Monoclonal antibodies to rat liver cytochrome P-450 2c/RLM5 that regiospecifically inhibit steroid metabolism

Biochem Pharmacol. 1989 Sep 15;38(18):3067-74. doi: 10.1016/0006-2952(89)90017-8.


Hybridomas were formed from myeloma cells and spleen cells derived from BALB/c female mice immunized with purified liver microsomal cytochrome P-450 2c/RLM5 (P-450 gene IIC11) isolated from untreated adult male rats. Six hybridoma clones produced monoclonal antibodies (MAbs) of the IgM(kappa) type. All the MAbs bound strongly to P-450 2c/RLM5 when measured by radioimmunoassay, and four of the six specifically immunoprecipitated P-450 2c/RLM5 in an Ouchterlony double-immunodiffusion test. These four MAbs also bound but did not immunoprecipitate P-450 RLM3. The MAbs that precipitated P-450 2c/RLM5 neither bound nor precipitated P-450 PB-B (gene IIB1) and P-450 BNF-B (gene IA1) of rats or P-450 LM2 and P-450 LM4 of rabbits. In contrast, mouse polyclonal anti-P-450 2c/RLM5 antibody strongly immunoprecipitated P-450 RLM3 as well as P-450 2c/RLM5 and to a lesser extent P-450 PB-B and P-450 LM2. The MAbs that precipitated P-450 2c/RLM5 also inhibited by more than 90% androstenedione 16 alpha-hydroxylase activity of untreated rat microsomes, but did not inhibit microsomal 6 beta- or 7 alpha-hydroxylation. In addition, complete inhibition of both androstenedione 16 alpha-hydroxylation and testosterone 16 alpha-hydroxylation was observed in a reconstituted system with P-450 2c/RLM5. Androstenedione 6 beta-hydroxylation catalyzed by P-450 2c/RLM5 was also inhibited, whereas P-450 3-catalyzed 7 alpha-hydroxylation was not inhibited by the MAbs. P-450 2c/RLM5 catalyzed 2 alpha-, 16 alpha- and 6 beta-hydroxylation of progesterone in a reconstituted system were also inhibited by the MAb by 60-80%. These MAbs should prove useful for "reaction phenotyping," i.e. for defining the contribution of microsomal P-450 2c/RLM5 to the oxidative metabolism of endogenous steroids and other P-450 substrates in animal and human tissues.

Publication types

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

MeSH terms

  • Androstenedione / metabolism
  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibody Specificity
  • Aryl Hydrocarbon Hydroxylases*
  • Cytochrome P-450 CYP2C8
  • Cytochrome P-450 CYP2C9
  • Cytochrome P-450 Enzyme System / immunology*
  • Cytochrome P450 Family 2
  • Female
  • Hydroxylation
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Microsomes, Liver / enzymology*
  • Progesterone / metabolism
  • Rats
  • Steroid 16-alpha-Hydroxylase
  • Steroids / metabolism*


  • Antibodies, Monoclonal
  • Steroids
  • Androstenedione
  • Progesterone
  • Cytochrome P-450 Enzyme System
  • CYP2C9 protein, human
  • Cytochrome P-450 CYP2C9
  • Aryl Hydrocarbon Hydroxylases
  • CYP2C11 protein, rat
  • CYP2C8 protein, human
  • Cytochrome P-450 CYP2C8
  • Cytochrome P450 Family 2
  • Steroid 16-alpha-Hydroxylase