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

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...

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Bibliographic Details
Published in:Archives of biochemistry and biophysics 1995-05, Vol.319 (1), p.157-167
Main Authors: Lee, C.A., Kadwell, S.H., Kost, T.A., Serabjitsingh, C.J.
Format: Article
Language:English
Subjects:
Animals
Baculoviridae - genetics
Catalysis
Cell Line
Cytochrome P-450 CYP3A
Cytochrome P-450 Enzyme Inhibitors
Cytochrome P-450 Enzyme System - genetics
Cytochrome P-450 Enzyme System - metabolism
Enzyme Activation
Gene Expression
Humans
In Vitro Techniques
Kinetics
Microsomes - enzymology
Microsomes, Liver - enzymology
Mixed Function Oxygenases - antagonists & inhibitors
Mixed Function Oxygenases - genetics
Mixed Function Oxygenases - metabolism
Moths
NADPH-Ferrihemoprotein Reductase - genetics
NADPH-Ferrihemoprotein Reductase - metabolism
Rabbits
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Spodoptera
Troleandomycin - pharmacology
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Summary: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 6β-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 μM), the extent of inhibition by 100 μM troleandomycin (>75%) in the presence of 25 μM testosterone, and the degree of P450 activation in the presence of 20 μM 7,8-benzoflavone. The coexpression of recombinant cytochrome b5 with CYP3A4OR did not result in an additional increase in CYP3A4OR activity.
ISSN:0003-9861
1096-0384
DOI:10.1006/abbi.1995.1278