Isolation and characterization of a cytochrome P450 of the IIA subfamily from human liver microsomes

Abstract

Antibodies raised against cytochrome P450, which is overexpressed in mouse hepatic tumors, (P450tu) crossreact with two human liver microsomal proteins (49 kDa and 52 kDa). We have quantified these proteins in 60 human liver samples and found great interindividual variability in both of them. The concentration of the 49-kDa protein varies up to 144 fold in the various samples and represents typically 10% of the total mincrosomal P450 content. Its immunologically determined concentration correlates well (R = 0.78) with the microsomal coumarin-7-hydroylase (COH) activity. This activity is strongly and completely inhibited by anti-P450tu antibody (IC50 = 0.13 mg IgG/mg microsomal protein). The crossreacting 49-kDa protein shows an unusually high substrate specificity towards coumarin; it presents all human COH and part of 7-ethoxycoumarin O-deethylase (ECOD). Besides these two activities, we did not find any activity with other typical P450 substrates. In primary cultures of human hepatocytes, it is inducible by phenobarbital and dexamethasone, but not by pyrazole and beta-naphthoflavone. We isolated this protein from human liver microsomes and purified it to homogeneity by a combination of aminooctyl-amino-Sepharose chromatography and immunoaffinity chromatography. The protein was identified as a cytochrome P450 of the IIA subfamily. Its N-terminal amino-acid sequence was identical with the first 20 residues deduced from the nucleotide sequence of P450IIA6.

Functional Characterization of Cytochrome P450 2A6 Allelic VariantsCYP2A6*15,CYP2A6*16,CYP2A6*21, andCYP2A6*22

“…Immunodetection with monoclonal mouse anti-human CYP2A6 revealed the presence of a molecular mass with approximately 49 kDa in all the recombinant mutant proteins (Fig. 3, lanes *15, *16, *21, and *22), which was in accordance with the reported molecular weight of CYP2A6 (Maurice et al, 1991). The levels of CYP2A6 mutant protein expressions appeared to be comparable to that of the wild-type CYP2A6, indicating consistency in the expression of these proteins in the bacterial membrane fragments.…”

In vivo evaluation of CYP2A6 and xanthine oxidase enzyme activities in the Serbian population

“…As observed in other populations [4], log-transformed 17U/17X ratios in Serbs were normally distributed. The level of interindividual variations in CYP2A6 activity found in Serbs corresponds to Caucasian data [11], while larger variability up to more than 100-fold has been reported in other populations [4,30,31]. Disease conditions and other modifying factors may CYP2A6*1A/*4, CYP2A6*1A/*9, CYP2A6*1B1/*4, CYP2A6*1B1/ *9; genotype group c: CYP2A6*4/*9, CYP2A6*9/*9 partly account for the observed wider interindividual variations in CYP2A6 activity [32].…”

Identification of the Cytochrome P450 Enzymes Involved in theN-Oxidation of Voriconazole

“…The remaining probe substrate activities used were as described previously in the literature. Coumarin 7-hydroxylase activity was determined at a substrate concentration of 100 M (Maurice et al, 1991), (S)-warfarin 7-hydroxylase activity was determined at a substrate concentration of 500 M (Doeke et al, 1991), (S)-mephenytoin 4-hydroxylase activity was determined at a substrate concentration of 500 M (Meier et al, 1985), bufuralol 1Ј-hydroxylase was determined at a substrate concentration of 10 M (Kronbach et al, 1987), 4-nitrophenol 3-hydroxylase activity was determined at a substrate concentration of 1000 M (Tassaneeyakul et al, 1993), and testosterone 6␤-hydroxylase activity was determined at a substrate concentration of 250 M (Funae and Imaoka, 1987).…”