Immunochemical characterization of a cytochrome P450 isozyme and a protein purified from liver microsomes of male guinea pigs and their roles in the oxidative metabolism of delta 9-tetrahydrocannabinol by guinea pig liver microsomes.
A protein (designated as protein-B) was purified from liver microsomes of adult male guinea pigs by an affinity chromatography with omega-aminooctyl Sepharose 4B, followed by HPLC using DEAE-5PW and hydroxyapatite columns which had been used to purify a cytochrome P450 (P450) isozyme (P450-A) from the same subcellular fraction (Narimatsu et al., Biochem Biophys Res Commun 172: 607-613, 1990). Protein-B had a molecular mass of 49 kDa in SDS-PAGE, but did not show absorbance at 417 nm for heme. Further, it did not show any oxidative activities towards aniline (AN), d-benzphetamine (d-BP), p-nitroanisole (p-NA) or delta 9-tetrahydrocannabinol (delta 9-THC) in a reconstituted system including dilauroylphosphatidylcholine, NADPH-P450 reductase, and cytochrome b5. However, antiserum against protein-B raised in rabbits suppressed liver microsomal oxidative activities towards d-BP and p-NA dose-dependently. The antibody decreased delta 9-THC oxidative activity most effectively, but did not decrease AN hydroxylation activity. Antiserum against P450-A suppressed all the activities towards these four substrates, especially towards delta 9-THC, in liver microsomes of male guinea pigs. Moreover, reconstitution with hemin made it possible for protein-B to produce some oxidative activity toward delta 9-THC. These results suggest that protein-B is also a cytochrome P450 isozyme which has lost a heme moiety during purification steps. Both P450-A and protein-B could have a role as cytochrome P450 isozymes in the oxidative metabolism of drugs, especially that of delta 9-THC by the liver microsomes of adult male guinea pigs.
Narimatsu
S
S
Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Japan.
Akutsu
Y
Y
Matsunaga
T
T
Watanabe
K
K
Yamamoto
I
I
Yoshimura
H
H
D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D000840:Anisoles / Q000378:metabolism; D000906:Antibodies / Q000276:immunology; D001589:Benzphetamine / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D013759:Dronabinol / Q000378:metabolism*; D006168:Guinea Pigs; D006418:Heme / Q000378:metabolism; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011506:Proteins / Q000302:isolation & purification*
D000814:Aniline Compounds; D000840:Anisoles; D000906:Antibodies; D007527:Isoenzymes; D011506:Proteins; D001589:Benzphetamine; D006418:Heme; D013759:Dronabinol; D003577:Cytochrome P-450 Enzyme System; C005306:4-nitroanisole; C023650:aniline