Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Feb 1;104(2):209–219. doi: 10.1083/jcb.104.2.209

Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies

PMCID: PMC2114413  PMID: 2433292

Abstract

Fifteen peptides, ranging in length from 6 to 31 amino acids and corresponding in sequence to portions of the major phenobarbital- inducible form of rat liver cytochrome P-450 (P-450 PB-4), were previously synthesized chemically and used to prepare site-specific rabbit antibodies (Frey, A. B., D.J. Waxman, and G. Kreibich, 1985, J. Biol. Chem., 260:15253-15265). The antipeptide antibodies were affinity purified using Sepharose resins derivatized with the respective peptides and 14 preparations were obtained that in an ELISA assay showed affinities to immobilized P-450 judged to be adequate for binding studies on intact rat liver microsomes. The binding of these antibodies to rough microsomes from the livers of phenobarbital treated rats was assessed using 125I-labeled IgG and by immunoelectron microscopy employing protein A-gold as a marker. It was found that many of the antibodies bound to the cytoplasmic surface of the membrane but none bound to the luminal face of ruptured or inverted microsomal vesicles or to contaminating membranes of other organelles present in the preparations. These observations eliminate previously proposed models for the transmembrane disposition of P-450 that postulate the existence of multiple transmembrane domains and the exposure of several polar segments of the polypeptide on the luminal side of the membrane. The fact that an antibody raised to the first 31 residues of P-450 bound well to the purified P-450 but very poorly to rough microsomes, whereas an antibody to a peptide comprising residues 24-38 showed relatively strong binding to intact microsomes, is consistent with the proposal that the amino terminal segment of P-450 extending approximately to residue 20 is embedded in the phospholipid bilayer and the immediately following segment is exposed on the cytoplasmic surface of the membrane. All these results favor a model in which the cytochrome P-450 molecule is largely exposed on the cytoplasmic surface of the endoplasmic reticulum membrane to which it is anchored by its short amino terminal hydrophobic segment.

Full Text

The Full Text of this article is available as a PDF (4.4 MB).

