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

D. E. Williams, S. E. Hale, R. T. Okita, B. S S Masters

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)14600-14608
Number of pages9
JournalJournal of Biological Chemistry
Volume259
Issue number23
StatePublished - 1984
Externally publishedYes

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Mixed Function Oxygenases
Cytochrome P-450 Enzyme System
Prostaglandins
Rabbits
Lung
Cytochromes b5
Hydroxylation
Microsomes
Electrophoresis
Sodium Dodecyl Sulfate
Purification
Alprostadil
Carbon Monoxide
Titration
Liver
Isoenzymes
Polyacrylamide Gel Electrophoresis
Pregnancy
Proteins
Swine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Williams, D. E., Hale, S. E., Okita, R. T., & Masters, B. S. S. (1984). A prostaglandin ω-hydroxylase cytochrome P-450 (P-450(PG-ω)) purified from lungs of pregnant rabbits. Journal of Biological Chemistry, 259(23), 14600-14608.

A prostaglandin ω-hydroxylase cytochrome P-450 (P-450(PG-ω)) purified from lungs of pregnant rabbits. / Williams, D. E.; Hale, S. E.; Okita, R. T.; Masters, B. S S.

In: Journal of Biological Chemistry, Vol. 259, No. 23, 1984, p. 14600-14608.

Research output: Contribution to journalArticle

Williams, DE, Hale, SE, Okita, RT & Masters, BSS 1984, 'A prostaglandin ω-hydroxylase cytochrome P-450 (P-450(PG-ω)) purified from lungs of pregnant rabbits', Journal of Biological Chemistry, vol. 259, no. 23, pp. 14600-14608.
Williams, D. E. ; Hale, S. E. ; Okita, R. T. ; Masters, B. S S. / A prostaglandin ω-hydroxylase cytochrome P-450 (P-450(PG-ω)) purified from lungs of pregnant rabbits. In: Journal of Biological Chemistry. 1984 ; Vol. 259, No. 23. pp. 14600-14608.
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abstract = "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.",
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N2 - 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.

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