The long term objective of this proposal is to understand the mechanism by which drug metabolism and microsomal biosynthetic reactions modulate one another. The process occurs via an interaction of the two microsomal electron transport chains of which cytochrome P-450 and cytochrome b5 are components. Evidence exists that such interaction occurs in microsomes and in a reconstituted system employing purified enzymes. My laboratory has demonstrated that cytochrome b5 is required both in microsomes and in a purified reconstituted cytochrome P-450 system for the metabolism of the volatile anesthetic methoxyflurane. Preliminary data from my laboratory also indicate that methoxyflurane inhibits the synthesis of cholesterol, a substance which is known to require cytochrome b5 with unknown but possibly substantial in vivo consequences. The immediate goals of this project are three-fold. First, the role of cytochrome b5 in the metabolism of the volatile anesthetics, halothane, enflurane and isoflurane will be explored. The haloacid metabolites of the volatile anesthetics will be derivatized; the haloacid derivatives will be separated from interfering substances by gas chromatography and quantitated by mass spectrometry. The second objective is to elucidate the chemical basis of the inactivation of cytochrome b5 by the protein modifying agent, diethylpyrocarbonate. The experiments will be designed to first determine which amino acid(s) is modified and then determine the exact location of the amino acid in the protein. Our third and final short term aim is to determine the molecular basis for our observation that antibodies to cytochrome b5 inhibit methoxyflurane metabolism much more readily in phenobarbital induced liver than in lung microsomes. Cytochrome b5 and cytochrome P-450 LM2 from liver and lung will be purified, characterized and compared. Completion of the previously outlined experiments will significantly contribute to our knowledge of the factors which govern the interactions between cytochrome P-450 and cytochrome b5.
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