title: Monkey hepatic microsomal alcohol oxygenase: purification and characterization of a cytochrome P450 enzyme catalyzing the stereoselective oxidation of 7alpha- and 7beta-hydroxy-delta8-tetrahydrocannabinol to 7-oxo-delta8-tetrahydrocannabinol.
abstract: The formation of 7-oxo-delta8-tetrahydrocannabinol (7-Oxo-delta8-THC) from 7alpha- or 7beta-hydroxy-delta8-THC (7alpha- or 7beta-OH-delta8-THC) was found in hepatic microsomes of monkeys. The activity in 7beta-OH-delta8-THC was stereoselectively 2.5- to 4.6-fold higher than that from 7alpha-OH-delta8-THC. The oxidative activities of 7alpha- and 7beta-OH-delta8-THC to 7-Oxo-delta8-THC were inhibited to 35% and 10%, respectively, of the control value by the antibody against P450GPF-B (CYP3A), a major enzyme responsible for the formation of 7-Oxo-delta8-THC in guinea pigs. In the Lineweaver-Burk double-reciprocal plot analysis, testosterone 6beta-hydroxylase activity was competitively inhibited by 7beta-OH-delta8-THC. Two cytochrome P450 enzymes, called P450JM-D and P450JM-E, were purified from hepatic microsomes of Japanese monkeys. P450JM-E, assumed to be CYP3A8, immunologically reacted with the antibody against P450GPF-B and showed high forming activity of 7-Oxo-delta8-THC from 7-OH-delta8-THC. On the other hand, 7-Oxo-delta8-THC forming activity of P450JM-D, assumed to be CYP2C, was less than 10% of that of P450JM-E (CYP3A8). Oxygen-18 (18O) derived from atmospheric oxygen was incorporated into about 40% of the corresponding ketone formed from 7alpha-OH-delta8-THC or 8beta-OH-delta9-THC by P450JM-E (CYP3A8), although the incorporation of the stable isotope into the oxidized metabolite from 7beta-OH-delta8-THC or 8alpha-OH-delta9-THC was negligible. These results indicate that P450JM-E (CYP3A8) is a major enzyme of the oxidation of 7-OH-delta8-THC in monkey hepatic microsomes. The oxidation mechanism may proceed as follows: the alpha- and beta-epimers of 7-OH-delta8-THC or 8-OH-delta9-THC may be converted to ketone through dehydration of an enzyme-bound gem-diol by P450JM-E (CYP3A8), although this stereoselective dehydration differentiates between two epimers.
authors.affiliation: Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Japan.
authors.forename: Tamihide
authors.initials: T
authors.lastname: Matsunaga
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Iwawaki
authors.forename: Akiko
authors.initials: A
authors.lastname: Komura
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Watanabe
authors.forename: Takashi
authors.initials: T
authors.lastname: Kageyama
authors.forename: Ikuo
authors.initials: I
authors.lastname: Yamamoto
mesh_terms: D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D013759:Dronabinol / Q000031:analogs & derivatives* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006213:Hallucinogens / Q000378:metabolism*; D066298:In Vitro Techniques; D008251:Macaca; D008253:Macaca mulatta; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D012727:Sex Characteristics; D013045:Species Specificity; D013237:Stereoisomerism
chemical_list: D019138:Antibodies, Blocking; D006213:Hallucinogens; D013759:Dronabinol; D003577:Cytochrome P-450 Enzyme System; D000429:Alcohol Oxidoreductases; C113764:Cyp2c13 protein, rat