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. 1984 Jul;81(14):4260–4264. doi: 10.1073/pnas.81.14.4260

On the amino acid sequence of cytochrome P-450 isozyme 4 from rabbit liver microsomes.

V S Fujita, S D Black, G E Tarr, D R Koop, M J Coon
PMCID: PMC345567  PMID: 6589592

Abstract

Isozyme 4 of rabbit liver microsomal cytochrome P-450 was shown earlier in this laboratory to contain multiple NH2-terminal residues, whereas isozymes 2, 3a, 3b, and 3c have single, unique NH2-terminal sequences. Similar results were obtained with isozyme 4 obtained from animals that were untreated, treated with phenobarbital (which does not induce this isozyme), or induced with beta-naphthoflavone or isosafrole. With the use of selective chemical blocking at seryl or at nonprolyl residues, the complexity of the NH2-terminal sequence has now been shown to be due to the presence of three forms of the cytochrome differing only in the absence of the first or the first two residues: NH2-Ala-Met-Ser-Pro-Ala-Ala-Pro-, NH2-Met-Ser-Pro-Ala-Ala-Pro-, and NH2-Ser-Pro-Ala-Ala-Pro-. These forms may result from variable biological processing. Peptides containing the seven cysteine residues were sequenced and compared with similar peptides reported for other P-450 cytochromes; homology was extensive with respect to two of the cysteine regions in isozyme 4, and a third cysteine region showed partial identity. The sequence of peptides representing about two-thirds of the amino acids in isosafrole-induced cytochrome P-450 isozyme 4 was determined. Comparison with phenobarbital-induced rabbit cytochrome P-450 isozyme 2 indicated about 25% homology. In contrast, comparison of isozyme 4 with rat cytochrome P-450d, which is also induced by isosafrole and for which the sequence has recently been deduced from cDNA [Kawajiri, K., Gotoh, O., Sogawa, K., Tagashira, Y., Muramatsu, M. & Fujii-Kuriyama, Y. (1984) Proc. Natl. Acad. Sci. USA 81, 1649-1653], showed about 70% homology.

