CDNA-DIRECTED EXPRESSION OF HUMAN CYTOCHROME-P450 CYP1A1 USING BACULOVIRUS - PURIFICATION, DEPENDENCY ON NADPH-P450 OXIDOREDUCTASE, AND RECONSTITUTION OF CATALYTIC PROPERTIES WITHOUT PURIFICATION

CDNA-DIRECTED EXPRESSION OF HUMAN CYTOCHROME-P450 CYP1A1 USING BACULOVIRUS - PURIFICATION, DEPENDENCY ON NADPH-P450 OXIDOREDUCTASE, AND RECONSTITUTION OF CATALYTIC PROPERTIES WITHOUT PURIFICATION

Buters J T M; Shou M G; Hardwick J P; Korzekwa K R; Gonzalez F J

Drug Metabolism and Disposition

1995

1995-07

A recombinant baculovirus containing the human cytochrome P450 (CYP) 1A1 cDNA was constructed and used to express CYP1A1 in Spodoptera frugiperda (SF9) insect cells (0.14+/-0.04 nmol/mg protein, 53+/-14 nmol/liter, N=30). The enzyme represented approximate to 1% of total cellular protein and was partially purified by a three-column procedure to specific content of 5.0 nmol/mg protein. Catalytic activity was reconstituted with both the purified enzyme using lipid and NADPH-P450 oxidoreductase, and the SF9 insect cell membrane fraction without purification using NADPH-P450 oxidoreductase and small amounts of detergent. Catalytic activity of the enzyme after reconstitution was optimum using molar ratios of CYP1A1 to NADPH-P450 oxidoreductase of 1:8. Cytochrome b(5) had no additional stimulating effect. The enzyme metabolized substrates characteristic for CYP1A1: benzo a pyrene (4.0+/-0.3 nmol/min/nmol CYP), 7-ethoxy-4-trifluoromethylcoumarin (36+/-2), ethoxyresorufin (37+/-1), but not pentoxyresorufin (0.77+/-0.02). Recombinant baculovirus expresses the highest amounts of all expression systems published to date of catalytically active CYP1A1. Because human CYP1A1 has never been isolated in a catalytically active state from human tissue, nor has recombinant unmodified human CYP1A1, this system is an excellent alternative for the isolation and characterization of this CYP.

rat; liver; cancer; Pharmacology & Pharmacy; metabolism; activation; enzymes; acid; oxidation; assay; yeast saccharomyces-cerevisiae

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Journal Article or Conference Abstract Publication

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Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).

696-701

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