Abstract

The multiplicity of phenobarbital-induced cytochromes P-450 in live microsomes from male rats was investigated by using two-dimensional gel electrophoresis, peptide fingerprinting, and immunoaffinity chromatography. Two colonies each of Holtzman and Long-Evans rats were studied. Four molecular forms of phenobarbital-induced cytochromes P-450 were distinguished as polypeptides (designated PB3, variant PB3, PB4, and PB5) which showed apparent immunochemical identity and greater than or equal to 95% fingerprint homology. Two of these polypeptides corresponded to cytochrome P-450b [Ryan, D., Thomas, P. E., Korzeniowski, D., & Levin, W. (1979) J. Biol. Chem. 254, 1365-1374] and cytochrome P-450e [Ryan, D., & Levin, W. (1981) Fed. Proc., Fed. Am. Soc. Exp. Biol. 40, 1640] which had been purified from Long-Evans rats (variant PB3 and PB5, respectively). Each rat colony was characterized by unique combinations of two or three of these immunochemically related forms of cytochromes P-450. Cytochrome P-450e was present in rats from all four colonies, but cytochrome P-450b was only found in Long-Evans rats. Polypeptide PB3 was only found in the two colonies of Holtzman rats, whereas polypeptide PB4 was present in one colony each of Holtzman and Long-Evans rats. In addition to these forms of cytochrome P-450, rats from each colony also evidenced three other major phenobarbital-induced polypeptides which gave unique fingerprints, and one of these was identified as representing epoxide hydrolase. Proteolytic digestion studies of intact microsomes demonstrated that the four immunochemically identical forms of cytochrome P-450 were partially exposed on the outer (cytoplasmic) surface of microsomes. However, polypeptide PB3 was characterized by the greatest rate of proteolytic degradation. These results clearly demonstrate that phenobarbital-induced cytochromes P-450 include microheterogeneous proteins which show remarkable variations related to rat strains and/or colony.

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