suggested colocalization of CTE-I and MTE-I in the kidney. The immunoreactivity was also found in various epithelial tissues in the body, including Harderian gland and sebaceous gland. These results demonstrated the distribution of CTE-I and MTE-I in a wide variety of rat tissues, primarily characterized by an epithelial localization, being consistent with their involvement in fatty acid metabolism.