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Selected References

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  1. Bhown A. S., Cornelius T. W., Volanakis J. E., Bennett J. C. A comparison of fluorescamine and o-phthaldialdehyde as effective blocking reagents in protein sequence analyses by the Beckman sequencer. Anal Biochem. 1983 Jun;131(2):337–340. doi: 10.1016/0003-2697(83)90179-3. [DOI] [PubMed] [Google Scholar]
  2. Black S. D., Coon M. J. Simple, rapid, and highly efficient separation of amino acid phenylthiohydantoins by reversed-phase high-performance liquid chromatography. Anal Biochem. 1982 Apr;121(2):281–285. doi: 10.1016/0003-2697(82)90480-8. [DOI] [PubMed] [Google Scholar]
  3. Black S. D., Tarr G. E., Coon M. J. Structural features of isozyme 2 of liver microsomal cytochrome P-450. Identification of a highly conserved cysteine-containing peptide. J Biol Chem. 1982 Dec 25;257(24):14616–14619. [PubMed] [Google Scholar]
  4. Botelho L. H., Ryan D. E., Yuan P. M., Kutny R., Shively J. E., Levin W. Amino-terminal and carboxy-terminal sequence of hepatic microsomal cytochrome P-450d, a unique hemoprotein from rats treated with isosafrole. Biochemistry. 1982 Mar 16;21(6):1152–1155. doi: 10.1021/bi00535a007. [DOI] [PubMed] [Google Scholar]
  5. Delaforge M., Koop D. R., Coon M. J. Role of isosafrole as complexing agent and inducer of P-450LM4 in rabbit liver microsomes. Biochem Biophys Res Commun. 1982 Sep 16;108(1):59–65. doi: 10.1016/0006-291x(82)91831-9. [DOI] [PubMed] [Google Scholar]
  6. Drapeau G. R. Cleavage at glutamic acid with staphylococcal protease. Methods Enzymol. 1977;47:189–191. doi: 10.1016/0076-6879(77)47023-x. [DOI] [PubMed] [Google Scholar]
  7. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  8. Fisher G. J., Fukushima H., Gaylor J. L. Isolation, purification, and properties of a unique form of cytochrome P-450 in microsomes of isosafrole-treated rats. J Biol Chem. 1981 May 10;256(9):4388–4394. [PubMed] [Google Scholar]
  9. Fontana A., Vita C., Toniolo C. Selective cleavage of the single tryptophanyl peptide bond in horse heart cytochrome c. FEBS Lett. 1973 May 15;32(1):139–142. doi: 10.1016/0014-5793(73)80757-4. [DOI] [PubMed] [Google Scholar]
  10. Friedman M., Krull L. H., Cavins J. F. The chromatographic determination of cystine and cysteine residues in proteins as s-beta-(4-pyridylethyl)cysteine. J Biol Chem. 1970 Aug 10;245(15):3868–3871. [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. Haniu M., Armes L. G., Yasunobu K. T., Shastry B. A., Gunsalus I. C. Amino acid sequence of the Pseudomonas putida cytochrome P-450. II. Cyanogen bromide peptides, acid cleavage peptides, and the complete sequence. J Biol Chem. 1982 Nov 10;257(21):12664–12671. [PubMed] [Google Scholar]
  13. Haugen D. A., Armes L. G., Yasunobu K. T., Coon M. J. Amino-terminal sequence of phenobarbital-inducible cytochrome P-450 from rabbit liver microsomes: similarity to hydrophobic amino-terminal segments of preproteins. Biochem Biophys Res Commun. 1977 Aug 8;77(3):967–973. doi: 10.1016/s0006-291x(77)80072-7. [DOI] [PubMed] [Google Scholar]
  14. Haugen D. A., Coon M. J. Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450. J Biol Chem. 1976 Dec 25;251(24):7929–7939. [PubMed] [Google Scholar]
  15. Haugen D. A., van der Hoeven T. A., Coon M. J. Purified liver microsomal cytochrome P-450. Separation and characterization of multiple forms. J Biol Chem. 1975 May 10;250(9):3567–3570. [PubMed] [Google Scholar]
  16. Heinemann F. S., Ozols J. The complete amino acid sequence of rabbit phenobarbital-induced liver microsomal cytochrome P-450. J Biol Chem. 1983 Apr 10;258(7):4195–4201. [PubMed] [Google Scholar]
  17. 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]
  18. Kawajiri K., Gotoh O., Sogawa K., Tagashira Y., Muramatsu M., Fujii-Kuriyama Y. Coding nucleotide sequence of 3-methylcholanthrene-inducible cytochrome P-450d cDNA from rat liver. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1649–1653. doi: 10.1073/pnas.81.6.1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Koop D. R., Morgan E. T., Tarr G. E., Coon M. J. Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits. J Biol Chem. 1982 Jul 25;257(14):8472–8480. [PubMed] [Google Scholar]
  20. Ozols J., Heinemann F. S., Johnson E. F. Amino acid sequence of an analogous peptide from two forms of cytochrome P-450. J Biol Chem. 1981 Nov 25;256(22):11405–11408. [PubMed] [Google Scholar]
  21. Previero A., Barry L. G., Coletti-Previero M. A. Specific O-acylation of hydroxylamino acids in presence of free amino groups. Biochim Biophys Acta. 1972 Mar 15;263(1):7–13. doi: 10.1016/0005-2795(72)90154-7. [DOI] [PubMed] [Google Scholar]
  22. Ryan D. E., Thomas P. E., Levin W. Hepatic microsomal cytochrome P-450 from rats treated with isosafrole. Purification and characterization of four enzymic forms. J Biol Chem. 1980 Aug 25;255(16):7941–7955. [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Tarr G. E., Crabb J. W. Reverse-phase high-performance liquid chromatography of hydrophobic proteins and fragments thereof. Anal Biochem. 1983 May;131(1):99–107. doi: 10.1016/0003-2697(83)90140-9. [DOI] [PubMed] [Google Scholar]
  26. Tarr G. E. Improved manual sequencing methods. Methods Enzymol. 1977;47:335–357. doi: 10.1016/0076-6879(77)47036-8. [DOI] [PubMed] [Google Scholar]
  27. Yuan P. M., Nakajin S., Haniu M., Shinoda M., Hall P. F., Shively J. E. Steroid 21-hydroxylase (cytochrome P-450) from porcine adrenocortical microsomes: microsequence analysis of cysteine-containing peptides. Biochemistry. 1983 Jan 4;22(1):143–149. doi: 10.1021/bi00270a021. [DOI] [PubMed] [Google Scholar]
  28. 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]

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