Characterization of the cytochrome P‐450IID subfamily in bovine liver

Abstract

To elucidate the molecular mechanisms underlying drug detoxification, the structures of the members of the microsomal cytochrome P-450IID subfamily were analyzed by isolating, mapping and sequencing cytochrome P-450IID (CYP2D) cDNA clones from bovine liver. The screening was performed under nonstringent conditions so that most of the P-450IID subfamily members could be obtained. 114 of the 147 positive clones were classified into four groups on the basis of their restrictionenzyme maps. The maps of the four groups were highly similar, however, the clones of one group contained an insertion of approximately 500 bp in the coding region. Analysis of partial nucleotide sequences of several representative clones from each group showed that the bovine P-450IID subfamily in liver consisted of several, not many, highly similar members, differing by less than 7% in their nucleotide sequences.The location of the insertion found in the minor group corresponded to intron 7 and the GT/AG rule was found at the exon/intron boundary, suggesting that intron 7 was retained in this group.The complete nucleotide sequences of two clones from the major group were examined to determine the structures of the P-450IID subfamily in bovine liver. A full-length cDNA clone (1615 bp) and a partial cDNA clone (1538 bp) contained open reading frames encoding 500 and 487 amino acid residues, respectively. The partial clone lacked the nucleotide sequence corresponding to the first 13 N-terminal amino acid residues. The deduced amino acid sequences of the two clones were 98% similar, and 80% and 68% similar to those from human CYP2D6 and rat CYP2D1, respectively. Comparisons of the amino acid sequences of the P-450IID subfamily members showed the highly conserved C-terminal region of their molecules and the high similarity between the members in one species, especially in cattle and man.

Isolation and Characterization of the Protein Components of the Liver Microsomal O2-insensitive NADH-Benzamidoxime Reductase

“…The amino acid sequences of three peptides isolated from a proteolytic digest of the purified protein are shown in Table III. The sequence of peptide 2 is identical to that of a sequence in P450 2D described by Tsuneoka et al (28), and the sequences of peptides 1 and 3 are similar to sequences of P450 2D (peptide 1, 66.7%; peptide 3, 83.3%) and P450 2D6 (peptide 1, 73.3%; peptide 3, 75%) (28,29). The sequence of peptide 3 also shows similarity to P450 isoenzymes of the subfamilies CYP2A (Յ57.8%), CYP1A (Յ57.8%) and CYP2J (Յ56.3%), while the sequence of peptide 2 shows a slight similarity to the subfamily CYP3A.…”

Cytochrome P450 expression and activities in rat, rabbit and bovine tongue

“…Consequently, the observed transcription of CYP2E1 in rabbit tongues may not translate to observable levels of 2E1 enzyme. This is significant in relation to potential interactions in the oral cavity involving ethanol consumption, since protein stabilization of 2E1 by alcohol is known to occur, and CYP2E1 is a major activator of carcinogenic nitrosamines found in tobacco product (Tsuneoka et al, 1992).…”

Cytochrome P450 Expression in the Liver of Food-Producing Animals

“…At least four proteins belonging to the CYP2D subfamily have been identified in cattle liver using cDNA techniques; these differed by less than 7% at the nucleotide level, and shared 80% and 68% amino acid sequence similarity with human CYP2D6 and rat CYP2D1 respectively [93]. Cattle liver microsomes displayed debrisoquine 4-hydroxylase and bufurarol 1'-hydroxylase activities that were inhibited by quinidine, an established CYP2D inhibitor ( Table 3) [94].…”