Purification of liver microsomal cytochrome P-450 isozymes 3a and 6 from imidazole-treated rabbits. Evidence for the identity of isozyme 3a with the form obtained by ethanol treatment.
Two forms of cytochrome P-450 have been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated with imidazole. Several criteria indicate that the cytochrome of higher electrophoretic mobility is identical with ethanol-inducible isozyme 3a. "Imidazole-3a" and "ethanol-3a" exhibit the same chromatographic characteristics and have identical electrophoretic mobilities upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, the two protein preparations have the same absorbance maxima and absorption coefficients in the oxidized, reduced, and reduced-CO states. A single immunoprecipitin band exhibiting complete identity was observed upon reaction of imidazole-3a and ethanol-3a with the immunoglobulin G fraction from sheep immunized with the latter protein. The amino acid composition and first 10 residues of the amino terminus of the two protein preparations are indistinguishable, as are the high-performance liquid chromatographic maps of the peptides obtained upon cleavage with trypsin, Staphylococcus aureus V8 protease, or Lys C endoproteinase . Furthermore, these preparations have very similar activities in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline. Evidence was obtained that the cytochrome of lower electrophoretic mobility isolated from imidazole-treated rabbits is probably identical with isozyme 6; the spectral characteristics, amino acid composition, and carboxyl-terminal sequence are described. As an inducer, imidazole has the advantage over ethanol of being less variable in its effects and requiring a shorter period of treatment. From the resulting liver microsomes, one can readily isolate, in addition to P-450 isozymes 3a and 6, isozymes 3c and 4 as well as epoxide hydrolase.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000302:isolation & purification*; D004790:Enzyme Induction; D000431:Ethanol / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology*; D005779:Immunodiffusion; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification*; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits
D000596:Amino Acids; D007093:Imidazoles; D007527:Isoenzymes; D000431:Ethanol; C029899:imidazole; D003577:Cytochrome P-450 Enzyme System