Purification and molecular characterization of beta-naphthoflavone-inducible cytochrome P-450 from rat epidermis.
This study was designed to characterize epidermal cytochrome P-450 (P-450) induced by skin application of beta-naphthoflavone (beta-NF). 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 comigrated 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 catalyzed-benzo(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.
Raza
H
H
Department of Dermatology, University Hospitals of Cleveland, Ohio.
Agarwal
R
R
Bickers
D R
DR
Mukhtar
H
H
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001571:Benzoflavones / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D004817:Epidermis / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism; D051381:Rats; D012867:Skin / Q000648:ultrastructure; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D015660:7-Alkoxycoumarin O-Dealkylase; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1