Hydroxylation of prostaglandins A1 and E1 by liver microsomal monooxygenase. Characteristics of the enzyme system in the guinea pig

D. Kupfer, Javier Navarro, D. E. Piccolo

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Abstract

Guinea pig liver microsomes in the presence of NADPH catalyzed the hydroxylation of prostaglandin A1 (PGA1) and prostaglandin E1 (PGE1) primarily (>95%) at the ω-1 and to a minor extent (<5%) at the ω position, yielding 19-hydroxy and 20-hydroxy derivatives. The identity of the 19-hydroxy metabolites was established in the form of 19-OH-PGB1 after isolation with the help of high pressure liquid chromatography (HPLC). The isolated product having characteristic UV absorption of PGB1 (λ(max) = 278 nm) was compared with authentic 19-OH-PGB1. The metabolites from PGA1 and PGE1 and authentic 19-OH-PGB1 exhibited identical retention times in HPLC as free acids and as the corresponding methyl esters. The corresponding t-butyldimethylsilyl ethers-methyl esters also exhibited identical retention times in the gas chromatography (GC) and similar fragmentations in GC/mass spectrometry. Both NADPH and NADH supported the hydroxylation of PGA1 and PGE1, NADH being less effective. There was no synergism by NADH of the NADPH-supported hydroxylation. Inhibitors of microsomal monooxygenase such as SKF 525A, metyrapone, nicotinamide, and carbon monoxide inhibited the hydroxylation of PGA1 and PGE1; similarly cytochrome c and antibodies to NADPH-cytochrome c reductase inhibited these reactions, indicating that the hydroxylation of these prostaglandins is catalyzed by a typical monooxygenase system. The kinetic constants for the hydroxylation of PGA1 and PGE1 were determined; the K(m) values were 2.1 x 10-4 M and 1.4 x 10-4 M, respectively. The V(max) values for the two prostaglandins were also similar, being for PGA1 and PGE1, 21.8 and 16.7 nmol/h/mg of protein, respectively.

Original languageEnglish (US)
Pages (from-to)2804-2811
Number of pages8
JournalJournal of Biological Chemistry
Volume253
Issue number8
StatePublished - 1978
Externally publishedYes

Fingerprint

Aryl Hydrocarbon Hydroxylases
Hydroxylation
Alprostadil
Liver
Guinea Pigs
Enzymes
NADP
High pressure liquid chromatography
NAD
Metabolites
Gas chromatography
Prostaglandins
Esters
High Pressure Liquid Chromatography
Proadifen
Metyrapone
Methyl Ethers
NADPH-Ferrihemoprotein Reductase
Niacinamide
Liver Microsomes

ASJC Scopus subject areas

  • Biochemistry

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Hydroxylation of prostaglandins A1 and E1 by liver microsomal monooxygenase. Characteristics of the enzyme system in the guinea pig. / Kupfer, D.; Navarro, Javier; Piccolo, D. E.

In: Journal of Biological Chemistry, Vol. 253, No. 8, 1978, p. 2804-2811.

Research output: Contribution to journalArticle

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abstract = "Guinea pig liver microsomes in the presence of NADPH catalyzed the hydroxylation of prostaglandin A1 (PGA1) and prostaglandin E1 (PGE1) primarily (>95{\%}) at the ω-1 and to a minor extent (<5{\%}) at the ω position, yielding 19-hydroxy and 20-hydroxy derivatives. The identity of the 19-hydroxy metabolites was established in the form of 19-OH-PGB1 after isolation with the help of high pressure liquid chromatography (HPLC). The isolated product having characteristic UV absorption of PGB1 (λ(max) = 278 nm) was compared with authentic 19-OH-PGB1. The metabolites from PGA1 and PGE1 and authentic 19-OH-PGB1 exhibited identical retention times in HPLC as free acids and as the corresponding methyl esters. The corresponding t-butyldimethylsilyl ethers-methyl esters also exhibited identical retention times in the gas chromatography (GC) and similar fragmentations in GC/mass spectrometry. Both NADPH and NADH supported the hydroxylation of PGA1 and PGE1, NADH being less effective. There was no synergism by NADH of the NADPH-supported hydroxylation. Inhibitors of microsomal monooxygenase such as SKF 525A, metyrapone, nicotinamide, and carbon monoxide inhibited the hydroxylation of PGA1 and PGE1; similarly cytochrome c and antibodies to NADPH-cytochrome c reductase inhibited these reactions, indicating that the hydroxylation of these prostaglandins is catalyzed by a typical monooxygenase system. The kinetic constants for the hydroxylation of PGA1 and PGE1 were determined; the K(m) values were 2.1 x 10-4 M and 1.4 x 10-4 M, respectively. The V(max) values for the two prostaglandins were also similar, being for PGA1 and PGE1, 21.8 and 16.7 nmol/h/mg of protein, respectively.",
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N2 - Guinea pig liver microsomes in the presence of NADPH catalyzed the hydroxylation of prostaglandin A1 (PGA1) and prostaglandin E1 (PGE1) primarily (>95%) at the ω-1 and to a minor extent (<5%) at the ω position, yielding 19-hydroxy and 20-hydroxy derivatives. The identity of the 19-hydroxy metabolites was established in the form of 19-OH-PGB1 after isolation with the help of high pressure liquid chromatography (HPLC). The isolated product having characteristic UV absorption of PGB1 (λ(max) = 278 nm) was compared with authentic 19-OH-PGB1. The metabolites from PGA1 and PGE1 and authentic 19-OH-PGB1 exhibited identical retention times in HPLC as free acids and as the corresponding methyl esters. The corresponding t-butyldimethylsilyl ethers-methyl esters also exhibited identical retention times in the gas chromatography (GC) and similar fragmentations in GC/mass spectrometry. Both NADPH and NADH supported the hydroxylation of PGA1 and PGE1, NADH being less effective. There was no synergism by NADH of the NADPH-supported hydroxylation. Inhibitors of microsomal monooxygenase such as SKF 525A, metyrapone, nicotinamide, and carbon monoxide inhibited the hydroxylation of PGA1 and PGE1; similarly cytochrome c and antibodies to NADPH-cytochrome c reductase inhibited these reactions, indicating that the hydroxylation of these prostaglandins is catalyzed by a typical monooxygenase system. The kinetic constants for the hydroxylation of PGA1 and PGE1 were determined; the K(m) values were 2.1 x 10-4 M and 1.4 x 10-4 M, respectively. The V(max) values for the two prostaglandins were also similar, being for PGA1 and PGE1, 21.8 and 16.7 nmol/h/mg of protein, respectively.

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