A prostaglandin ω-hydroxylase cytochrome P-450 (P-450(PG-ω)) purified from lungs of pregnant rabbits

Cytochrome P-450-dependent prostaglandin ω-hydroxylation is induced over 100-fold during late gestation in rabbit pulmonary microsomes Purification of cytochromes P-450 from lung microsomes of pregnant rabbits yielded three fractions. Two of these fractions correspond to rabbit lung P-450(I) (LM₂) and P-450(II) (LM₅), which together constitute 70-97% of total cytochrome P-450 in lung microsomes from nonpregnant rabbits. The third form, which we designate rabbit cytochrome P-450(PG-ω), regioselectively hydroxylates prostaglandins at the ω-position in reconstituted systems with a turnover of 1-5 min^-1. Titration with purified pig liver cytochrome b₅, demonstrated a 4-fold maximum stimulation at a cytochrome b₅ to a P-450 molar ratio. of 1-2. Rabbit lung P-450(PG-ω) formed a typical type I binding spectrum upon the addition of prostaglandin E₁ with a calculated K(s) of 1 μM, which agreed reasonably well with the kinetically calculated K(m) of 3 μM. Cytochrome P-450(PG-ω) was isolated as a low-spin isozyme with a λ(max) (450 nm) in the CO-difference spectrum distinguishable from P-450(I) (451 nm) and P-450(II) (449 nm). Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis demonstrated that although purified P-450(PG-ω) had a relatively low specific content (12.1 nmolmg^-1), it appeared homogeneous with a calculated minimum M(r) of 56,000, intermediate between rabbit LM₄ and LM₆. When lung microsomes from pregnant and nonpregnant rabbit were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a protein band, with a M(r) identical to P-450(PG-ω), was observed in the pregnant rabbit, whereas this band appeared to be very faint or absent in microsomes from the nonpregnant rabbit. Purification of cytochromes P-450 from nonpregnant rabbit lung yielded only P-450(I) and P-450(II). P-450(PG-ω) appears to be a novel rabbit P-450, possessing high activity towards ω-hydroxylation of prostaglandins, and is greatly induced during pregnancy in rabbit lung.