Purification and characterization of liver microsomal cytochromes p-450: electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or beta-naphthoflavone

Biochemistry. 1982 Nov 9;21(23):6019-30. doi: 10.1021/bi00266a045.


Eight different forms of cytochrome P-450 (P-450) were purified to electrophoretic homogeneity by a common procedure from liver microsomes of rats treated with phenobarbital or beta-naphthoflavone. Antibodies were prepared to seven of these forms in rabbits. The eight P-450s were distinguished by spectral properties of the ferric, ferrous, and ferrous carbonyl forms, apparent monomeric molecular weights, peptide mapping, immunological reactivity as discerned by double-diffusion immunoprecipitin analysis and crossed immunoelectrophoresis, and catalytic activities toward the substrates acetanilide, aminopyrine, aniline, benzo[a]-pyrene, d-benzphetamine, N,N-dimethylnitrosamine, 7-ethoxycoumarin, 7-ethoxyresorufin, ethylmorphine, p-nitroanisole, testosterone, and (R)- and (S)-warfarin. Crossed sodium dodecyl sulfate-polyacrylamide gel immunoelectrophoresis was used to estimate the levels of each of the eight forms of P-450 present in the liver microsomes of untreated rats and rats treated with phenobarbital, 5,6-benzoflavone, pregnenolone-16 alpha-carbonitrile, isosafrole, or the polychlorinated biphenyl mixture Aroclor 1254. In each situation, the sum of the levels of these eight P-450s was at least as high as the spectrally determined P-450 content. The results clearly demonstrate that individual forms of P-450 can be induced by different compounds and that a single compound can lower the level of one form of P-450 while inducing one or more other forms of P-450. Catalytic activities toward each of the substrates observed with microsomal preparations are compared to rates predicted on the basis of the content of each of the eight P-450s. These studies provide a basis for further studies on the regulation of individual P-450s, the physical properties of the different P-450s, and the metabolic consequences of changes in the forms of P-450 in rat liver models.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Benzoflavones / pharmacology*
  • Cytochrome P-450 Enzyme System / isolation & purification*
  • Cytochrome P-450 Enzyme System / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Induction / drug effects
  • Flavonoids / pharmacology*
  • Immunologic Techniques
  • Isoenzymes / isolation & purification*
  • Isoenzymes / metabolism
  • Male
  • Microsomes, Liver / enzymology*
  • Phenobarbital / pharmacology*
  • Rats
  • Rats, Inbred Strains
  • Spectrophotometry
  • Substrate Specificity
  • beta-Naphthoflavone


  • Benzoflavones
  • Flavonoids
  • Isoenzymes
  • beta-Naphthoflavone
  • Cytochrome P-450 Enzyme System
  • Phenobarbital