Liposomes pp 11-20 | Cite as

The Use of Liposomes in the Study of Drug Metabolism: A Method to Incorporate the Enzymes of the Cytochrome P450 Monooxygenase System into Phospholipid, Bilayer Vesicles

  • James R. ReedEmail author
Part of the Methods in Molecular Biology™ book series (MIMB, volume 606)


Although lipids are essential for the optimal activity of the cytochromes P450 monooxygenase system, relatively little is known about the membrane environment in which these enzymes function. One approach used to mimic the structural arrangement of lipids and enzymes within the endoplasmic reticulum is to physically incorporate the cytochromes P450 and their redox partners in a vesicle bilayer of phospholipids. Several methods have been devised for this purpose. This chapter describes a method in which the P450 monooxygenase system is incorporated by first, solubilizing the enzymes and lipid with sodium glycocholate. After the protein and lipid aggregates are dispersed, the detergent is removed by adsorption using BioBeads SM-2 resin which leads to the formation of bilayer vesicles of phospholipid containing incorporated cytochrome P450 and NADPH cytochrome P450 reductase. This procedure requires relatively a short preparation time, provides concentrated reconstituted systems that can be used in a wide range of applications, allows for several enzyme samples to be prepared simultaneously so that different conditions can be compared, and results in minimal loss of active enzyme.

