Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Jun 1;284(Pt 2):483–490. doi: 10.1042/bj2840483

Interaction of 7-n-alkoxycoumarins with cytochrome P-450(2) and their partitioning into liposomal membranes. Assessment of methods for determination of membrane partition coefficients.

M Vermeir 1, N Boens 1, K P Heirwegh 1
PMCID: PMC1132664  PMID: 1599434

Abstract

A study was made of the binding of 7-ethoxy-, 7-n-propoxy- and 7-n-pentoxy-coumarin to cytochrome P-450(2) reconstituted into large unilamellar liposomes composed of a mixture of egg L-alpha-phosphatidylcholine, egg phosphatidylethanolamine and dipalmitoyl phosphatidic acid (2:1:0.06, by weight). The apparent spectral dissociation constants Ksapp. increased linearly with increasing proteoliposomal concentration. When both cytochrome P-450(2) and NADPH:cytochrome P-450 reductase were reconstituted into liposomes, the apparent Michaelis constants Kmapp. for O-dealkylation of 7-methoxy-, 7-ethoxy- and 7-n-propoxy-coumarin showed a similar dependence on the proteoliposomal concentration. The results were in accordance with models for kinetic or equilibrium processes in biphasic systems containing membrane-bound catalytic or acceptor sites, in which a linear solute partition in the bilayer membrane is postulated. The methyl, ethyl and n-propyl ether were readily dealkylated. However, the O-dealkylation rate of 7-n-butoxycoumarin was low and became very small for longer alkyl ethers. Both the effective dissociation constants and effective Michaelis constants decreased with elongation of the alkyl side chain of the coumarins. From plots of the apparent dissociation constants and apparent Michaelis constants against the lipid volume fraction of the proteoliposomes, the membrane partition coefficients for several homologues were calculated. When protein-free liposomes were added to 7-n-alkoxycoumarin solutions, the fluorescence intensity of the coumarins decreased and eventually became negligible in the presence of an excess of liposomal material. On the assumption that the overall fluorescence can be ascribed exclusively to the fraction of 7-n-alkoxycoumarin molecules present in the aqueous phase, partition coefficients for liposomal accumulation of the test compounds could be determined directly. For several coumarin ethers, comparable values were derived for the membrane partition coefficients from binding, kinetic and fluorescence intensity measurements. The change in free energy per methylene group of the 7-n-alkoxycoumarins for partitioning between n-octanol and buffer was significantly different from the value for liposome partitioning.

