Benzo[a]pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from beta-naphthoflavone-fed rainbow trout.
We have purified five isozymes of liver microsomal (LM) P-450 from beta-naphthoflavone-fed rainbow trout. Four forms (LM3, LM1, LM4a and LMx) were resolved on DEAE-Sepharose. Chromatography on hydroxylapatite further resolved LMx into two components, LM2 and LM4b. This latter form, obtained in highest yield (5%), had an apparent minimum molecular weight (Mr), as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), of 58,000, a specific content of 11.9 nmoles/mg, a lambda max in the carbon monoxide-ligated, reduced difference spectrum of 447.0 nm, and was active towards benzo[a]pyrene in a reconstituted system. A second form, LM4a, obtained in a final yield of 2%, had a specific content of 10.3 and was indistinguishable from Lm4b by Mr, lambda max, or activity towards benzo[a]pyrene. Form LM2 (2% yield) had a specific content of 10.8, a Mr of 54,000, a lambda max of 449.5 nm, and was not effective in reconstitution of benzo[a]pyrene-hydroxylase. In addition, two other forms with lower specific contents were obtained, LM1 and LM3. Neither LM1 nor LM3 was active towards benzo[a]pyrene. The properties of LM2, LM4a and LM4b were further examined with the aid of antibodies prepared from rabbits. Antibodies to LM4a and LM4b each cross-reacted with the other antigen and formed lines of identity on Ouchterlony plates, and both IgGs exhibited some cross-reaction to P-448 from rat. Neither antibody cross-reacted with trout LM2, and LM2-IgG did not cross-react with any other purified P-450. Benzo[a]pyrene-hydroxylase, catalyzed by either LM4a or LM4b, was inhibited by LM4b-IgG but not by LM4a-IgG, suggesting that these antibodies recognize different antigenic sites. Further comparison of LM4a and LM4b by amino acid composition, peptide mapping, kinetic properties, sensitivity to alpha-naphthoflavone, and regioselectivity towards benzo[a]pyrene-dihydrodiol formation indicates that these forms are highly similar in structure and function.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000936:Antigen-Antibody Complex; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001571:Benzoflavones / Q000494:pharmacology*; D001579:Benzopyrene Hydroxylase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology*; D007106:Immune Sera; D005779:Immunodiffusion; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D014337:Trout; D019324:beta-Naphthoflavone
D000596:Amino Acids; D000936:Antigen-Antibody Complex; D001571:Benzoflavones; D005419:Flavonoids; D007106:Immune Sera; D007527:Isoenzymes; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D001579:Benzopyrene Hydroxylase; D001189:Aryl Hydrocarbon Hydroxylases