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. 1977 Dec;74(12):5477–5481. doi: 10.1073/pnas.74.12.5477

Hepatic mitochondrial cytochrome P-450: Isolation and functional characterization

R Sato *, Y Atsuta †, Y Imai *, S Taniguchi †, K Okuda †
PMCID: PMC431771  PMID: 271969

Abstract

A CO-binding heme protein was solubilized and partially purified from the inner membrane fraction of rat liver mitochondria by a modification of a method [Imai, Y. & Sato, R. (1974) Biochem. Biophys. Res. Commun. 60, 8-14] developed to purify cytochrome P-450 from liver microsomes. The partially purified preparation contained protoheme and its spectral properties are characteristic of the heme proteins of the cytochrome P-450 family. The isolated cytochrome P-450 preparation could reconstitute a CO-sensitive, NADPH-dependent 26-hydroxylation activity for 5β-cholestane-3α,7α,-12α-triol when supplemented with NADPH-adrenodoxin reductase and adrenodoxin, both purified from bovine adrenocortical mitochondria. Unlike a cytochrome P-450 purified from liver microsomes of drug-untreated rats, however, the liver mitochondrial cytochrome P-450 could not catalyze NADPH-dependent benzphetamine N-demethylation in the presence of adrenodoxin reductase and adrenodoxin or function with the purified microsomal NADPH-cytochrome c reductase plus Emulgen 913 as an electron-donating system. It is concluded that the rat liver inner mitochondrial membrane houses a species of cytochrome P-450 functional in 5β-cholestane-3α,7α,12α-triol 26-hydroxylation.

Keywords: heme protein, liver mitochondria, bile acid, cholestanetriol 26-hydroxylase

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Selected References

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

  1. Björkhem I., Danielsson H., Wikvall K. Side chain hydroxylations in biosynthesis of cholic acid. 25- and 26-Hydroxylation of 5beta-cholestane-3alpha, 7alpha, 12alpha-triol by reconstituted systems from rat liver microsomes. J Biol Chem. 1976 Jun 10;251(11):3495–3495. [PubMed] [Google Scholar]
  2. Björkhem I., Gustafsson J. Mitochondrial omega-hydroxylation of cholesterol side chain. J Biol Chem. 1974 Apr 25;249(8):2528–2535. [PubMed] [Google Scholar]
  3. Björkhem I., Gustafsson J. Omega-hydroxylation of steriod side-chain in biosynthesis of bile acids. Eur J Biochem. 1973 Jul 2;36(1):201–212. doi: 10.1111/j.1432-1033.1973.tb02902.x. [DOI] [PubMed] [Google Scholar]
  4. Cronholm T., Johansson G. Oxidation of 5 beta-cholestane-3alpha, 7alpha, 12alpha-triol by rat liver microsomes. Eur J Biochem. 1970 Oct;16(2):373–381. doi: 10.1111/j.1432-1033.1970.tb01091.x. [DOI] [PubMed] [Google Scholar]
  5. Ghazarian J. G., Jefcoate C. R., Knutson J. C., Orme-Johnson W. H., DeLuca H. F. Mitochondrial cytochrome p450. A component of chick kidney 25-hydrocholecalciferol-1alpha-hydroxylase. J Biol Chem. 1974 May 25;249(10):3026–3033. [PubMed] [Google Scholar]
  6. 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]
  7. Imai Y., Sato R. A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits. Biochem Biophys Res Commun. 1974 Sep 9;60(1):8–14. doi: 10.1016/0006-291x(74)90164-8. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Lu A. Y., Levin W., Kuntzman R. Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. VII. Stimulation of benzphetamine N-demethylation by lipid and detergent. Biochem Biophys Res Commun. 1974 Sep 9;60(1):266–272. doi: 10.1016/0006-291x(74)90200-9. [DOI] [PubMed] [Google Scholar]
  10. Nishibayashi H., Sato R. Preparation of hepatic microsomal particles containing P-450 as the sole heme constituent and absolute spectra of P-450. J Biochem. 1968 Jun;63(6):766–779. doi: 10.1093/oxfordjournals.jbchem.a128842. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Okuda K., Hoshita N. Oxidation of 5-beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol by rat-liver mitochondria. Biochim Biophys Acta. 1968 Oct 22;164(2):381–388. [PubMed] [Google Scholar]
  13. Okuda K., Weber P., Ullrich V. Photochemical action spectrum of the co-inhibited 5beta-cholestane- 3alpha, 7alpha, 12alpha-triol 26-hydroxylase system. Biochem Biophys Res Commun. 1977 Feb 7;74(3):1071–1076. doi: 10.1016/0006-291x(77)91627-8. [DOI] [PubMed] [Google Scholar]
  14. Pedersen J. I., Ghazarian J. G., Orme-Johnson N. R., DeLuca H. F. Isolation of chick renal mitochondrial ferredoxin active in the 25-hydroxyvitamin D3-1alpha-hydroxylase system. J Biol Chem. 1976 Jul 10;251(13):3933–3941. [PubMed] [Google Scholar]
  15. Suhara K., Ikeda Y., Takemori S., Katagiri M. The purification and properties of NADPH-adrenodoxin reductase from bovine adrenocortical mitochondria. FEBS Lett. 1972 Nov 15;28(1):45–47. doi: 10.1016/0014-5793(72)80673-2. [DOI] [PubMed] [Google Scholar]
  16. Suhara K., Takemori S., Katagiri M. Improved purification of bovine adrenal iron-sulfur protein. Biochim Biophys Acta. 1972 Apr 15;263(2):272–278. doi: 10.1016/0005-2795(72)90079-7. [DOI] [PubMed] [Google Scholar]
  17. Taniguchi S., Hoshita N., Okuda K. Enzymatic characteristics of CO-sensitive 26-hydroxylase system for 5beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol in rat-liver mitochondria and its intramitochondrial localization. Eur J Biochem. 1973 Dec 17;40(2):607–617. doi: 10.1111/j.1432-1033.1973.tb03233.x. [DOI] [PubMed] [Google Scholar]

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