Full text

PDF
483

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Al-Gailany K. A., Bridges J. W., Netter K. J. The dealkylation of some rho-nitrophenylalkylethers and their alpha-deuterated analogues by rat liver microsomes. Biochem Pharmacol. 1975 Apr 15;24(8):867–870. doi: 10.1016/0006-2952(75)90156-2. [DOI] [PubMed] [Google Scholar]
  2. Al-Gailany K. A., Houston J. B., Bridges J. W. The role of substrate lipophilicity in determining type 1 microsomal P450 binding characteristics. Biochem Pharmacol. 1978 Mar 1;27(5):783–788. doi: 10.1016/0006-2952(78)90521-x. [DOI] [PubMed] [Google Scholar]
  3. Backes W. L., Hogaboom M., Canady W. J. The true hydrophobicity of microsomal cytochrome P-450 in the rat. Size dependence of the free energy of binding of a series of hydrocarbon substrates from the aqueous phase to the enzyme and to the membrane as derived from spectral binding data. J Biol Chem. 1982 Apr 25;257(8):4063–4070. [PubMed] [Google Scholar]
  4. Backes W. L., Means M., Canady W. J. Competition between hydrocarbon and barbiturate for spectral binding to hepatic cytochrome P-450. Inferences concerning spin state of the enzyme. J Biol Chem. 1984 Aug 25;259(16):10092–10099. [PubMed] [Google Scholar]
  5. Barbieri R. L., Canick J. A., Ryan K. J. High-affinity steroid binding to rat testis 17 alpha-hydroxylase and human placental aromatase. J Steroid Biochem. 1981 Apr;14(4):387–393. doi: 10.1016/0022-4731(81)90158-8. [DOI] [PubMed] [Google Scholar]
  6. Born J. L., Hadley W. M. Ligand interactions of 4-pyridylethers with cytochrome P-450. Proc West Pharmacol Soc. 1980;23:259–262. [PubMed] [Google Scholar]
  7. Brooke D. N., Dobbs A. J., Williams N. Octanol:water partition coefficients (P): measurement, estimation, and interpretation, particularly for chemicals with P greater than 10(5). Ecotoxicol Environ Saf. 1986 Jun;11(3):251–260. doi: 10.1016/0147-6513(86)90099-0. [DOI] [PubMed] [Google Scholar]
  8. Brown C. A., Black S. D. Membrane topology of mammalian cytochromes P-450 from liver endoplasmic reticulum. Determination by trypsinolysis of phenobarbital-treated microsomes. J Biol Chem. 1989 Mar 15;264(8):4442–4449. [PubMed] [Google Scholar]
  9. Burke M. D., Mayer R. T. Differential effects of phenobarbitone and 3-methylcholanthrene induction on the hepatic microsomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (alkoxyresorufins). Chem Biol Interact. 1983 Jul 15;45(2):243–258. doi: 10.1016/0009-2797(83)90072-8. [DOI] [PubMed] [Google Scholar]
  10. Burke M. D., Thompson S., Elcombe C. R., Halpert J., Haaparanta T., Mayer R. T. Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochem Pharmacol. 1985 Sep 15;34(18):3337–3345. doi: 10.1016/0006-2952(85)90355-7. [DOI] [PubMed] [Google Scholar]
  11. Bösterling B., Stier A., Hildebrandt A. G., Dawson J. H., Trudell J. R. Reconstitution of cytochrome P-450 and cytochrome P-450 reductase into phosphatidylcholine-phosphatidylethanolamine bilayers: characterization of structure and metabolic activity. Mol Pharmacol. 1979 Jul;16(1):332–342. [PubMed] [Google Scholar]
  12. Cleland W. W. Statistical analysis of enzyme kinetic data. Methods Enzymol. 1979;63:103–138. doi: 10.1016/0076-6879(79)63008-2. [DOI] [PubMed] [Google Scholar]
  13. Cohen G. M., Mannering G. J. Involvement of a hydrophobic site in the inhibition of the microsomal p-hydroxylation of aniline by alcohols. Mol Pharmacol. 1973 May;9(3):383–397. [PubMed] [Google Scholar]
  14. De Lemos-Chiarandini C., Frey A. B., Sabatini D. D., Kreibich G. Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies. J Cell Biol. 1987 Feb;104(2):209–219. doi: 10.1083/jcb.104.2.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ebel R. E., O'Keeffe D. H., Peterson J. A. Substrate binding to hepatic microsomal cytochrome P-450. Influence of the microsomal membrane. J Biol Chem. 1978 Jun 10;253(11):3888–3897. [PubMed] [Google Scholar]
  16. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  17. French J. S., Coon M. J. Properties of NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes. Arch Biochem Biophys. 1979 Jul;195(2):565–577. doi: 10.1016/0003-9861(79)90383-7. [DOI] [PubMed] [Google Scholar]
