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Purification and Characterization of the Rat Liver CYP2D1 and Utilization of Reconstituted CYP2D1 in Caffeine Metabolism

Chung, Woon-Gye  
  • 초록

    In order to assess the possibility whether CYP2D is involved in caffeine metabolism, we have purified and characterized the rat liver microsomal cytochrome P4502D1 (CYP2D1), equivalent to CYP2D6 in human liver, and have utilized the reconstituted CYP2D1 in the metabolism of 4 primary caffeine (1, 3, 7-trimethylxanthine) metabolites such as paraxanthine (1, 7-dimethylxanthine), 1, 3, 7-trimethylurate, theophylline (1, 3-dimethylxanthine) and theobromine (3, 7-dimethylxanthine). Rat liver CYP 2D1 has been purified to a specific content of 8.98 nmole/mg protein (13.4fold purification, 1.5% yield) using $\omega$ -aminooctylagarose, hydroxlapatite, and DE52 columns in a sequential manner. As judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the purified CYP2D1 was apparently homogeneous. Molecular weight of the purified CYP2D1 was found to be 51, 000 Da. Catalytic activity of the purified and then reconstituted CYP2D1 was confirmed by using bufuralol, a known subsFate of CYP2D1. The reconstituted CYP2D1 was found to produce to 1-hydroxylbufuralol at a rate of 1.43 $\pm$ 0.13 nmol/min/nmol P450. The kinetic analysis of bufuralol hydroxylation indicated that Km and Vmax values were 7.32 $\mu M$ and 1.64 nmol/min/nmol P450, respectively. The reconstituted CYP2D1 could catalyze the 7-demethylation of PX to 1-methylxanthine at a rate of 12.5 pmol/min/pmol, and also the 7- and 3- demethylations of 1, 3, 7-trimethylurate to 1, 3-dimethylurate and 1, 7-dimethylurate at 6.5 and 12.8 pmol/min/pmol CYP2D1, respectively. The reconstituted CYP2D1 could also 3-demethylate theophylline to 1-methylxanthine at 5 pmol/min/pmol and hydroxylate the theophylline to 1, 3-dimethylurate at 21.8 pmol/min/pmol CYP2D1. The reconstituted CYP2D1, however, did not metabolize TB at all (detection limits were 0.03 pmol/min/pmol). This study indicated that CYP2D1 is involved in 3-and 7-demethylations of paraxanthine and theophylline and suggested that CYP2D6 (equivalent to CYP2D1 in rat liver) present in human liver may be involved in the secondary metabolism of the primary metabolites of caffeine.


  • 주제어

    Purified CYP2D1 .   Caffeine metabolism.  

  • 참고문헌 (21)

