Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1)

Halvorson, Michael R.; Greenway, Denise; Eberhart, Delmont; Fitzgerald, Kathleen; Parkinson, Andrew

doi:10.1016/0003-9861(90)90566-h

Cited by 74 publications

(39 citation statements)

References 46 publications

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“…To avoid this difficulty, heterologously expressed P450 can also be purified to be used in reconstituted systems (Brian et al, 1990), with the advantage of the presence of only one isoform. However, the reorganization of the monooxygenase complex within added lipids is not always correctly achieved and many authors have reported problems in the particular case of P450 species from the 3A subfamily (Waxman et al, 1985;Kawano et al, 1987;Imaoka et al, 1988;Gemzik et al, 1990;Halvorson et al, 1990;Brian et al, 1990). This has been explained by progressive inactivation of P450 3A forms during the purification protocol.…”

Section: Discussionmentioning

confidence: 99%

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b₅

Peyronneau

Renaud

Truan

et al. 1992

European Journal of Biochemistry

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Human liver P450 NF25 (CYP3A4) had been previously expressed in Succhuromyces cerevisiue using the inducible GALIO-CYCl promoter and the phosphoglycerate kinase gene terminator [Renaud, J. p., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194,. The use of an improved expression vector [Urban, P., Cullin, C. and Pompon, D. (1990) Biochimie 72,463 -4721 increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25 -iron-metabolite complexes. %fold, g-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6@-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5. Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b, always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450-NF25 -iron-metabolite complex. A P450 Fe(I1)-(RNO) complex was obtained upon oxidation of Nhydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase. These results show that specific activities of yeast-expressed P450 NF25 may be artificially low, owing to limiting amounts of the associated microsomal redox proteins and emphasize the importance of controlling the amounts of the different components of the monooxygenase complex in order to optimize these catalytic activities, especially when the expression system is to be used for demonstrating metabolic capacities towards new substrates.

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Section: Discussionmentioning

confidence: 99%

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b₅

Peyronneau

Renaud

Truan

et al. 1992

European Journal of Biochemistry

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“…For lauric acid and testosterone, both engendering more than one metabolite, the 100% reference activity is chosen as being the highest activity among the P450 isoforms and among all the produced metabolites. (data adapted from Dutton et al, 1987;Waxman et al, 1987;Halvorson et al, 1990;Liu et al, 1996;Nguyen et al, 1999;Rowlands et al, 2000;Wang et al, 2000;Kobayashi et al, 2002;Warrington et al, 2004). No rat recombinant CYP2G1 and CYP4B1 have been developed until now.…”

Section: Downloaded Frommentioning

confidence: 99%

Characterization of Microsomal Cytochrome P450-Dependent Monooxygenases in the Rat Olfactory Mucosa

Minn¹,

Pelczar²,

Denizot³

et al. 2005

Drug Metab Dispos

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ABSTRACT:Nasal administration of a drug ensures therapeutic action by rapid systemic absorption and/or the entry of some molecules into the brain through different routes. Many recent studies have pointed out the presence of xenobiotic-metabolizing enzymes in rat olfactory mucosa (OM). Nevertheless, very little is known about the precise identity of isoforms of cytochrome P450 (P450)-dependent monooxygenases (P450) and their metabolic function in this tissue. Therefore, we evaluated mRNA expression of 19 P450 isoforms by semiquantitative reverse transcriptase-polymerase chain reaction and measured their microsomal activity toward six model substrates. For purposes of comparison, studies were conducted on OM and the liver. Specific activities toward phenacetin, chlorzoxazone, and dextromethorphan are higher in OM than in the liver; those toward lauric acid and testosterone are similar in both tissues, and that toward tolbutamide is much lower in OM. There are considerable differences between the two tissues with regard to mRNA expression of P450 isoforms. Some isoforms are expressed in OM but not in the liver (CYP1A1, 2G1, 2B21, and 4B1), whereas mRNA of others (CYP2C6, 2C11, 2D2, 3A1, 3A2, and 4A1) is present only in hepatic tissue. Although expression of CYP1A2, 2A1, 2A3, 2B2, 2D1, 2D4, 2E1, 2J4, and 3A9 is noticed in both tissues, there are a number of quantitative differences. On the whole, our results strongly suggest that CYP1A1, 1A2, 2A3, 2E1, 2G1, and 3A9 are among the main functional isoforms present in OM, at least regarding activities toward the six tested substrates. The implication of olfactory P450-dependent monooxygenases in toxicology, pharmacology, and physiology should be further investigated.

