Original Article

Journal of Investigative Dermatology (1992) 98, 233–240; doi:10.1111/1523-1747.ep12556034

Purification and Molecular Characterization of bold beta-Naphthoflavone–Inducible Cytochrome P-450 from Rat Epidermis

Haider Raza1,2,3, Rajesh Agarwal1,2,3, David R Bickers1,2,3 and Hasan Mukhtar1,2,3

  1. 1Department of Dermatology, University Hospitals of Cleveland, Ohio, U.S.A.
  2. 2Skin Diseases Research Center, Case Western Reserve University, Ohio, U.S.A.
  3. 3Department of Veterans Affairs Medical Center, Cleveland, Ohio, U.S.A.

Received 16 July 1991; Accepted 22 October 1991.

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Abstract

This study was designed to characterize epidermal cytochrome P-450 (P-450) induced by skin application of beta-naphthoflavone (beta-NP). Topical application of beta-NF (40 mg/kg) to rats resulted in a 2.6-times increase in epidermal P-450 content and a 3-14-times increase in epidermal monooxygenase activities. The purified epidermal P-450 showed a major band at 54 kDa on SDS-PAGE, which co-migrated with hepatic P-4501A1 and cross-reacted with monoclonal and polyclonal antibodies specific to P-4501A1. The specific content of purified epidermal P-450 was 1.53 nmol/mg protein, representing 42-times purification. HPLC analysis of the purified epidermal P-450 showed similar elution profile and retention time as that of hepatic P-4501A1. The purified preparation efficiently catalyzedbenzo(a)pyrene hydroxylation when reconstituted with purified NADPH-P-450 reductase and phospholipid. Peptide fingerprint analysis of the purified epidermal P-450 and hepatic P-4501A1 showed similar monoclonal antibody 1-7-1 reacting epitopes. Partial N-terminal amino acid sequence analysis of purified epidermal P-450 showed complete homology with the known sequence of P-4501A1. Similarly, HPLC analysis of tryptic digest of purified epidermal P-450 and hepatic P-4501A1 showed identical peptide peaks with comparable retention times. N-terminal amino acid sequence analysis of three randomly selected tryptic peptides showed complete homology with the known sequence of P-4501A1. These results indicate that rat epidermal P-450 induced by beta-NF is similar to hepatic P-4501A1.

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