Catalytic properties of the human cytochrome P450 2E1 produced by cDNA expression in mammalian cells

Abstract

A full-length cDNA encoding human cytochrome P450 2E1 was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2E1 and comigrated with P450 2E1 from human liver microsomes. P450 2E1 expressed in Hep G2 cells, a human cell line which contains both cytochrome b 5 and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates: N-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent K m and V max values for NDMA demethylation were 22 μ m and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK 143 cells, which do not contain b 5, displayed K m and V max values of 31 μ m and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b 5 into TK −143 microsomes increased the V max 2.2-fold and decreased the K m to 22 μ m. Addition of b 5 to Hep G2 microsomes resulted in a 1.6-fold increase in V max, but showed no effect on the K m . P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a K m of 47 μ m and V max of 213 pmol/min/mg microsomal protein. Addition of b 5 lowered the K m to 27 μ m, but had no effect on V max. These results demonstrate conclusively that P450 2E1 is responsible for the low K m forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b 5.