Multiplicity, strain differences, and topology of phenobarbital-induced cytochromes P-450 in rat liver microsomes

Abstract

The multiplicity of phenobarbital-induced cytochromes P-450 in liver microsomes from male rats was investigated using 2-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 .gtoreq. 95% fingerprint homology. Two of these polypeptides corresponded to cytochrome P-450b and cytochrome P-450e which were purified from Long-Evans rats (variant PB3 and PB5, respectively). Each rat colony was characterized by unique combinations of 2 or 3 of these immunochemically related forms of cytochrome P-450. Cytochrome P-450e was present in rats from all 4 colonies, but cytochrome P-450b was only found in Long-Evans rats. Polypeptide PB3 was only found in the 2 colonies of Holtzman rats, whereas polypeptide PB4 was present in 1 colony each of Holtzman and Long-Evans rats. Rats from each colony also evidenced 3 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 4 immunochemically identical forms of cytochrome P-450 were partially exposed on the outer (cytoplasmic) surface of microsomes. Polypeptide PB3 was characterized by the greatest rate of proteolytic degradation. Evidently, phenobarbital-induced cytochromes P-450 include microheterogeneous proteins which show remarkable variations related to rat strain and/or colony.