Isolation and characterization of two constitutive forms of microsomal cytochrome P-450 from a single bovine liver.
Two constitutive forms of cytochrome P-450 isozyme were isolated from microsomes prepared from a single bovine liver. The two highly purified isozymes were electrophoretically homogeneous on SDS-polyacrylamide gel and their apparent minimum molecular weights were estimated to be 50 000 and 55 000. The isozyme of smaller molecular weight, designated cytochrome P-450A, and the one of large molecular weight, designated cytochrome P-450B, were distinct proteins by the criteria, SDS-polyacrylamide gel electrophoresis, peptide maps, amino acid contents. To reveal the immunochemical relation between these two isozymes, antibodies to each isozyme was raised in rabbit. Antibodies to cytochrome P-450A gave a single precipitin line against its antigen in Ouchterlony double-diffusion plates, but did not cross-react against cytochrome P-450B. On the other hand, antibodies to cytochrome P-450B formed a single precipitin line with its antigen and did not show any cross-reactivity against cytochrome P-450B. These results indicate that two isozymes are immunochemically distinct. This conclusion was supported by the results from immunochemical staining of the SDS-polyacrylamide gel electrophoretogram of the purified isozymes and detergent-solubilized bovine liver microsomes transferred to the nitrocellulose sheet. Both cytochromes P-450 showed high catalytic activities toward (+)-benzphetamine and aminopyrine in reconstituted systems, indicating that both enzymes have a high turnover number for N-demethylation.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002248:Carbon Monoxide; D002417:Cattle; D002845:Chromatography; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D013053:Spectrophotometry; D014018:Tissue Distribution
D000596:Amino Acids; D007527:Isoenzymes; D010446:Peptide Fragments; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System