Key words

Phospholipid vesicles Cytochromes P450 Reconstituted systems Drug metabolism 


  1. 1.
    Porter TD, Coon MJ (1991) Cytochrome P-450. Multiplicity of isoforms, substrates, and catalytic and regulatory mechanisms. J Biol Chem 266:13469-13472PubMedGoogle Scholar
  2. 2.
    Guengerich FP (2001) Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem Res Toxicol 14:611-650CrossRefPubMedGoogle Scholar
  3. 3.
    Weng Y, Fang C, Turesky RJ, Behr M, Kaminsky LS, Ding X (2007) Determination of the role of target tissue metabolism in lung carcinogenesis using conditional cytochrome P450 reductase-null mice. Cancer Res 67:7825-7832CrossRefPubMedGoogle Scholar
  4. 4.
    Iyanagi T (2007) Molecular mechanism of phase I and phase II drug-metabolizing enzymes: implications for detoxification. Int Rev Cytol 260:35-112CrossRefPubMedGoogle Scholar
  5. 5.
    Rooney PH, Telfer C, McFadyen MC, Melvin WT, Murray GI (2004) The role of cytochrome P450 in cytotoxic bioactivation: future therapeutic directions. Curr Cancer Drug Targets 4:257-265CrossRefPubMedGoogle Scholar
  6. 6.
    Hannemann F, Bichet A, Ewen KM, Bernhardt R (2007) Cytochrome P450 systems-biological variations of electron transport chains. Biochim Biophys Acta 1770:330-344PubMedGoogle Scholar
  7. 7.
    Guengerich FP (1989) Characterization of human microsomal cytochrome P-450 enzymes. Annu Rev Pharmacol Toxicol 29:241-264CrossRefPubMedGoogle Scholar
  8. 8.
    West SB, Lu AYH (1972) Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. V. Competition between cytochromes P-450 and P-448 for reductase in 3, 4-benzpyrene hydroxylation. Arch Biochem Biophys 153:298-303CrossRefPubMedGoogle Scholar
  9. 9.
    Saine SE, Strobel HW (1976) Drug metabolism in liver tumors. Resolution of components and reconstitution of activity. Mol Pharmacol 12:649-657PubMedGoogle Scholar
  10. 10.
    Strobel HW, Lu AYH, Heidema J, Coon MJ (1970) Phosphatidylcholine requirement in the enzymatic reduction of hemoprotein P-450 and in fatty acid, hydrocarbon, and drug hydroxylation. J Biol Chem 245:4851-4854PubMedGoogle Scholar
  11. 11.
    Ingelman-Sundberg M (1977) Phospholipids and detergents as effectors in the liver microsomal hydroxylase system. Biochim Biophys Acta 488:225-234PubMedGoogle Scholar
  12. 12.
    Taniguchi H, Pyerin W (1988) Phospholipid bilayer membranes play decisive roles in the cytochrome P-450-dependent monooxygenase system. J Cancer Res Clin Oncol 114:335-340CrossRefPubMedGoogle Scholar
  13. 13.
    Causey KM, Eyer CS, Backes WL (1990) Dual role of phospholipid in the reconstitution of cytochrome P- 450 LM2-dependent activities. Mol Pharmacol 38:134-142PubMedGoogle Scholar
  14. 14.
    Balvers WG, Boersma MG, Veeger C, Rietjens IM (1993) Kinetics of cytochromes P-450 IA1 and IIB1 in reconstituted systems with dilauroyl- and distearoyl-glycerophosphocholine. Eur J Biochem 215:373-381CrossRefPubMedGoogle Scholar
  15. 15.
    Autor AP, Kaschnitz RM, Heidema JK, Coon MJ (1973) Sedimentation and other properties of the reconstituted liver microsomal mixed-function oxidase system containing cytochrome P-450, reduced triphosphopyridine nucleotide-cytochrome P-450 reductase, and phosphatidylcholine. Mol Pharmacol 9:93-104PubMedGoogle Scholar
  16. 16.
    French JS, Guengerich FP, Coon MJ (1980) Interactions of cytochrome P-450, NADPH-cytochrome P-450 reductase, phospholipid, and substrate in the reconstituted liver microsomal enzyme system. J Biol Chem 255:4112-4119PubMedGoogle Scholar
  17. 17.
    Reed JR, Kelley RW, Backes WL (2006) An evaluation of methods for the reconstitution of cytochromes P450 and NADPH P450 reductase into lipid vesicles. Drug Metab Dispos 34:660-666CrossRefPubMedGoogle Scholar
  18. 18.
    Taniguchi H, Imai Y, Iyanagi T, Sato R (1979) Interaction between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membrane of phosphatidylcholine vesicles. Biochim Biophys Acta 550:341-356CrossRefPubMedGoogle Scholar
  19. 19.
    Ingelman-Sundberg M, Glaumann H (1980) Incorporation of purified components of the rabbit liver microsomal hydroxylase system into phospholipid vesicles. Biochim Biophys Acta 599:417-435CrossRefPubMedGoogle Scholar
  20. 20.
    Schwarz D, Gast K, Meyer HW, Lachmann U, Coon MJ, Ruckpaul K (1984) Incorporation of the cytochrome P-450 monooxygenase system into large unilamellar liposomes using octylglucoside, especially for measurements of protein diffusion in membranes. Biochem Biophys Res Commun 121:118-125CrossRefPubMedGoogle Scholar
  21. 21.
    Ingelman-Sundberg M, Blanck J, Smettan G, Ruckpaul K (1983) Reduction of cytochrome P-450 LM2 by NADPH in reconstituted phospholipid vesicles is dependent on membrane charge. Eur J Biochem 134:157-162CrossRefPubMedGoogle Scholar
  22. 22.
    Bosterling B, Trudell JR, Galla HJ (1981) Phospholipid interactions with cytochrome P-450 in reconstituted vesicles. Preference for negatively-charged phosphatidic acid. Biochim Biophys Acta 643:547-556CrossRefPubMedGoogle Scholar
  23. 23.
    Kawato S, Gut J, Cherry RJ, Winterhalter KH, Richter C (1982) Rotation of cytochrome P-450. I. Investigations of protein-protein interactions of cytochrome P-450 in phospholipid vesicles and liver microsomes. J Biol Chem 257:7023-7029PubMedGoogle Scholar
  24. 24.
    Schwarz D, Pirrwitz J, Ruckpaul K (1982) Rotational diffusion of cytochrome P-450 in the microsomal membrane-evidence for a clusterlike organization from saturation transfer electron paramagnetic resonance spectroscopy. Arch Biochem Biophys 216:322-328CrossRefPubMedGoogle Scholar
  25. 25.
    Taniguchi H, Imai Y, Sato R (1987) Protein-protein and lipid-protein interactions in a reconstituted cytochrome P-450 dependent microsomal monooxygenase. Biochem 26:7084-7090CrossRefGoogle Scholar
  26. 26.
    Hjelmeland LM (1990) Solubilization of native membrane proteins. Meth Enzymol 182:253-264CrossRefPubMedGoogle Scholar
  27. 27.
    Jackson ML, Schmidt CF, Lichtenberg D, Litman BJ, Albert AD (1982) Solubilization of phosphatidylcholine bilayers by octyl glucoside. Biochem 21:4576-4582CrossRefGoogle Scholar
  28. 28.
    Bayerl TM, Werner G-D, Sackmann E (1989) Solubilization of DMPC and DPPC vesicles by detergents below their critical midellization concentration: high-sensitivity differential scanning calorimetry, Fourier transform infared spectroscopy and freeze-fracture electron microscopy reveal two interaction sites of detergents in vesicles. Biochim Biophys Acta 984:214-224CrossRefPubMedGoogle Scholar
  29. 29.
    Reed JR, Brignac-Huber LM, Backes WL (2008) Physical incorporation of NADPH-cytochrome P450 reductase and cytochrome P450 into phospholipid vesicles using glycocholate and Bio-Beads. Drug Metab Dispos 36:582-588CrossRefPubMedGoogle Scholar
  30. 30.
    Pike LJ (2004) Lipid rafts: heterogeneity on the high seas. Biochem J 378:281-292CrossRefPubMedGoogle Scholar
  31. 31.
    Browman DT, Resek ME, Zajchowski LD, Robbins SM (2006) Erlin-1 and erlin-2 are novel members of the prohibitin family of proteins that define lipid-raft-like domains of the ER. J Cell Sci 119:3149-3160CrossRefPubMedGoogle Scholar
  32. 32.
    Holloway PW (1973) A simple procedure for removal of Triton X-100 from protein samples. Anal Biochem 53:304-308CrossRefPubMedGoogle Scholar
  33. 33.
    Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370-2378PubMedGoogle Scholar
  34. 34.
    Phillips AH, Langdon RG (1962) Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization, and kinetic studies. J Biol Chem 237:2652-2660PubMedGoogle Scholar
  35. 35.
    Stewart JC (1980) Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal Biochem 104:10-14CrossRefPubMedGoogle Scholar
  36. 36.
    Antonian L, Deb S, Spivak W (1990) Critical self-association of bile lipids studied by infrared spectroscopy and viscometry. J Lipid Res 31:947-951PubMedGoogle Scholar
  37. 37.
    Levy D, Bluzat A, Seigneuret M, Rigaud JL (1990) A systematic study of liposome and proteoliposome reconstitution involving Bio-Bead-mediated Triton×-100 removal. Biochim Biophys Acta 1025:179-190CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of PharmacologyLouisiana State University Health Science CenterNew OrleansUSA

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