Selected References

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

  1. Adelman M. R., Blobel G., Sabatini D. D. An improved cell fractionation procedure for the preparation of rat liver membrane-bound ribosomes. J Cell Biol. 1973 Jan;56(1):191–205. doi: 10.1083/jcb.56.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adesnik M., Atchison M. Genes for cytochrome P-450 and their regulation. CRC Crit Rev Biochem. 1986;19(3):247–305. doi: 10.3109/10409238609084657. [DOI] [PubMed] [Google Scholar]
  3. Bar-Nun S., Kreibich G., Adesnik M., Alterman L., Negishi M., Sabatini D. D. Synthesis and insertion of cytochrome P-450 into endoplasmic reticulum membranes. Proc Natl Acad Sci U S A. 1980 Feb;77(2):965–969. doi: 10.1073/pnas.77.2.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Black S. D., Coon M. J. Structural features of liver microsomal NADPH-cytochrome P-450 reductase. Hydrophobic domain, hydrophilic domain, and connecting region. J Biol Chem. 1982 May 25;257(10):5929–5938. [PubMed] [Google Scholar]
  5. Black V. H., Cornacchia L., 3rd Stereological analysis of peroxisomes and mitochondria in intestinal epithelium of patients with peroxisomal deficiency disorders: Zellweger's syndrome and neonatal-onset adrenoleukodystrophy. Am J Anat. 1986 Sep;177(1):107–118. doi: 10.1002/aja.1001770112. [DOI] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. Cooper M. B., Craft J. A., Estall M. R., Rabin B. R. Asymmetric distribution of cytochrome P-450 and NADPH--cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver. Biochem J. 1980 Sep 15;190(3):737–746. doi: 10.1042/bj1900737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Frey A. B., Kreibich G., Wadhera A. B., Clarke L., Waxman D. J. 3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine photolabels a substrate-binding site of rat hepatic cytochrome P-450 form PB-4. Biochemistry. 1986 Aug 26;25(17):4797–4803. doi: 10.1021/bi00365a012. [DOI] [PubMed] [Google Scholar]
  9. Frey A. B., Waxman D. J., Kreibich G. The structure of phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4. Production and characterization of site-specific antibodies. J Biol Chem. 1985 Dec 5;260(28):15253–15265. [PubMed] [Google Scholar]
  10. Friedlander M., Blobel G. Bovine opsin has more than one signal sequence. 1985 Nov 28-Dec 4Nature. 318(6044):338–343. doi: 10.1038/318338a0. [DOI] [PubMed] [Google Scholar]
  11. Fujii-Kuriyama Y., Mizukami Y., Kawajiri K., Sogawa K., Muramatsu M. Primary structure of a cytochrome P-450: coding nucleotide sequence of phenobarbital-inducible cytochrome P-450 cDNA from rat liver. Proc Natl Acad Sci U S A. 1982 May;79(9):2793–2797. doi: 10.1073/pnas.79.9.2793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gotoh O., Tagashira Y., Iizuka T., Fujii-Kuriyama Y. Structural characteristics of cytochrome P-450. Possible location of the heme-binding cysteine in determined amino-acid sequences. J Biochem. 1983 Mar;93(3):807–817. doi: 10.1093/jb/93.3.807. [DOI] [PubMed] [Google Scholar]
  13. Heinemann F. S., Ozols J. The covalent structure of rabbit phenobarbital-induced cytochrome P-450. Partial amino acid sequence and order of cyanogen bromide peptides. J Biol Chem. 1982 Dec 25;257(24):14988–14999. [PubMed] [Google Scholar]
  14. Hopp T. P., Woods K. R. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3824–3828. doi: 10.1073/pnas.78.6.3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Horisberger M., Rosset J. Colloidal gold, a useful marker for transmission and scanning electron microscopy. J Histochem Cytochem. 1977 Apr;25(4):295–305. doi: 10.1177/25.4.323352. [DOI] [PubMed] [Google Scholar]
  16. Inouye M., Halegoua S. Secretion and membrane localization of proteins in Escherichia coli. CRC Crit Rev Biochem. 1980;7(4):339–371. doi: 10.3109/10409238009105465. [DOI] [PubMed] [Google Scholar]
  17. Kaiser E. T., Kézdy F. J. Amphiphilic secondary structure: design of peptide hormones. Science. 1984 Jan 20;223(4633):249–255. doi: 10.1126/science.6322295. [DOI] [PubMed] [Google Scholar]
  18. Kreibich G., Sabatini D. D., Adesnik M. Biosynthesis of hepatocyte endoplasmic reticulum proteins. Methods Enzymol. 1983;96:530–542. doi: 10.1016/s0076-6879(83)96046-9. [DOI] [PubMed] [Google Scholar]
  19. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Lewis J. A., Sabatini D. D. Accessibility of proteins in rat liver-free and membrane-bound ribosomes to lactoperoxidase-catalyzed iodination. J Biol Chem. 1977 Aug 10;252(15):5547–5555. [PubMed] [Google Scholar]
  22. Lu A. Y., West S. B. Multiplicity of mammalian microsomal cytochromes P-45. Pharmacol Rev. 1979 Dec;31(4):277–295. [PubMed] [Google Scholar]
  23. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  24. Masters B. S., Okita R. T. The history, properties, and function of NADPH-cytochrome P-450 reductase. Pharmacol Ther. 1980;9(2):227–244. doi: 10.1016/s0163-7258(80)80020-9. [DOI] [PubMed] [Google Scholar]
