RESUMEN
Oxidative activity of 9-anthraldehyde (9-AA) to 9-anthracenecarboxylic acid in monkey liver was mainly located in microsomes. The reaction required NADPH as an essential cofactor and was significantly inhibited by SKF 525-A, metyrapone, disulfiram, and CO, potent inhibitors of microsomal aldehyde oxygenase (MALDO). Two cytochrome P450 isozymes, named P450JM-A and P450JM-C, which mediate the oxidative biotransformation of 9-AA were purified from hepatic microsomes of untreated male and female Japanese monkeys, respectively. These isozymes each showed a single band of molecular mass 51,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal amino acid sequences of P450JM-A and P450JM-C are highly homologous with those of several P450s belonging to the CYP2A and CYP2B subfamilies, respectively. The anti-P450JM-C antibody significantly suppressed 9-AA MALDO activity in monkey liver, but anti-P450JM-A antibody did not. The antibody against CYP2C11, which is a major isozyme responsible for 9-AA MALDO in male rat liver, also inhibited the activity. These results indicate that P450JM-C and isozyme(s) immunologically related to CYP2C11 predominantly possess MALDO activity toward 9-AA.
Asunto(s)
Antracenos/metabolismo , Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Sistema Enzimático del Citocromo P-450/metabolismo , Hígado/enzimología , Oxigenasas/aislamiento & purificación , Oxigenasas/metabolismo , Secuencia de Aminoácidos , Animales , Citocromo P-450 CYP2A6 , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/química , Familia 2 del Citocromo P450 , Inhibidores Enzimáticos/farmacología , Femenino , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Macaca , Macaca mulatta , Masculino , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Datos de Secuencia Molecular , Peso Molecular , NAD/metabolismo , NADP/metabolismo , Oxidación-Reducción , Oxigenasas/antagonistas & inhibidores , Oxigenasas/química , Análisis de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The most abundant metabolite of delta 9-tetrahydrocannabinol (delta 9-THC) formed with hepatic microsomes of monkeys was 11-OH-delta 9-THC, followed by 8 alpha-OH-, 8 beta-OH- and 3'-OH-delta 9-THCs. Two cytochrome P450 isozymes, P450RM-A and P450JM-C, were purified from monkey hepatic microsomes and found to have a molecular weight of 51,000. In the reconstituted system, the activities of P450RM-A towards formation of 11-OH-, 8 alpha-OH-, 8 beta-OH- and 3'-OH-delta 9-THCs were 19-, 40-, 22- and 10-fold higher, respectively, than the corresponding activities of the hepatic microsomes. The activity of P450JM-C towards formation of 3'-OH-delta 9-THC was 10-fold higher than that of P450RM-A, while the activities of both isozymes for 11- and 8 alpha-hydroxylation were not so much different and the 8 beta-hydroxylation activity was 14-fold higher in P450RM-A than in P450JM-C. Antibodies against P450RM-A and P450JM-C markedly inhibited the microsomal formation of 11-OH- and 8 alpha-OH-delta 9-THCs, and 3'-OH-delta 9-THC, respectively. These results suggest that P450RM-A and P450JM-C are major isozymes responsible for the formation of 11-OH- and 8 alpha-OH-delta 9-THCs, and 3'-OH-delta 9-THC, respectively, in monkeys.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Dronabinol/metabolismo , Isoenzimas/metabolismo , Microsomas Hepáticos/metabolismo , Secuencia de Aminoácidos , Animales , Sistema Enzimático del Citocromo P-450/química , Femenino , Isoenzimas/química , Macaca , Macaca mulatta , Masculino , Datos de Secuencia Molecular , Oxidación-ReducciónAsunto(s)
Humanos , Femenino , Preescolar , Hipersensibilidad a los Alimentos , Vigna , Alérgenos , Glycine max , Técnicas de Laboratorio ClínicoRESUMEN
Microsomal aldehyde oxygenase (MALDO) activity for 9-anthraldehyde (9-AA) was significantly higher in the male than in the female adult rat liver. 9-AA MALDO activity was also significantly enhanced by pretreatment with dexamethasone and phenobarbital, whereas it was not significantly changed by 3-methylcholanthrene or acetone. Several cytochrome P450 isozymes purified from rat hepatic microsomes were able to catalyze the oxidation of 9-AA to 9-anthracene carboxylic acid (9-ACA) in the presence of NADPH, NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. Under the ordinary conditions of the reconstituted system, the catalytic activities (nmol/min/nmol P450) of cytochrome P450s, 2A1, 2B2, 2C6, 2C11 and 3A2 were 1.53 (1.37 in the presence of cytochrome b5), 1.20 (2.06), 4.87 (7.75), 18.0 (21.6) and 0.90 (1.17), respectively. Cytochrome P450 2C11 (CYP 2C11) showed the highest catalytic activity of the cytochromes examined. In the reconstituted system using the lipids extracted from microsomes, CYP 3A2 more effectively catalyzed the oxidation of 9-AA to 9-ACA, and its catalytic activity (nmol/min/nmol P450) was 3.33 or 6.61 in the absence or presence of cytochrome b5, respectively. The antibody against CYP 2C11 inhibited by 90% the hepatic microsomal oxidation of 9-AA MALDO activity in adult male rats, but the activity was not inhibited by antibody against CYP 3A2. These results show that the individual forms of cytochrome P450 have a catalytic activity for the oxidation of 9-AA to 9-ACA, and that CYP 2C11 is the major constitutive catalyst of 9-AA MALDO activity in untreated adult male rat liver.