Identification of CYP2E1 in marmoset monkey.
CYP2E1 is the main enzyme responsible for chlorzoxazone 6-hydroxylase activity in human liver. Here, it is shown that marmoset monkey liver microsomal fraction catalyses this reaction at a similar rate and with a similar Km to human liver and that the activity is increased 4-fold in marmosets treated with isoniazid, a known inducer of CYP2E1. This indicates that CYP2E1 is present in marmoset liver. However conversely, an anti-peptide antibody targeted against the C-terminus of human and cynomolgus monkey CYP2E1 (Val-Ile-Pro-Arg-Ser) failed to bind to marmoset monkey hepatic microsomal fraction. To investigate if there is a difference in the C-terminus of CYP2E1 in these species, this region of marmoset CYP2E1 was sequenced following amplification of marmoset liver cDNA with primers selected according to conserved regions identified in human and cynomolgus monkey CYP2E1. It was found that the deduced amino acid sequence of marmoset CYP2E1 in this region is very similar to human CYP2E1, but due to two base differences in the marmoset nucleic acid sequence, the C-terminus of marmoset CYP2E1 is extended by 2 amino acids, i.e. Val-Ile-Pro-Arg-Ser-Ser-Val. This difference is sufficient to prevent the binding of an antibody raised against the C-terminus of human CYP2E1. The expression of CYP2E1 in the marmoset was confirmed by raising an antibody against the deduced C-terminus of marmoset CYP2E1 (Pro-Arg-Ser-Ser-Val). In immunoblotting, this antibody bound to a single protein of 54 kDa in marmoset liver microsomal fraction. The intensity of the band was increased in isoniazid-treated marmosets, consistent with induction of CYP2E1. The antibody did not recognise human or cynomolgus monkey CYP2E1. This was expected since the immunising peptide sequence does not occur in these enzymes. The results demonstrate the presence of CYP2E1 in marmoset liver and illustrate the importance of the C-terminus for the production of specific antibodies against P450 enzymes.
Schulz
T G
TG
Section on Clinical Pharmacology, Imperial College School of Medicine, Hammersmith Hospital, London, UK. tschulz1@gwdg.de
Thiel
R
R
Davies
D S
DS
Edwards
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D002753:Chlorzoxazone / Q000378:metabolism*; D019392:Cytochrome P-450 CYP2E1 / Q000276:immunology / Q000378:metabolism*; D000882:Haplorhini; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D010446:Peptide Fragments; D006899:Mixed Function Oxygenases; D019392:Cytochrome P-450 CYP2E1; D002753:Chlorzoxazone