  25. Matsuura S., Fujii-Kuriyama Y., Tashiro Y. Immunoelectron microscope localization of cytochrome P-450 on microsomes and other membrane structures of rat hepatocytes. J Cell Biol. 1978 Aug;78(2):503–519. doi: 10.1083/jcb.78.2.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matsuura S., Fujii-Kuriyama Y., Tashiro Y. Quantitative immunoelectron-microscopic analyses of the distribution of cytochrome P-450 molecules on rat liver microsomes. J Cell Sci. 1979 Apr;36:413–435. doi: 10.1242/jcs.36.1.413. [DOI] [PubMed] [Google Scholar]
  27. Matsuura S., Masuda R., Sakai O., Tashiro Y. Immunoelectron microscopy of the outer membrane of rat hepatocyte nuclear envelopes in relation to the rough endoplasmic reticulum. Cell Struct Funct. 1983 Mar;8(1):1–9. doi: 10.1247/csf.8.1. [DOI] [PubMed] [Google Scholar]
  28. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  29. Nilsson O. S., DePierre J. W., Dallner G. Investigation of the transverse topology of the microsomal membrane using combinations of proteases and the non-penetrating reagent diazobenzene sulfonate. Biochim Biophys Acta. 1978 Jul 20;511(1):93–104. doi: 10.1016/0005-2736(78)90067-6. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Rodriguez Boulan E., Sabatini D. D., Pereyra B. N., Kreibich G. Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins. J Cell Biol. 1978 Sep;78(3):894–909. doi: 10.1083/jcb.78.3.894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Roth J., Bendayan M., Orci L. Ultrastructural localization of intracellular antigens by the use of protein A-gold complex. J Histochem Cytochem. 1978 Dec;26(12):1074–1081. doi: 10.1177/26.12.366014. [DOI] [PubMed] [Google Scholar]
  33. Ryan D. E., Thomas P. E., Levin W. Purification of characterization of a minor form of hepatic microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls. Arch Biochem Biophys. 1982 Jun;216(1):272–288. doi: 10.1016/0003-9861(82)90212-0. [DOI] [PubMed] [Google Scholar]
  34. Ryan D. E., Thomas P. E., Reik L. M., Levin W. Purification, characterization and regulation of five rat hepatic microsomal cytochrome P-450 isozymes. Xenobiotica. 1982 Nov;12(11):727–744. doi: 10.3109/00498258209038947. [DOI] [PubMed] [Google Scholar]
  35. Sabatini D. D., Kreibich G., Morimoto T., Adesnik M. Mechanisms for the incorporation of proteins in membranes and organelles. J Cell Biol. 1982 Jan;92(1):1–22. doi: 10.1083/jcb.92.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schiffer M., Edmundson A. B. Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys J. 1967 Mar;7(2):121–135. doi: 10.1016/S0006-3495(67)86579-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shinnick T. M., Sutcliffe J. G., Green N., Lerner R. A. Synthetic peptide immunogens as vaccines. Annu Rev Microbiol. 1983;37:425–446. doi: 10.1146/annurev.mi.37.100183.002233. [DOI] [PubMed] [Google Scholar]
  38. Steiner D. F., Quinn P. S., Chan S. J., Marsh J., Tager H. S. Processing mechanisms in the biosynthesis of proteins. Ann N Y Acad Sci. 1980;343:1–16. doi: 10.1111/j.1749-6632.1980.tb47238.x. [DOI] [PubMed] [Google Scholar]
  39. Suwa Y., Mizukami Y., Sogawa K., Fujii-Kuriyama Y. Gene structure of a major form of phenobarbital-inducible cytochrome P-450 in rat liver. J Biol Chem. 1985 Jul 5;260(13):7980–7984. [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. Thomas P. E., Lu A. Y., West S. B., Ryan D., Miwa G. T., Levin W. Accessibility of cytochrome P450 in microsomal membranes: inhibition of metabolism by antibodies to cytochrome P450. Mol Pharmacol. 1977 Sep;13(5):819–831. [PubMed] [Google Scholar]
  42. Waxman D. J., Walsh C. Phenobarbital-induced rat liver cytochrome P-450. Purification and characterization of two closely related isozymic forms. J Biol Chem. 1982 Sep 10;257(17):10446–10457. [PubMed] [Google Scholar]
  43. Welton A. F., Aust S. D. The effects of 3-methylcholanthrene and phenobarbital induction on the structure of the rat liver endoplasmic reticulum. Biochim Biophys Acta. 1974 Dec 10;373(2):197–210. doi: 10.1016/0005-2736(74)90145-x. [DOI] [PubMed] [Google Scholar]
  44. West S. B., Huang M. T., Miwa G. T., Lu A. Y. A simple and rapid procedure for the purification of phenobarbital-inducible cytochrome P-450 from rat liver microsomes. Arch Biochem Biophys. 1979 Mar;193(1):42–50. doi: 10.1016/0003-9861(79)90006-7. [DOI] [PubMed] [Google Scholar]
  45. Yuan P. M., Ryan D. E., Levin W., Shively J. E. Identification and localization of amino acid substitutions between two phenobarbital-inducible rat hepatic microsomal cytochromes P-450 by micro sequence analyses. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1169–1173. doi: 10.1073/pnas.80.5.1169. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES