Microsomal Alcohol Oxygenase: Purification and Characterization of a Cytochrome P450 Responsible for Oxidation of 7-Hydroxy-Δ8-tetrahydrocannabinol to 7-Oxo-Δ8-tetrahydrocannabinol in Guinea Pig Liver

Matsunaga, Tamihide; Tanaka, Hiroyuki; Komura, Akiko; Watanabe, Kazuhito; Yamamoto, Ikuo; Yoshimura, Hidetoshi

doi:10.1006/abbi.1997.0390

Cited by 10 publications

(13 citation statements)

References 43 publications

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“…6,7,[29][30][31] The P450-dependent monooxygenase system preferentially requires NADPH as a source of reducing equivalents; the NADPH-P450 reductase transfers two electrons from this nucleotide to the P450. In the presence of NADPH, however, NADH can function as the source of the second electron, transferred to the P450 through cytochrome b 5 reductase, following incorporation of the oxygen to the enzyme-substrate complex.…”

Section: Discussionmentioning

confidence: 99%

“…[2][3][4] We have previously reported that a hepatic microsomal enzyme, named microsomal alcohol oxygenase (MALCO), is able to oxidize 7a-and 7b-hydroxy-D 8 -THC to 7-oxo-D 8 -THC in the presence of NADPH and molecular oxygen. 5,6) We have purified CYP3A8 as a major enzyme of MALCO from hepatic microsomes of monkey.7) The activity was stereoselective and the rate of conversion of 7b-hydroxy-D 8 -THC to 7-oxo-D 8 -THC was higher than that from 7a-hydroxy-D 8 -THC. 7,8) In general, the P450-dependent monooxygenase system has an absolute requirement for NADPH.…”

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“…5,6) We have purified CYP3A8 as a major enzyme of MALCO from hepatic microsomes of monkey. 7) The activity was stereoselective and the rate of conversion of 7b-hydroxy-D 8 -THC to 7-oxo-D 8 -THC was higher than that from 7a-hydroxy-D 8 -THC.…”

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Effective NADH-Dependent Oxidation of 7.BETA.-Hydroxy-.DELTA.8-tetrahydrocannabinol to the Corresponding Ketone by Japanese Monkey Hepatic Microsomes

Matsunaga

Higuchi

Watanabe

et al. 2005

Biological & Pharmaceutical Bulletin

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Tetrahydrocannabinol (THC) is a major psychoactive constituent of marijuana, Cannabis sativa L. It is well known that THC is oxidized to a number of metabolites in the liver of mammals.1) Recent studies have clarified that P450 plays a major role in the oxidation of THC. [2][3][4] We have previously reported that a hepatic microsomal enzyme, named microsomal alcohol oxygenase (MALCO), is able to oxidize 7a-and 7b-hydroxy-D 8 -THC to 7-oxo-D 8 -THC in the presence of NADPH and molecular oxygen. 5,6) We have purified CYP3A8 as a major enzyme of MALCO from hepatic microsomes of monkey.7) The activity was stereoselective and the rate of conversion of 7b-hydroxy-D 8 -THC to 7-oxo-D 8 -THC was higher than that from 7a-hydroxy-D 8 -THC. 7,8) In general, the P450-dependent monooxygenase system has an absolute requirement for NADPH. A few studies have indicated that NADH can also accelerate the oxidative metabolism of p-nitrophenetole 9) and p-nitroanisole, 10,11) and the bioactivation of 2-acetylaminofluorene 12) and nitrosoamines. 13,14) During our studies on the microsomal oxidative metabolism of D 8 -THC, we observed that 7b-hydroxy-D 8 -THC was effectively biotransformed to 7-oxo-D 8 -THC by using NADH as a cofactor, without NADPH (Fig. 1).The present study was undertaken to define the basic characteristics of the microsomal NADH-mediated oxidation of 7b-hydroxy-D 8 -THC to 7-oxo-D 8 -THC in the liver of monkeys and compare it to the NADPH-dependent systems. -THC-4Ј-oic acid 17) were prepared by the methods previously reported. The purity of the cannabinoids was verified to be more than 98% by gas chromatography. Microsomal lipids were extracted from hepatic microsomes with chloroform-methanol (2 : 1) and the solvent was evaporated to dryness in vacuo. Other chemicals and solvents used were of the highest quality commercially available. MATERIALS AND METHODS MaterialsAnimals and Preparation of Microsomes Liver samples from Japanese monkeys were provided by the Primate Research Institute, Kyoto University (Inuyama, Japan). Liver tissue homogenized using a Teflon-homogenizer was centrifuged to obtain the microsomes as described previously. 18)The microsomal pellets obtained were suspended in 100 mM potassium phosphate buffer (pH 7.4) containing 20% glycerol and 5 mM EDTA.Enzyme Assays In the study of the microsomal fraction, * To whom correspondence should be addressed. The NADH-dependent activity by hepatic microsomes of Japanese monkeys for 7-oxo-D D

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Effective NADH-Dependent Oxidation of 7.BETA.-Hydroxy-.DELTA.8-tetrahydrocannabinol to the Corresponding Ketone by Japanese Monkey Hepatic Microsomes

Matsunaga

Higuchi

Watanabe

et al. 2005

Biological & Pharmaceutical Bulletin

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“…Samples containing 1 mg protein were diluted in an equal volume of sample buffer and separated on 10% polyacrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as previously reported. 23,24) SUPERSOMES expressed CYP3A7 (BD Genetest, Franklin Lakes, NJ, U.S.A.) was used as the reference standard. Western blotting analysis was carried out according to the method reported previously.…”