  18. Greinert R., Finch S. A., Stier A. Cytochrome P-450 rotamers control mixed-function oxygenation in reconstituted membranes. Rotational diffusion studied by delayed fluorescence depolarization. Xenobiotica. 1982 Nov;12(11):717–726. doi: 10.3109/00498258209038946. [DOI] [PubMed] [Google Scholar]
  19. Guengerich F. P., Martin M. V. Purification of cytochrome P-450, NADPH-cytochrome P-450 reductase, and epoxide hydratase from a single preparation of rat liver microsomes. Arch Biochem Biophys. 1980 Dec;205(2):365–379. doi: 10.1016/0003-9861(80)90119-8. [DOI] [PubMed] [Google Scholar]
  20. Heirwegh K. P., Meuwissen J. A., Vermeir M., De Smedt H. Liposomes as carriers of poorly water-soluble substrates: linear modelling of membrane systems with catalytic or binding sites of different facedness. Significance of experimental membrane partition coefficients and of kinetic and equilibrium parameters. Biochem J. 1988 Aug 15;254(1):101–108. doi: 10.1042/bj2540101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hudecek J., Anzenbacher P. Secondary structure prediction of liver microsomal cytochrome P-450; proposed model of spatial arrangement in a membrane. Biochim Biophys Acta. 1988 Aug 10;955(3):361–370. doi: 10.1016/0167-4838(88)90216-6. [DOI] [PubMed] [Google Scholar]
  22. Hunter W. H., Wilson P. The hydroxylation and dealkylation of some naphthyl alkyl ethers by rat liver microsomes. Xenobiotica. 1981 Mar;11(3):179–188. doi: 10.3109/00498258109045290. [DOI] [PubMed] [Google Scholar]
  23. Kominami S., Higuchi A., Takemori S. Interaction of steroids with adrenal cytochrome P-450 (P-450(17)alpha,lyase) in liposome membranes. Biochim Biophys Acta. 1988 Jan 13;937(1):177–183. doi: 10.1016/0005-2736(88)90239-8. [DOI] [PubMed] [Google Scholar]
  24. Kominami S., Itoh Y., Takemori S. Studies on the interaction of steroid substrates with adrenal microsomal cytochrome P-450 (P-450C21) in liposome membranes. J Biol Chem. 1986 Feb 15;261(5):2077–2083. [PubMed] [Google Scholar]
  25. Komori M., Imai Y., Sato R. Purification and characterization of cytochrome P-450 with high affinity for 7-alkoxycoumarins. J Biochem. 1984 May;95(5):1379–1388. doi: 10.1093/oxfordjournals.jbchem.a134745. [DOI] [PubMed] [Google Scholar]
  26. Kühn-Velten N., Meyer I., Staib W. Specificity of steroid binding to testicular microsomal cytochrome P-450. Relation of steroid structure to type-I spectral responses after correction for hydrophobic association with the membrane. J Steroid Biochem. 1989 Jul;33(1):33–39. doi: 10.1016/0022-4731(89)90354-3. [DOI] [PubMed] [Google Scholar]
  27. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  28. Lu A. Y., Jerina D. M., Levin W. Liver microsomal epoxide hydrase. J Biol Chem. 1977 Jun 10;252(11):3715–3723. [PubMed] [Google Scholar]
  29. Morgan E. T., Koop D. R., Coon M. J. Catalytic activity of cytochrome P-450 isozyme 3a isolated from liver microsomes of ethanol-treated rabbits. Oxidation of alcohols. J Biol Chem. 1982 Dec 10;257(23):13951–13957. [PubMed] [Google Scholar]
  30. Nebert D. W., Gelboin H. V. Substrate-inducible microsomal aryl hydroxylase in mammalian cell culture. I. Assay and properties of induced enzyme. J Biol Chem. 1968 Dec 10;243(23):6242–6249. [PubMed] [Google Scholar]
  31. Newman G. C., Huang C. Structural studies on phophatidylcholine-cholesterol mixed vesicles. Biochemistry. 1975 Jul 29;14(15):3363–3370. doi: 10.1021/bi00686a012. [DOI] [PubMed] [Google Scholar]
  32. OMURA T., SATO R. THE CARBON MONOXIDE-BINDING PIGMENT OF LIVER MICROSOMES. I. EVIDENCE FOR ITS HEMOPROTEIN NATURE. J Biol Chem. 1964 Jul;239:2370–2378. [PubMed] [Google Scholar]
  33. Ozols J., Heinemann F. S., Johnson E. F. The complete amino acid sequence of a constitutive form of liver microsomal cytochrome P-450. J Biol Chem. 1985 May 10;260(9):5427–5434. [PubMed] [Google Scholar]
