CDNA-DIRECTED EXPRESSION OF HUMAN CYTOCHROME-P450 CYP1A1 USING BACULOVIRUS - PURIFICATION, DEPENDENCY ON NADPH-P450 OXIDOREDUCTASE, AND RECONSTITUTION OF CATALYTIC PROPERTIES WITHOUT PURIFICATION
rat; liver; cancer; Pharmacology & Pharmacy; metabolism; activation; enzymes; acid; oxidation; assay; yeast saccharomyces-cerevisiae
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.
Buters J T M; Shou M G; Hardwick J P; Korzekwa K R; Gonzalez F J
Drug Metabolism and Disposition
1995
1995-07
Journal Article or Conference Abstract Publication
n/a
CDNA-DIRECTED EXPRESSION OF HUMAN CYTOCHROME-P450 CYP3A4 USING BACULOVIRUS
pharmacokinetics; Pharmacology & Pharmacy; metabolism; polymorphism; identification; reductase; oxidation; vaccinia virus; human-liver; catalytic activities; hydroxylation
A recombinant baculovirus containing the human CYP3A4 cDNA was constructed and used to express CYP3A4 in SF9 insect cells (0.46 +/- 0.13 nmol/mg protein, 103 +/- 29 nmol/liter, N = 15). The enzyme represented similar to 2-3% of total cellular protein and could be purified by a two column procedure to a specific content of 12.7 nmol/mg protein. Catalytic activity of the purified enzyme after reconstitution was optimum using molar ratios of CYP3A4 to cytochrome b(5) to NADPH-P450 oxidoreductase of 1:3:20, respectively. The enzyme metabolized cortisol, erythromycin, testosterone, and (R)-warfarin. Recombinant baculovirus expresses the highest amounts of all expression systems published to date of catalytically intact CYP3A4. This system is an excellent alternative for the isolation and characterization of P450 forms from human liver.
Buters J T M; Korzekwa K R; Kunze K L; Omata Y; Hardwick J P; Gonzalez F J
Drug Metabolism and Disposition
1994
1994-09
Journal Article or Conference Abstract Publication
n/a
Catalysis of the cysteine conjugation and protein binding of acetaminophen by microsomes from a human lymphoblast line transfected with the cDNAs of various forms of human cytochrome P450
activation; ethanol; hepatic microsomes; hepatocytes; inhibitors; monoclonal-antibodies; oxidation; para-benzoquinone imine; Pharmacology & Pharmacy; rat-liver; reactive metabolite formation
We have previously found that for acetaminophen kinetic differences exist between the hepatic microsomal catalyzed protein binding and cysteine conjugation. We have also observed that the protein binding of acetaminophen is only to intralumenal proteins. Together these data suggested that two pools of the reactive metabolite, N-acetyl-p-benzoquinone imine (NABQI), are formed during the oxidative metabolism of acetaminophen: one on the cytosolic surface and the other within the lumen of the microsomes. This would indicate that some of forms of cytochrome P450 (CYP) catalyzing NABQI formation have their active site on the cytosolic surface and others on the lumenal surface. We have examined this question by comparing the rates of cysteine conjugation and protein binding of acetaminophen by microsomes from lymphoblasts tranfected with the cDNAs for human CYPs. We found that CYP2D6 catalyzed only cysteine conjugation; CYP1A2 and 3A4 catalyzed only protein binding; CYP2E1 catalyzed both; and CYP1A1, CYP2A6 and CYP2B6 catalyzed neither. These data suggest that CYP2D6 has its active site only on the cytosolic surface; CYP1A2 and CYP3A4 only on the lumenal surface; and CYP2E1 has catalytic sites on both the lumenal and cytosolic surfaces of the membrane. In mouse studies we have found that ethanol administration increased acetaminophen protein binding by 265% but cysteine conjugation by only 61%. CYP2E1 and CYP2B increased, whereas CYP3A decreased and the others did not change. These data suggest that in control mice CYP2E1 catalyzes the bulk of protein binding, whereas CYP2D catalyzes slightly more cysteine conjugation than does CYP2E1.
Zhou L X; Erickson R R; Hardwick J P; Park S S; Wrighton S A; Holtzman J L
Journal of Pharmacology and Experimental Therapeutics
1997
1997-05
Journal Article
n/a
Vitamin B-12 and Redox Homeostasis: Cob(II)alamin Reacts with Superoxide at Rates Approaching Superoxide Dismutase (SOD)
binding; chaperone; Chemistry; cobalamin; free-radicals; mechanism; nitric-oxide; oxidation; state
We report a kinetic study of the reaction between superoxide and an important intracellular form of vitamin B-12, cob(II)alamin. Superoxide is implicated in the pathophysiology of many inflammatory diseases, whereas vitamin B,2 derivatives are often beneficial in their treatment. We found that cob(II)alamin reacts with superoxide at rates approaching those of superoxide dismutase itself, suggesting a probable mechanism by which vitamin B-12. protects against chronic inflammation and modulates redox homeostasis.
Suarez-Moreira E; Yun J; Birch C S; Williams J H H; McCaddon A; Brascht N E
Journal of the American Chemical Society
2009
2009-10
Journal Article
<a href="http://doi.org/10.1021/ja904670x" target="_blank" rel="noreferrer noopener">10.1021/ja904670x</a>