RESUMO
Diacylglycerol (DG) lipase, which hydrolyses 1-stearoyl-2-arachidonyl-sn-glycerol to produce an endocannabinoid, 2-arachidonoylglycerol, was purified from the soluble fraction of rat brain lysates. DG lipase was purified about 1,200-fold by a sequential column chromatographic procedure. Among proteins identified by mass spectrometry analysis in the partially purified DG lipase sample, only DDHD domain containing two (DDHD2), which was formerly regarded as a phospholipase A1, exhibited significant DG lipase activity. Rat DDHD2 expressed in Chinese hamster ovary cells showed similar enzymatic properties to partially purified DG lipase from rat brain. The source of DG lipase activity in rat brain was immunoprecipitated using anti-DDHD2 antibody. Thus, we concluded that the DG lipase activity in the soluble fraction of rat brain is derived from DDHD2. DDHD2 is distributed widely in the rat brain. Immunohistochemical analysis revealed that DDHD2 is expressed in hippocampal neurons, but not in glia.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Hipocampo/enzimologia , Lipase Lipoproteica , Proteínas do Tecido Nervoso , Neurônios/enzimologia , Animais , Ácidos Araquidônicos/genética , Ácidos Araquidônicos/metabolismo , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , Endocanabinoides/genética , Endocanabinoides/metabolismo , Glicerídeos/genética , Glicerídeos/metabolismo , Lipase Lipoproteica/biossíntese , Lipase Lipoproteica/química , Lipase Lipoproteica/genética , Lipase Lipoproteica/isolamento & purificação , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Neuroglia/enzimologia , Domínios Proteicos , RatosRESUMO
CASE HISTORY: A 3-month-old infant was found dead in his bed. A postmortem computed tomography (CT) scan suggested fatty attenuation in the liver parenchyma, but no other potentially fatal changes were found. To clarify the cause of death, a medicolegal autopsy was carried out. AUTOPSY FINDINGS: Internal examination confirmed the presence of liver steatosis as well as hepatomegaly. There were no other significant findings including encephalitis or brain edema. MASS SPECTROMETRY ANALYSIS: To clarify the mechanism underlying lipid accumulation in the liver, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) analysis was conducted. This indicated a significant accumulation of C14:1 acylcarnitine in the liver of the deceased, suggesting very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency. GENETIC ANALYSIS: To find the cause of the VLCAD deficiency, genetic analysis of the responsible gene, acyl-CoA dehydrogenase, very long chain (ACADVL), was performed. This revealed two novel mutations that may have accounted for the disease. CONCLUSION: A combination of these data revealed that the liver steatosis in this case might have been caused by VLCAD deficiency based on genetic mutations of ACADVL. Thus, the deceased might have been vulnerable to energy crisis and sudden infant death. The present findings show that MALDI-IMS analysis as well as genetic analysis can be useful for elucidating the cause of death.
Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Fígado Gorduroso/genética , Erros Inatos do Metabolismo Lipídico/diagnóstico , Doenças Mitocondriais/diagnóstico , Doenças Musculares/diagnóstico , Acil-CoA Desidrogenase de Cadeia Longa/genética , Povo Asiático/genética , Sequência de Bases , Síndrome Congênita de Insuficiência da Medula Óssea , Fígado Gorduroso/patologia , Genética Forense , Patologia Legal , Hepatomegalia/patologia , Humanos , Lactente , Japão , Masculino , Dados de Sequência Molecular , Mutação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca(2+) for its maximum activity. The K(M) values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 microM, and the V(max) values were 27.3 and 1.62 micromol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.
Assuntos
Tioléster Hidrolases/metabolismo , Linhagem Celular , Cromatografia Líquida , Humanos , Cinética , Lisofosfatidilcolinas/metabolismo , Lisofosfolipase/metabolismo , Espectrometria de Massas , Proteínas Mutantes/metabolismo , Peptídeos/química , Transporte Proteico , Frações Subcelulares/enzimologia , Tioléster Hidrolases/isolamento & purificaçãoRESUMO
Monoclonal antibodies have been elicited against an achiral rhodium complex and this complex was used in the presence of a resultant antibody, 1G8, for the catalytic hydrogenation of 2-acetamidoacrylic acid to produce N-acetyl-L-alanine in high (>98%) enantiomeric excess.