Cytochrome P-450 isozyme 1 from phenobarbital-induced rat liver: purification, characterization, and interactions with metyrapone and cytochrome b5

Biochemistry. 1983 Sep 27;22(20):4846-55. doi: 10.1021/bi00289a035.


Cytochrome P-450 isozyme 1 (PB-1) (Mr congruent to 53 000) was purified to apparent homogeneity from phenobarbital (PB)-induced rat liver microsomes, and its spectral, structural, immunochemical, and catalytic properties were determined. PB-1, present in significant amounts in uninduced rat liver microsomes, is induced approximately 2-4-fold by phenobarbital, as compared to the greater than 30-fold induction typical of the major PB isozymes characterized previously. PB-1 was distinguished from the major PB-induced isozymes PB-4 and PB-5 [Waxman, D. J., & Walsh, C. (1982) J. Biol. Chem. 257, 10446-10457] by the absence of a Fe2+-metyrapone P446 complex, by its unique NH2-terminal sequence and distinct peptide maps, by the lack of immuno-cross-reactivity to PB-4, and by its characteristic substrate-specificity profile. Metyrapone effected a saturable enhancement of several PB-1-catalyzed reactions in the reconstituted system [Km(metyrapone) congruent to 200 microM], which varied in magnitude with the substrate, with a maximal stimulation of 5-8-fold in the case of acetanilide 4-hydroxylation. That metyrapone enhanced the corresponding microsomal activities only in cases where the metyrapone-sensitive PB-4 did not catalyze the same reaction at significant rates suggested that PB-1 is probably responsible for the substrate-dependent stimulatory effects of metyrapone on microsomal monooxygenations. In contrast to PB-4 and PB-5, PB-1 was characterized by a marked, but not absolute, dependence on cytochrome b5 (b5) for catalytic activity, with 4-7-fold stimulations typically effected by inclusion of stoichiometric b5 in the reconstituted system. That these b5-stimulations were lipid dependent and were abolished with specific proteolytic fragments lacking b5's COOH-terminal membranous segment evidenced the importance of this segment for efficient, b5-mediated electron transfer to P-450 PB-1 in the reconstituted monooxygenase system.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cytochrome P-450 Enzyme System / isolation & purification
  • Cytochrome P-450 Enzyme System / metabolism*
  • Cytochrome b Group / metabolism*
  • Cytochromes b5
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism
  • Kinetics
  • Metyrapone / pharmacology*
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology*
  • Peptide Fragments / analysis
  • Phenobarbital / pharmacology*
  • Rabbits
  • Rats
  • Rats, Inbred Strains
  • Species Specificity
  • Substrate Specificity


  • Cytochrome b Group
  • Isoenzymes
  • Peptide Fragments
  • Cytochromes b5
  • Cytochrome P-450 Enzyme System
  • Phenobarbital
  • Metyrapone