Obligatory role of cytochrome b5 in the microsomal metabolism of methoxyflurane.
Cytochrome b5 has recently been shown to be required in the reconstituted cytochrome P-450 system for the metabolism of the volatile anesthetic methoxyflurane [E. Canova-Davis and L. A. Waskell, J. biol. Chem. 259, 2541 (1984)]. To determine whether this observation in the reconstituted system was merely dependent on the particular ratios of the various components or some other fortuitous, unknown factor, or whether cytochrome b5 plays a role in the liver microsomal metabolism of methoxyflurane, the following studies were undertaken. Antibody to rabbit holocytochrome b5 was raised in guinea pigs. The antibody to cytochrome b5 was able to inhibit 75% of the microsomal metabolism of methoxyflurane. This same antibody also inhibited methoxyflurane metabolism in the reconstituted system. When the antibody to cytochrome b5 was treated with purified cytochrome b5 before addition to the microsomes, it did not inhibit methoxyflurane metabolism. Furthermore, the antibody to cytochrome b5 did not inhibit the microsomal metabolism of benzphetamine. This suggests that cytochrome b5 was required for the microsomal metabolism of methoxyflurane. It is possible that cytochrome b5 functioned in the metabolism of methoxyflurane by retaining a specific conformation of cytochrome P-450 and not by transferring the second electron to cytochrome P-450. To explore this possibility, cytochrome b5 was reconstituted with Mn3+-protoporphyrin IX. The Mn3+-protoporphyrin IX derivative retained the conformation of cytochrome b5 but not its electron transfer properties. This manganese derivative of cytochrome b5 was unable to stimulate the metabolism of methoxyflurane. The study demonstrated that cytochrome b5 was obligatory for the microsomal metabolism of methoxyflurane, whereas it was not required for the microsomal N-demethylation of benzphetamine. Moreover, the heme moiety of cytochrome b5 functioned to transfer electrons in this reaction.
Canova-Davis
E
E
Chiang
J Y
JY
Waskell
L
L
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000494:pharmacology; D003573:Cytochrome b Group / Q000276:immunology / Q000502:physiology*; D003574:Cytochrome c Group / Q000494:pharmacology; D015786:Cytochromes b5; D003640:Dealkylation; D007074:Immunoglobulin G / Q000276:immunology; D008297:Male; D008733:Methoxyflurane / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D009243:NAD / Q000494:pharmacology; D010634:Phenobarbital / Q000494:pharmacology; D011817:Rabbits
D003573:Cytochrome b Group; D003574:Cytochrome c Group; D007074:Immunoglobulin G; D009243:NAD; D008733:Methoxyflurane; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital