Purification and characterization of human liver microsomal cytochrome P-450 2A6.
Cytochrome P-450 (P-450) 2A6 was purified by chromatography of human liver microsomes. The final preparation was electrophoretically homogeneous and contained 16 nmol of P-450/mg of protein. The amino-terminal amino acid sequence of the protein (first 13 residues) matched that of the reported cDNA exactly. The UV-visible spectrum indicated that the isolated hemoprotein was in the low-spin form. The protein was recognized by rabbit antibodies raised against rat P-450 2A1, and a rabbit antiserum against the P-450 2A6 preparation was also prepared. With these antibodies, it was estimated that P-450 2A6 accounted for a maximum of 1% of the total P-450 present in the human liver microsomes; the level varied greater than 100-fold among the 20 samples examined. Purified P-450 2A6 catalyzed coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation at rates similar to those measured in the human liver sample used to prepare P-450 2A6, and these two microsomal activities were strongly inhibited by the antibodies. The purified P-450 2A6 enzyme also catalyzed low levels of 4,4'-methylene-bis(2-chloroaniline) (MOCA) N-oxidation and activation of aflatoxin B1, 6-aminochrysene, 2-amino-3-methylimidazo[4,5-f]quinoline, and 2-amino-3,5-dimethylimidazo [4,5-f]quinoline to genotoxic products; the antibody inhibited the activity of purified P-450 2A6 towards aflatoxin B1 and 6-aminochrysene but did not inhibit these reactions in human liver microsomes (MOCA N-oxidation was inhibited approximately 20%). Human P-450 2A6 did not catalyze testosterone 7 alpha-hydroxylation, a characteristic activity of the related rat P-450 2A1 protein. These results emphasize the need to characterize individual P-450 enzymes in order to understand their functions in the context of more complex systems.
Yun
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Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Shimada
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Guengerich
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FP
D015660:7-Alkoxycoumarin O-Dealkylase / Q000037:antagonists & inhibitors; D016604:Aflatoxin B1 / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001711:Biotransformation; D002911:Chrysenes / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000302:isolation & purification* / Q000502:physiology; D015786:Cytochromes b5 / Q000494:pharmacology; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats
D002911:Chrysenes; D003374:Coumarins; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D015660:7-Alkoxycoumarin O-Dealkylase; C100101:6-chrysenamine