1. CYP3A4 expressed by insect cells infected with a recombinant baculovirus containing both CYP3A4 and human NADPH-cytochrome P450 reductase is catalytically similar to human liver microsomal CYP3A4.
- Author
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Lee CA, Kadwell SH, Kost TA, and Serabjit-Singh CJ
- Subjects
- Animals, Baculoviridae genetics, Catalysis, Cell Line, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme Inhibitors, Enzyme Activation, Gene Expression, Humans, In Vitro Techniques, Kinetics, Microsomes enzymology, Microsomes, Liver enzymology, Mixed Function Oxygenases antagonists & inhibitors, Moths, NADPH-Ferrihemoprotein Reductase metabolism, Rabbits, Recombinant Proteins genetics, Recombinant Proteins metabolism, Spodoptera, Troleandomycin pharmacology, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, Mixed Function Oxygenases genetics, Mixed Function Oxygenases metabolism, NADPH-Ferrihemoprotein Reductase genetics
- Abstract
Human cytochrome CYP3A4 is the most abundant of all the P450s in human liver and is involved in the metabolism of many environmental toxicants and drugs. Kinetic studies with CYP3A4 have been hampered due to low activity of this enzyme obtained from recombinant gene expression systems or difficulty in reconstituting activity with the native enzyme purified from human liver. To overcome these obstacles, we have expressed high levels of catalytically active CYP3A4 and human NADPH-cytochrome P450 reductase (CYPOR) together in two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T.ni), via a single recombinant baculovirus carrying both cDNAs (CYP3A4-OR). Microsomes containing recombinant CYP3A4-OR from these cell lines were up to 50-times more active in testosterone 6 beta-hydroxylase activity than recombinant CYP3A4 expressed alone and supplemented with purified rabbit CYPOR. The spectral P450 content of CYP3A4-OR T.ni microsomes was 107 pmol/mg microsomal protein and the cytochrome c reductase activity was 3904 units/mg. Recombinant CYP3A4-OR was catalytically similar to human liver CYP3A4 based on similarities in the testosterone metabolite profile, time course of metabolite formation, Vmax and Km values (for CYP3A4-OR, Vmax was 8.8 nmol/min/mg microsomal protein [70 nmol/min/nmol CYP3A4] and Km was 33 microM), the extent of inhibition by 100 microM troleandomycin (> 75%) in the presence of 25 microM testosterone, and the degree of P450 activation in the presence of 20 microM 7,8-benzoflavone. The coexpression of recombinant cytochrome b5 with CYP3A4-OR did not result in an additional increase in CYP3A4-OR activity.
- Published
- 1995
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