A novel species of cytochrome P-450 (P-450(ib)) specific for the small intestine of rabbits: cDNA cloning and its expression in COS cells

We have isolated a cDNA clone for a P-450, designated P-450(ib) (Ichihara, K., Kusunose, E., Kaku, M., Yamamoto, S., and Kusunose, M. (1985) Biochim. Biophys. Acta 831, 99-105), from a cDNA library of rabbit small intestine mucosa by using synthetic DNA fragment by the polymerase chain reaction, as a hybridization probe. The cDNA with a 1,829-base pair insert encodes a polypeptide of 501 amino acids. The deduced amino acid sequence contains all of the sequences of the NH₂-terminal and 14 tryptic fragments from purified P-450(ib). As the NH₂-terminal methionine was not found in the sequence from the purified protein, the apoprotein of P-450(ib) is composed of 500 amino acids with a molecular weight of 57,193. P-450(ib) shows 35-41% sequence similarity with several members of 8 subfamilies in the P-450 II family, whereas it has a less than 30% sequence similarity with other P-450 families, suggesting that this P-450 is the first member of a novel subfamily within the P-450 II family. RNA blot analysis shows that mRNA hybridized to the cDNA is expressed in the small intestine, but not significantly in other tissues including liver, colon, kidney, lung, spleen, brain, stomach, and cecum, indicating that P-450(ib) is a P-450 specific to the small intestine. The protein expressed in COS-7 cells using the cDNA in an expression vector, pKCRH₂, shows benzphetamine N-demethylase activity and gives a band identical with that of P-450(ib) in its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.