  34. Parry G., Palmer D. N., Williams D. J. Ligand partitioning into membranes: its significance in determining Km and Ks values for cytochrome P-450 and other membrane bound receptors and enzymes. FEBS Lett. 1976 Aug 15;67(2):123–129. doi: 10.1016/0014-5793(76)80348-1. [DOI] [PubMed] [Google Scholar]
  35. Sakaguchi M., Mihara K., Sato R. A short amino-terminal segment of microsomal cytochrome P-450 functions both as an insertion signal and as a stop-transfer sequence. EMBO J. 1987 Aug;6(8):2425–2431. doi: 10.1002/j.1460-2075.1987.tb02521.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schenkman J. B., Remmer H., Estabrook R. W. Spectral studies of drug interaction with hepatic microsomal cytochrome. Mol Pharmacol. 1967 Mar;3(2):113–123. [PubMed] [Google Scholar]
  37. Schwarz D., Pirrwitz J., Ruckpaul K. 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. 1982 Jun;216(1):322–328. doi: 10.1016/0003-9861(82)90217-x. [DOI] [PubMed] [Google Scholar]
  38. Seybert D. W., Lancaster J. R., Jr, Lambeth J. D., Kamin H. Participation of the membrane in the side chain cleavage of cholesterol. Reconstitution of cytochrome P-450scc into phospholipid vesicles. J Biol Chem. 1979 Dec 10;254(23):12088–12098. [PubMed] [Google Scholar]
  39. Sklar L. A. The partition of cis-parinaric acid and trans-parinaric acid among aqueous, fluid lipid, and solid lipid phases. Mol Cell Biochem. 1980 Nov 20;32(3):169–177. doi: 10.1007/BF00227444. [DOI] [PubMed] [Google Scholar]
  40. Tarr G. E., Black S. D., Fujita V. S., Coon M. J. Complete amino acid sequence and predicted membrane topology of phenobarbital-induced cytochrome P-450 (isozyme 2) from rabbit liver microsomes. Proc Natl Acad Sci U S A. 1983 Nov;80(21):6552–6556. doi: 10.1073/pnas.80.21.6552. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ullrich V., Weber P. The O-dealkylation of 7-ethoxycoumarin by liver microsomes. A direct fluorometric test. Hoppe Seylers Z Physiol Chem. 1972 Jul;353(7):1171–1177. doi: 10.1515/bchm2.1972.353.2.1171. [DOI] [PubMed] [Google Scholar]
  42. Uvarov VYu, Tretiakov V. E., Leshchenko A. V., Rukavishnikov I. G., Dzhuzenova C. S., Tretiakova L. Z., Archakov A. I. Effect of the microenvironment on the tertiary structure of cytochrome P-450 LM2. Eur J Biochem. 1989 May 1;181(2):391–396. doi: 10.1111/j.1432-1033.1989.tb14737.x. [DOI] [PubMed] [Google Scholar]
  43. Van De Straat R., De Vries J., Kulkens T., Debets A. J., Vermeulen N. P. Paracetamol, 3-monoalkyl- and 3,5-dialkyl derivatives. Comparison of their microsomal cytochrome P-450 dependent oxidation and toxicity in freshly isolated hepatocytes. Biochem Pharmacol. 1986 Nov 1;35(21):3693–3699. doi: 10.1016/0006-2952(86)90653-2. [DOI] [PubMed] [Google Scholar]
  44. Vergères G., Winterhalter K. H., Richter C. Identification of the membrane anchor of microsomal rat liver cytochrome P-450. Biochemistry. 1989 May 2;28(9):3650–3655. doi: 10.1021/bi00435a005. [DOI] [PubMed] [Google Scholar]
  45. Vermilion J. L., Coon M. J. Purified liver microsomal NADPH-cytochrome P-450 reductase. Spectral characterization of oxidation-reduction states. J Biol Chem. 1978 Apr 25;253(8):2694–2704. [PubMed] [Google Scholar]
  46. Waterman M. R., Ullrich V., Estabrook R. W. Effect of substrate on the spin state of cytochrome P-450 in hepatic microsomes. Arch Biochem Biophys. 1973 Apr;155(2):355–360. doi: 10.1016/0003-9861(73)90124-0. [DOI] [PubMed] [Google Scholar]
  47. Wessel D., Flügge U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. doi: 10.1016/0003-2697(84)90782-6. [DOI] [PubMed] [Google Scholar]
  48. Yih T. D., van Rossum J. M. K5 values of some homologous series of barbiturates and the relationship with the lipophilicity and metabolic clearance. Biochem Pharmacol. 1977 Nov 15;26(22):2117–2120. doi: 10.1016/0006-2952(77)90261-1. [DOI] [PubMed] [Google Scholar]
  49. Zumbuehl O., Weder H. G. Liposomes of controllable size in the range of 40 to 180 nm by defined dialysis of lipid/detergent mixed micelles. Biochim Biophys Acta. 1981 Jan 8;640(1):252–262. doi: 10.1016/0005-2736(81)90550-2. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES