Purification and characterization of a microsomal cytochrome P-450 IIB enzyme from sheep lung.

1. Cytochrome P-450 was purified to apparent homogeneity from pulmonary microsomes of female sheep. 2. Sheep lung P-450 was isolated in the low spin state with a lambda max in the CO-reduced vs CO difference spectrum of 450nm with no detectable absorbance at 420 nm. 3. The minimum mol. wt, determined by SDS-PAGE, was 51,400 daltons. Examination of substrate specificity of sheep lung P-450 indicated N-demethylation of benzphetamine, cocaine and N-dimethylnitrosamine. Nicotine was also metabolized by sheep lung P-450, but little turnover was observed with benzo[a]pyrene. 4. The amino acid composition and N-terminal sequence was highly similar to rat or rabbit members of the P-450 IIB gene subfamily. Antibodies raised in rabbit to sheep lung P-450 cross-reacted on Western blots with rabbit lung P-450 IIB4. 5. The P-450 isolated in this study comprises 75% of total P-450 in sheep pulmonary microsomes. Small amounts of this isozyme were also detected in sheep liver. These results show that, as is the case in rabbit and rat, the major constitutive P-450 in sheep is a member of the IIB gene subfamily.