Purification and characterization of cytochrome P-450 with high affinity for 7-alkoxycoumarins.
A unique form of cytochrome P-450 (called P-450(2] with high affinity for 7-alkoxycoumarins was purified from liver microsomes of phenobarbital-treated rabbits with an overall yield of about 0.6%. The purified preparation had a specific cytochrome P-450 content of 15-16 nmol per mg of protein and was homogenous on sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The apparent molecular weight estimated for P-450(2) was 50,500. In the oxidized state its absorption spectrum was characteristic of low-spin cytochrome P-450. The activity of purified P-450(2) to catalyze O-dealkylation of several 7-alkoxycoumarins in the reconstituted system was compared with those of P-450(1), the major phenobarbital-inducible form in rabbit liver microsomes, and rat P-448(2), purified from liver microsomes of beta-naphthoflavone-treated rats. With 7-ethoxycoumarin as substrate, the pH optimum for P-450(2) was 6.5, whereas those for P-450(1) and rat P-448(2) were 7.5-8.0. P-450(2) showed Km values of 0.38 to 1.4 microM for the five alkoxycoumarins tested, whereas the corresponding Km values determined for P-450(1) and rat P-448(2) were 40- to 700-fold higher. The Km values of P-450(2) and P-450(1) were relatively unaffected by the length of the alkyl chain in the alkoxycoumarins, whereas those of rat P-448(2), decreased markedly as the length of the alkyl chain increased. For all the three forms of cytochrome P-450, the highest Vmax values were observed with 7-ethoxycoumarin, and Vmax values tended to decrease with increase in the alkyl chain length in the substrate. Only P-450(2) catalyzed 7-hydroxylation of coumarin, though at a low rate. Progressive and irreversible inactivation of P-450(2), but not P-450(1) and rat P-448(2), was observed during the catalysis of 7-methoxycoumarin O-demethylation, and this inactivation was considerably prevented by inclusion of cytochrome b5 in the reconstituted system.
D015660:7-Alkoxycoumarin O-Dealkylase; D000818:Animals; D003374:Coumarins / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010105:Oxygenases / Q000302:isolation & purification* / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D013379:Substrate Specificity
D003374:Coumarins; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D015660:7-Alkoxycoumarin O-Dealkylase; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital