Xenobiotic-metabolizing enzymes in the liver play key roles in the detoxification of lipophilic drugs, pollutants and toxins, and the bioactivation of procarcinogens to carcinogens. We propose to study the structures and active-site relationships of several cytochromes P450 isolated from rat liver microsomes, and the cytochrome P450 reductase isolated from pig and rabbit liver microsomes. The cytochrome P450 isozymes which form the basis of this study are the two induced by 3-methylcholanthrene, P450c and P450d, both of which are immunologically related. The structural studies include the determination of their primary sequences and the characterization of their shared antigenic determinants. Active site studies will be performed by labeling the enzymes with bromoacetoxy derivatives of substrates, and in the case of P450c, labeling the isozyme with the specific inhibitor acetonitrophenacyl bromide. The active site studies on the cytochrome P450 reductase include the affinity labeling of the FMN, FAD, and NADPH binding sites with the appropriate arylazido photoactive derivatives. We will study the interaction of cytochrome P450 with its reductase using antibody and peptide inhibitor probes directed at their postulated binding sites. Preliminary results suggests that these approaches will form a basis for an understanding of the structure-function relationships in these important enzyme systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034426-03
Application #
3285356
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1985-09-16
Project End
1989-06-30
Budget Start
1987-09-01
Budget End
1989-06-30
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
City of Hope/Beckman Research Institute
Department
Type
DUNS #
City
Duarte
State
CA
Country
United States
Zip Code
91010
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Liu, X F; Yuan, H; Haniu, M et al. (1989) Reaction of rat liver DT-diaphorase (NAD(P)H:quinone acceptor reductase) with 5'-[p-(fluorosulfonyl)benzoyl]-adenosine. Mol Pharmacol 35:818-22
Haniu, M; Yuan, H; Chen, S A et al. (1988) Structure-function relationship of NAD(P)H:quinone reductase: characterization of NH2-terminal blocking group and essential tyrosine and lysine residues. Biochemistry 27:6877-83
Haniu, M; Shively, J E (1988) Microsequence analysis of peptides and proteins. IX. Manual gas-phase microsequencing of multiple samples. Anal Biochem 173:296-306
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Wrighton, S A; Thomas, P E; Molowa, D T et al. (1986) Characterization of ethanol-inducible human liver N-nitrosodimethylamine demethylase. Biochemistry 25:6731-5
Haniu, M; Iyanagi, T; Miller, P et al. (1986) Complete amino acid sequence of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes. Biochemistry 25:7906-11