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“…The testosterone 6b-hydroxylase activity of P450JM-E is considerablely higher than that of CYP3A8 (P450CMLc) purified from hepatic microsomes of cynomolgus monkey as reported by Ohmori et al 37) The reason may be attributable to the use of microsomal lipids instead of dilauroylphosphatidylcholine as the lipid in the reconstituted system of P450JM-E, because a number of studies have shown that the catalytic activities of CYP3A enzymes are very low in a reconstituted system using only dilauroylphosphatidylcholine as the lipid. 17,[39][40][41] P450JM-E showed comparable activity to CYP3A1, 40) CYP3A4, 41) and CYP3A11 11) for testosterone 6b-hydroxylase. In the reconstituted system of P450JM-E, the MALCO activity for 7b-OH-D 8 -THC was about 6-fold higher than that for 7a-OH-D 8 -THC.…”

Section: Discussionmentioning

confidence: 90%

Monkey Hepatic Microsomal Alcohol Oxygenase: Purification and Characterization of a Cytochrome P450 Enzyme Catalyzing the Stereoselective Oxidation of 7.ALPHA.- and 7.BETA.-Hydroxy-.DELTA.8-tetrahydrocannabinol to 7-Oxo-.DELTA.8-tetrahydrocannabinol.

Matsunaga

Iwawaki

Komura

et al. 2002

Biological & Pharmaceutical Bulletin

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Tetrahydrocannabinol (THC) is the major psychoactive constituent of marijuana. It is well known that D 8 -THC is oxidized to numerous metabolites in the liver of various mammals including monkeys and humans.1,2) Most metabolites have been suggested to contribute to the psychoactivity of the parent compound. 7-Oxo-D 8 -THC shows almost equivalent pharmacological activity to D 8 -THC in mice, whereas 7a-and 7b-hydroxy-D 8 -THC (7a-and 7b-OH-D 8 -THC) were without significant activity.3) Several recent studies have clarified that cytochrome P450 (P450) plays a major role in the oxidation of THC. [4][5][6][7][8] However, the metabolic reaction is complicated and the isoforms responsible for specific metabolic reactions have not been completely elucidated. We have previously found that a guinea pig hepatic microsomal enzyme, called microsomal alcohol oxygenase (MALCO), is able to oxidize 7-OH-D 8 -THC to 7-Oxo-D 8-THC (Fig. 1), 9) and have recently clarified that P450 isoforms belonging to the 3A subfamily are the major enzymes of 7-OH-D 8 -THC MALCO in guinea pigs (P450GPF-B), 10) mice (CYP3A11), 11) rats (CYP3A1 and CYP3A2), 12) and humans (CYP3A4). 13)CYP3A is a highly expressed enzyme in rodents, monkeys, and humans and it has been demonstrated to play a prominent role in the metabolism of many clinically important drugs. 14) CYP3A shows selectivity for the chemical substituents rather than selectivity for substrates. 15,16) For example, the allylic positions, in spite of structurally unrelated compounds, are the major site of oxidation catalyzed by CYP3A.17-20) Species-related differences exist in the catalytic properties of P450 enzymes belonging to the same family or subfamily, but it is difficult to elucidate the properties of the enzyme responsible for a specific reaction across species. 12,21) To our best knowledge, no detailed study has been reported with respect to the specific isoform(s) involved in the formation of 7-oxo-D 8 -THC from 7a-and 7b-OH-D 8 -THC in monkeys.In the present study, we purified a P450 enzyme catalyzing the stereoselective oxidation of 7a-and 7b-OH-D 8 -THC to 7-Oxo-D 8 -THC from hepatic microsomes of Japanese monkeys and considered the oxidation mechanisms of 7-OH-D 8 -THC and 8-OH-D 9 -THC using oxygen-18 gas. The formation of 7-oxo-D D 8 -tetrahydrocannabinol ( -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. MATERIALS AND METHODS Materials

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Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1)

Cited by 74 publications

References 46 publications

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b₅

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b₅

Characterization of Microsomal Cytochrome P450-Dependent Monooxygenases in the Rat Olfactory Mucosa

Monkey Hepatic Microsomal Alcohol Oxygenase: Purification and Characterization of a Cytochrome P450 Enzyme Catalyzing the Stereoselective Oxidation of 7.ALPHA.- and 7.BETA.-Hydroxy-.DELTA.8-tetrahydrocannabinol to 7-Oxo-.DELTA.8-tetrahydrocannabinol.

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Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1)

Cited by 74 publications

References 46 publications

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b5

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b5

Characterization of Microsomal Cytochrome P450-Dependent Monooxygenases in the Rat Olfactory Mucosa

Monkey Hepatic Microsomal Alcohol Oxygenase: Purification and Characterization of a Cytochrome P450 Enzyme Catalyzing the Stereoselective Oxidation of 7.ALPHA.- and 7.BETA.-Hydroxy-.DELTA.8-tetrahydrocannabinol to 7-Oxo-.DELTA.8-tetrahydrocannabinol.

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Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b₅

Optimization of yeast‐expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH‐cytochrome P450 reductase and cytochrome b₅