Cytochrome P-450 Related to P-4504 from Phenobarbital-Treated Rabbit Liver: Molecular Cloning of cDNA and Characterization of Cytochrome P-450 Obtained by Its Expression in Yeast Cells1

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Abstract
CDNA complementary to mRNA coding for a minor form of cytochrome P-450 from phenobarbital-treated rabbit liver (pHP3) was isolated using cDNA for the major phenobarbital-inducible cytochrome P-450 of rat liver as a probe in the first screening of a cDNA library. The nucleotide sequence of pHP3 was determined and contained a continuous reading frame encoding 490 amino acids. The deduced amino acid sequence of pHP3 protein exhibited about 50% homology with the major cytochrome P-450 from phenobarbital-treated rabbit liver, while the homology was as high as 80% between two minor cytochrome forms, pHP2 and pHP3. Two expression plasmids, pAHF3 and pAHΔN3, were constructed by insertion of pHP3 fragment between yeast alcohol dehydrogenase 1 (ADH1) promoter and terminator regions. pAHF3 contained the entire coding sequence of pHP3, but nucleotide sequences for the N-terminal region of pHP3 protein (from the 2nd to the 23rd amino acid) were deleted in pAHΔN3. On introduction of the constructed plasmids into Saccharomyces cerevisiae AH22 cells, the absorption spectrum of cytochrome P-450 was detected in the microsomal fraction from the transformed cells carrying pAHF3. On the other hand, cytochrome P-450 could not be detected spectro-photometrically in any subcellular fractions from the yeast cells carrying pAHΔN3, although the transcript of pHP3 insert was detected in RNA blot analysis. These results suggest that the N-terminal region of pHP3 protein plays an important role in accumulation of the newly synthesized pHP3 protein in yeast cells. Cytochrome P-450 (pHP3) was solubilized from microsomal membranes of the transformed yeast cells and purified partially on an aminooctyl Sepharose column (specific content, about 6 nmol per mg of protein). In the oxidized state the cytochrome preparation exhibited an absorption spectrum characteristic of a low-spin ferric cytochrome P-450. The reduced CO complex of the cytochrome showed a Soret absorption maximum at 450 nm. The monooxygenase activity of cytochrome P-450 (pHP3) was examined in a reconstituted system containing the cytochrome preparation and NADPH-cytochrome P-450 reductase. Cytochrome P-450 (pHP3) catalyzed N -demethylation of benzphetamine and aminopyrine and denitrification of 1-nitropropane. Addition of cytochrome b5 to the reconstituted system resulted in stimulation of the N -demethylation activities but inhibition of the denitrification activity. Neither 7-ethoxycoumarin O -deethylation activity nor acetanilide p -hydroxylation activity was detected, either in the presence or absence of cytochrome b5 . This substrate specificity closely resembles that of P-450 4 which has been purified from liver microsomes of drug-untreated rabbits.