Section: Methodsmentioning

confidence: 99%

Cytochrome P450 3As Gene Expression and Testosterone 6.BETA.-Hydroxylase Activity in Human Fetal Membranes and Placenta at Full Term

Maezawa

Matsunaga

Takezawa

et al. 2010

Biological & Pharmaceutical Bulletin

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Cytochrome P450 represent a superfamily of heme proteins that play a critical role in the oxidative metabolism of xenobiotics and endogenous substances, and there are several isoforms. The human CYP3A subfamily contains mainly three isoforms, CYP3A4, CYP3A5, and CYP3A7.3)CYP3A4 is a major isoform that accounts for about 30% of the amount of total CYP in human adult livers and is responsible for approximately 50% of the metabolism by CYPs of known drugs in humans. 4,5) CYP3A5 is expressed polymorphically throughout development in the liver.6,7) CYP3A7 is a major CYP isoform in the human fetal and newborn liver. 8)It is known that the human placenta contains multiple CYP isoforms in the mitochondria and endoplasmic reticulum of trophoblastic cells.9) The type and amount of expressed CYPs varies depending on the period of gestation and maternal health status. 10,11) In general, it appears that the placenta expresses a wider variety of CYP isoforms in the first trimester than at term. 12,13) Thus it has been suggested that xenobiotic metabolism in the placenta may be feto-protective during the critical embryogenic and organogenesis stage prior to the second trimester, when it is most susceptible to the effects of teratogens, the expression of CYP mRNAs is maximal.14) However, the central nervous system develops throughout the pregnancy and may also be affected in the later stages of pregnancy. Even though, as described above, various studies of enzymes related to pharmacokinetics in human placenta have been reported, there is only limited research in fetal membranes. The fetal membranes, which extend from the placenta, are an important barrier between the fetus and mother. The membranous structure that surrounds the developing fetus and forms the amniotic cavity is derived from fetal tissue except for decidua and is composed of three layers: amnion (inner layer), chorion leave (middle layer) and decidua (outer layer). The amnion is a translucent structure adjacent to the amniotic fluid. The chorion leave is a more opaque membrane that is attached to the decidua (i.e., maternal tissue that lines the uterus during pregnancy). The amnion and chorion leave are separated by the exocelomic cavity until approximately three months gestation, when they become fused. These membranes regulate the access of substances including drugs and other harmful chemicals to and from the amniotic fluid and, hence, the fetus.15) In recent years, it has been reported that the multidrug resistance/P-glycoprotein gene (MDR1/P-gp) and breast cancer resistance protein (BCRP), belong to the ATP-binding cassette transporter family are expressed in human fetal membranes including decidua. 16,17) It is considered that the role of these transporters is to discharge drugs in amniotic fluid and to keep out transfer of drugs from the maternal side via the uterus. It is well established that P-glycoprotein transports many drugs that are metabolized by CYP3A4 and many modulators of P-glycoprotein also modulate the CYP3A gene family.18) To the best of o...

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Tailored nanodisc immobilization for one‐step purification and reconstitution of cytochrome P450: A tool for membrane proteins’ hard cases

Zhao

Tao

Huang

et al. 2021

J of Separation Science

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A novel nanodisc-based immobilization method was developed for high-efficient purification and reconstitution of cytochrome P450 in one step. Using membrane scaffold protein containing a histidine tag, charged-nanodiscs were prepared in the form of self-assembly of lipid-protein nanoparticles. Their properties including the particle diameter and its distribution and Zeta potential were controlled well by adjusting molar ratios of phospholipids to membrane scaffold protein. At an optimum lipid-to-membrane scaffold protein molar ratio of 60:1, uniformly regular-shaped and discoidal nanodiscs with an average particle diameter of 10 nm and Zeta potential of -19 mV were obtained. They can be well fractionated by size exclusion chromatography. Charged-nanodiscs were successfully immobilized onto Ni-chelating microspheres via histidine tags with a density of 6.6 mg membrane scaffold protein/mL gel. After being packed in a column, chromatography studies demonstrated that this nanodisc-immobilized chromatographic medium had a specific binding to cytochrome P450 in rat liver microsome. Nanodiscs containing cytochrome P450 can be furthermore eluted from the column with a diameter of about 87.0 nm and height of about 8.0 nm, respectively. The purity of cytochrome P450 after purification increased 25 folds strikingly. This nanodisc-immobilized chromatography method is promising for the one-step purification and reconstitution of membrane protein.

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Microsomal Alcohol Oxygenase: Purification and Characterization of a Cytochrome P450 Responsible for Oxidation of 7-Hydroxy-Δ8-tetrahydrocannabinol to 7-Oxo-Δ8-tetrahydrocannabinol in Guinea Pig Liver

Cited by 10 publications

References 43 publications

Effective NADH-Dependent Oxidation of 7.BETA.-Hydroxy-.DELTA.8-tetrahydrocannabinol to the Corresponding Ketone by Japanese Monkey Hepatic Microsomes

Effective NADH-Dependent Oxidation of 7.BETA.-Hydroxy-.DELTA.8-tetrahydrocannabinol to the Corresponding Ketone by Japanese Monkey Hepatic Microsomes

Cytochrome P450 3As Gene Expression and Testosterone 6.BETA.-Hydroxylase Activity in Human Fetal Membranes and Placenta at Full Term

Tailored nanodisc immobilization for one‐step purification and reconstitution of cytochrome P450: A tool for membrane proteins’ hard cases

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