RECOMBINANT MOUSE CYTOCHROMES P-1-450 AND P-3-450 - ENZYMATIC CHARACTERIZATION OF THE HEMOPROTEIN EXPRESSED IN HUMAN-CELLS INFECTED WITH RECOMBINANT VACCINIA VIRUS

We expressed mouse cytochrome P-1-450 and P-3-450 using recombinant va ccinia virus gene expression system in HeLa cells that were devoid of significant basal levels of P-450. HeLa cells were infected with the r ecombinant vaccinia virus containing either mouse cytochrome P-1-450 o r P-3-450 cDNA, and the cell lysates were analyzed for the kinetics of P-450 enzyme activity and protein expression at the same time. 7-Etho xycoumarin O-deethylase and ethoxyresorufin O-deethylase activities we re 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 24h time point, and were kept for 36h. Western immunoblot analysis showed that these cytochrome P-450 protiens were detected at 16h and reached maximum quantity at 24h after infection. These data showed a good corr elation between cytochrome P-450 enzyme activity and protein concentra tion throughout the process of P-450 gene expression by vaccinia virus vector, suggesting a complete formation of cytochrome P-450 holoenzym e from the early stage of the protein expression.