Recombinant mouse cytochromes P1-450 and P3-450: enzymatic characterization of the hemoprotein expressed in human cells infected with recombinant vaccinia virus.
We expressed mouse cytochrome P1-450 and P3-450 using recombinant vaccinia virus gene expression system in HeLa cells that were devoid of significant basal levels of P-450. HeLa cells were infected with the recombinant vaccinia virus containing either mouse cytochrome P1-450 or P3-450 cDNA, and the cell lysates were analyzed for the kinetics of P-450 enzyme activity and protein expression at the same time. 7-Ethoxycoumarin O-deethylase and ethoxyresorufin O-deethylase activities were measured as an expression of the cytochrome P-450 enzyme activities. Both cell lines began to express these enzyme activities as early as 12h after infection. The activities increased linearly up to the 24 h time point, and were kept for 36 h. Western immunoblot analysis showed that these cytochrome P-450 proteins were detected at 16 h and reached maximum quantity at 24 h after infection. These data showed a good correlation between cytochrome P-450 enzyme activity and protein concentration throughout the process of P-450 gene expression by vaccinia virus vector, suggesting a complete formation of cytochrome P-450 holoenzyme from the early stage of the protein expression.
Nouso
K
K
First Department of Internal Medicine, Okayama University Medical School, Japan.
Battula
N
N
Thorgeirsson
S S
SS
Higashi
T
T
Tsuji
T
T
D015660:7-Alkoxycoumarin O-Dealkylase / Q000378:metabolism; D000818:Animals; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D010088:Oxidoreductases / Q000378:metabolism; D011994:Recombinant Proteins; D011995:Recombination, Genetic; D014018:Tissue Distribution; D014615:Vaccinia / Q000201:enzymology*; D014616:Vaccinia virus / Q000235:genetics
D018076:DNA, Complementary; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D015660:7-Alkoxycoumarin O-Dealkylase; D019363:Cytochrome P-450 CYP1A1