Novel exogenous heme-dependent expression of mammalian cytochrome P450 using baculovirus.
Rat P450 IIA1 is a membrane-bound hemoprotein that catalyzes the 7 alpha- and 6 alpha-hydroxylation of testosterone. A recombinant baculovirus, containing the cDNA encoding IIA1, was constructed and used to infect Spodoptera frugiperda cells. Infected cells contained up to 10% IIA1 protein as quantified by Western blot analysis; however, only a small percentage of this protein contained heme and was catalytically active. Addition of hemin to the culture medium during the course of viral infection resulted in a marked sixfold increase in both testosterone hydroxylase activity and heme-containing IIA1 protein. When the exogenously added protoheme was substituted with modified heme molecules containing hydrogen or ethyl groups at the 2 and 4 positions of the protoporphyrin IX, enzymes with only marginal changes in catalytic properties were produced. These data indicate that baculovirus can be used as a system to produce high levels of mammalian cytochrome P450s containing custom modified heme moieties.
Asseffa
A
A
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
Smith
S J
SJ
Nagata
K
K
Gillette
J
J
Gelboin
H V
HV
Gonzalez
F J
FJ
D000818:Animals; D001059:Apoproteins / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D005822:Genetic Vectors; D006418:Heme / Q000031:analogs & derivatives / Q000494:pharmacology*; D006427:Hemin / Q000494:pharmacology; D007304:Insect Viruses / Q000235:genetics*; D051381:Rats; D011995:Recombination, Genetic; D013053:Spectrophotometry; D013250:Steroid Hydroxylases / Q000378:metabolism
D001059:Apoproteins; D006418:Heme; D006427:Hemin; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C022724:testosterone 7-alpha-hydroxylase, hamster