    1. Differential roles of cytochromes P450 2D1, 2C11, and 1A1/2 in the hydroxylation of bufuralol by rat liver microsomes , Mimura M.;Yamazaki H.;Sugahara C.;Hiro T.;Funae Y.;Shimada T. , Biochem. Pharmacol. / v.47,pp.1957-1963,
    2. Human cytochrome $P-450_{PA}$ (P-450IA2), the phenacetin O-deethylase, is primarily responsible for the hepatic 3-demethylation of caffeine and N-oxidation of carcinogenic arylamines , Butler M.A.;Iwasaki M.;Guengerich P.F.;Kadlubar F.F. , Proc. Natl. Acad. Sci. / v.86,pp.7696-7700,
    3. Use of caffeine metabolites ratios to explore CYP1A2 and xanthine oxidase activities , Kalow W.;Tang B. , Clin. Pharmaco. Ther. / v.50,pp.508-519,
    4. The P450 superfamily: Update on new sequences, gene mapping, accession numbers, early trial names of enzymes and nomenclature , Nelson D.R.;Kamataki T.;Waxman D.J.;Guengerich P.F.;Estabrook R.W.;Feyereisen R.;Gonzalez F.J.;Coon M.J.;Gunsalus I.C.;Gotoh O.;Okuda K.;Nerbert D.W. , DNA Cell Biol. / v.12,pp.1-51,
    5. Accelerated caffeine metabolism after omeprazole treatment is indicated by urinary metabolite ratios: Coincidence with plasma clearance and breath test , Rost K.L.;Roots I. , Clin. Pharmaco. Ther. / v.55,pp.402-411,
    6. Prevalence of CYP2D6 gene duplication and its repercussion on the oxidative phenotype in a white population , Agundez J.A.;Ledesma M.C.;Ladero J.M.;Benitez J. , Clin. Pharmaco. Ther. / v.57,pp.265-269,
    7. Determination of CYP1A2 and NAT2 phenotype in human populations by analysis of caffeine urinary metabolites , Butler M.A.;Lang N.P.;Young J.F.;Caporaso N.E.;Vineis P.;Hayes R.B.;Teitel C.H.;Massengill J.P.;Lawsen M.F.;Kadlubar F.F. , Pharmacogenetics / v.2,pp.116-127,
    8. Phenotyping of CYP1A2 in Japanese population by analysis of caffeine urinary metabolites: Absence of mutation prescribing the phenotype in the CYP1A2 gene , Nakajima M.;Yokoi T.;Mizutani M.;Shin S.;Kadlubar F.F.;Kamataki T. , Cancer Epidemiol. Biomarkers Prev. / v.3,pp.413-421,
    9. Protein measurement with folin phenol reagent , Lowry O.H.;Rosebrough N.J.;Farr A.L.;Randall R.J. , J. Biol. Chem. / v.193,pp.256-275,
    10. Debrisoquine/sparteine-type polymorphism of drug oxidation , Gut J.;Catin T.;Dayer P.;Kronbach T.;Zanger U.;Meyer U.A. , J. Biol. Chem. / v.261,pp.11734-11743,
    11. Pronounced differences between native Chinese and Swedish population in the polymorphic hydroxylations of debrisoquine and S-mephenytoin , Bertilsson L.;Lou Y-Q.;Du Y-L.;Kuang T-Y.;Liao X-M.;Wang K-Y.;Reviriego J.;Iselius L.;Sjoqvist F. , Clin. Pharmaco. Ther. / v.51,pp.388-397,
    12. Biotransformation of caffeine and theophylline in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms , Fuhr U.;Doehmer J.;Battula N.;Wolfel C.;Kudla C.;Keita Y.;Staib A.H. , Biochem. Pharmacol. / v.43,pp.225-235,
    13. Polymorphic hydroxylation of debrisoquine in man , Mahgoub A.;Idle J.R.;Dring L.G.;Lancaster R.;Smith R.L. , Lancet / v.2,pp.584-586,
    14. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature , Omura R.;Sato T. , J. Biol. Chem. / v.239,pp.2370-2378,
    15. Caffeine urinary metabolite ratios as markers of enzyme activity: A theoretical assessment , Rostami-Hodjegan A.;Nurminen S.;Jackson P.R.;Tucker G.T. , Pharmacogenetics / v.6,pp.121-149,
    16. Differential inhibition of flavin-containing monooxygenase with grapefruit juice , Chung W.G.;Kang J.H.;Roh H.K.;Cha Y.N. , The Toxicologist / v.36,pp.1789,
    17. The CYP2D gene subfamily: Analysis of the molecular basis of the debrisoquine 4-hydroxylase deficiency in DA rats , Matsunaga E.;Zanger U.M.;Hardwick J.P.;Gelboin H.V.;Meyer U.A.;Gonzalez F.J. , Biochem. / v.28,pp.7349-7355,
    18. Pharmacogentic phenotyping and genotyping , Gonzalez F.J.;Idle J.R. , Clin. Pharmacokinet. / v.26,pp.59-70,
    19. Characterization of human cytochrome P450 involved in theophylline 8-hydroxylation , Zhang Z. Y.;Kaminsky L.S. , Biochem. Pharmacol. / v.50,pp.205-211,
    20. Caffeine metabolism by human hepatic cytochrome P450: Contributions of 1A2, 2E1 and 3A isoforms , Tassaneeyakul W.;Birkett D.J.;McManus M.E.;Tassaneeyakul W.;Veronese M.E.;Andersson T.;Tukey R.T.;Miners J.O. , Biochem. Pharmacol. / v.47,pp.1767-1776,
    21. Evidence for the involvement of several cytochrome P-450 in the first steps of caffeine metabolism by human liver microsomes , Berthou F.;Flinois J.;Ratanasavanh D.;Beaune P.;Riche C.;Guillouzo A. , Drug Metab. Dispos. / v.19,pp.561-567,

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    1. 1997 "한국인에서 CYP1A2의존성 Caffeine 대사에 대한 자몽과즙의 억제효과" 臨床藥理學會誌 = The journal of Korean society for Clinical Pharmacology and Therapeutics 5 (1): 26~34    
    2. 1999 "Assessment of Flavin-containing Monooxygenase (FMO) Activity by Determining Urinary Ratio of Theobromine and Caffeine in a Korean Population after Drinking a Cup of Coffee" The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology 3 (2): 207~213    
    3. 2001 "Phenotyping of Flavin-Containing Monooxygenase (FMO) Activity and Factors Affecting FMO Activity in Korean" Journal of toxicology and public health : an official journal of the Korean Society of Toxicology 17 (0): 127~133    
    4. 2002 "마우스 및 랫드에서 자양강장제 황제공진보의 단회투여독성시험" Journal of toxicology and public health : an official journal of the Korean Society of Toxicology 18 (1): 73~77    
    5. 2002 "덱스트로메토르판에 대한 한국인의 표현형 및 유전자형 분석" 약학회지 = Yakhak hoeji 46 (3): 179~184    
    6. 2003 "Alteration of Substrate Specificity by Common Variants, E158K/E308G and V257M, in Human Hepatic Drug-metabolizing Enzyme, Flavin-containing Monooxygenase 3" The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology 7 (3): 157~162    
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