Comparisons of highly purified hepatic microsomal cytochromes P-450 from Holtzman and Long-Evans rats.
The present study describes the purification and characterization of strain variant forms of a major phenobarbital-inducible microsomal hemoprotein, cytochrome P-450b, from Holtzman and Long-Evans rats. The strain variant hemoproteins cannot be resolved by sodium dodecyl sulfate gel electrophoresis, but can be partially separated in two-dimensional isoelectric focusing SDS gels. If, however, sodium tetradecyl sulfate is incorporated into the one-dimensional gel system, separation of the cytochromes P-450b is achieved. Minor structural differences are detected in the peptides of the cytochromes P-450b following limited proteolysis by Staphylococcus aureus V8 protease, cleavage by cyanogen bromide, or reverse-phase high-pressure liquid chromatography of tryptic peptides. The strain variant cytochromes P-450b are immunochemically and spectrally indistinguishable. The optical spectra of the ferric and ferrous hemoproteins are identical, as are the CO- and ethylisocyanide-reduced difference spectra. Ferrous cytochromes P-450b from both rat strains effectively bind metyrapone with equivalent affinities. In addition, the cytochromes P-450b do not differ in their catalytic activities toward benzphetamine, hexobarbital, benzo [a]pyrene, zoxazolamine, 7-ethoxycoumarin, estradiol-17 beta and testosterone. Cytochrome P-450c, the predominant isozyme inducible in rat liver by 3-methylcholanthrene, was purified from Holtzman and Long-Evans rats. Cytochromes P-450c from both rat strains are indistinguishable based on electrophoretic, immunological, spectral and catalytic properties. Minor structural differences in the cytochromes P-450c were revealed in the reverse-phase high-pressure liquid chromatographic profiles of the tryptic peptides of these hemoproteins, but not in the peptides generated by limited proteolysis or cleavage with cyanogen bromide.
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D007527:Isoenzymes / Q000302:isolation & purification; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity; D013379:Substrate Specificity
D007527:Isoenzymes; D010446:Peptide Fragments; D003577:Cytochrome P-450 Enzyme System