A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450

1. We have constructed a full-length human liver cytochrome P450IIA cDNA from a partial-length clone by oligonucleotide-directed mutagenesis, and subcloned it into the monkey kidney (COS-7) cell expression vector, pSVL. 2. The cDNA encodes a 49 kDa protein with coumarin 7-hydroxylase (COH) activity which cross-reacts with antisera to the mouse cytochrome P-450 isoenzyme responsible for COH activity and comigrates with a human liver microsomal protein. 3. Western blot analysis of a panel of human livers indicates that the level of the 49 kDa protein, detected using antisera to either the mouse COH P-450 or rat P450IIA1 protein, correlates very highly with COH activity. 4. Antisera to the rat P450IIA1 protein can inhibit COH activity in human liver microsomes. Taken together, these data indicate that a member of the P450IIA subfamily is responsible for most, if not all, of the COH activity in human liver.

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Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)89213-x ◽

1986 ◽

Vol 261 (11) ◽

pp. 5051-5060

Author(s):

F P Guengerich ◽

M V Martin ◽

P H Beaune ◽

P Kremers ◽

T Wolff ◽

...

Keyword(s):

Genetic Polymorphism ◽

Drug Metabolism ◽

Human Liver ◽

Microsomal Cytochrome ◽

Cytochrome P 450 ◽

Oxidative Drug Metabolism

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Expression and alternative splicing of the cytochrome P-450 CYP2A7

Biochemical Journal ◽

10.1042/bj3060161 ◽

1995 ◽

Vol 306 (1) ◽

pp. 161-166 ◽

Cited By ~ 63

Author(s):

S Ding ◽

B G Lake ◽

T Friedberg ◽

C R Wolf

Keyword(s):

Alternative Splicing ◽

Cell Line ◽

Human Liver ◽

Fibroblast Cell ◽

Protein Product ◽

Cdna Clones ◽

Fibroblast Cell Line ◽

Rt Pcr ◽

Hydroxylase Activity ◽

Cytochrome P 450

In order to investigate the relative levels of expression of human cytochrome P-450 (P-450) CYP2A genes and determine how this relates to polymorphism in coumarin hydroxylase activity, cDNA clones for members of the CYP2A gene family were isolated. These clones were CYP2A6, CYP2A7 and an alternatively spliced version of CYP2A7 (CYP2A7AS). The latter clone was missing exon 2, but contained a 10 bp segment of intron 1. Translation of CYP2A7AS resulted in an in-frame deletion of 51 amino acids. The expression of these cDNAs in COS-7 cells showed that both CYP2A6 and CYP2A7 generated a protein of molecular mass 49 kDa, whereas the protein product of CYP2A7AS was about 44 kDa. Only the CYP2A6 had coumarin hydroxylase activity. The relative level of CYP2A7 and CYP2A7AS mRNA was investigated by reverse transcription followed by PCR (RT-PCR) using human liver RNAs and an RNA sample from a human skin fibroblast cell line. In one of five liver RNAs studied, the aberrantly spliced CYP2A7 mRNA was 3-4-fold more abundant than the normal mRNA. The other samples contained very low levels of this mRNA species. Interestingly, CYP2A7AS mRNA was the major CYP2A7 mRNA detected in the fibroblast cell line. In this case only a protein band of 44 kDa was observed by Western-blot analysis. The relative of mRNA encoding CYP2A6 and CYP2A7 was established in seven human liver samples by RT-PCR and found to range between 1:0.5 and 1:3. These data strength the previous findings that alternative splicing is an important factor in determining the levels of many human P-450s and that this may be subject to tissue-specific effects. Whether in this case the protein product has some function remains to be determined.

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