Hydroxylation of prostaglandin E1 by kidney cortex microsomal monooxygenase in the guinea pig

Javier Navarro, Daniel E. Piccolo, David Kupfer

Research output: Contribution to journalArticle

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Abstract

The incubation of [5,6-3H]prostaglandin E1 ([3H]PGE1) with guinea pig kidney cortex microsomes in the presence of NADPH in an atmosphere of air, resulted in chromatographically polar metabolites. The incubation products were treated with base which converted PGE1 derivatives into PGB1 derivatives, with a λmax = 278 nm and the products were analyzed by TLC and high pressure-liquid chromatography (HPLC). Based on UV absorption, mobility on TLC and retention time in HPLC, as compared with authentic compounds, it was concluded that the two polar UV-absorbing peaks in HPLC represented 19-hydroxy-PGB1 (19-OH-PGB1) and 20-hydroxy-PGB1 (20-OH-PGB1). Further identification of the metabolites was obtained by derivatizing the incubation products as methyl esters and t-butyldimethylsilyl ethers, followed by co-injection with similarly derivatized authentic compounds in HPLC and gas chromatography. Finally, the derivatized metabolites were identified by comparing their mass fragmentation with that of similarly derivatized authentic compounds. There was an absolute requirement for NADPH, and NADH did not significantly support the hydroxylation of PGE1. Inhibitors of microsomal monooxygenase (SKF 525A, metyrapone, and cytochrome c) inhibited the hydroxylation of PGE1 by kidney cortex microsomes. By contrast, carbon monoxide at a CO:O2 ratio of 5:1 did not inhibit the hydroxylation of PGE1, pointing to a low or lack of CO sensitivity of the hydroxylation of PGE1. The addition of PGE1 or laurate to guinea pig kidney cortex microsomes elicited Type I spectral changes. The spectral dissociation constant (Ks) for PGE1 was 2.4 × 10-4 m. The kinetic constants for 19- and 20-hydroxylations of PGE1 were determined. The KM values for the 19- and 20-hydroxylation pathways were found to be identical, being 3.3 × 10-4 m, suggesting that the same enzyme is involved in both hydroxylations; however, the Vmax values for 19-hydroxylation and 20-hydroxylation of PGE1 were 50 nmol/hr and 20.8 nmol/hr respectively. These results demonstrate that PGE1 is a substrate for the kidney cortex microsomal monooxygenase. The similarities and differences of the kidney monooxygenase in the guinea pig with that in the rat are discussed.

Original languageEnglish (US)
Pages (from-to)125-133
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume191
Issue number1
DOIs
StatePublished - 1978
Externally publishedYes

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Aryl Hydrocarbon Hydroxylases
Kidney Cortex
Hydroxylation
Alprostadil
Guinea Pigs
High pressure liquid chromatography
Carbon Monoxide
Metabolites
Microsomes
High Pressure Liquid Chromatography
NADP
Laurates
Proadifen
Derivatives
Metyrapone
Ethers
Mixed Function Oxygenases
Cytochromes c
Atmosphere
Gas chromatography

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Hydroxylation of prostaglandin E1 by kidney cortex microsomal monooxygenase in the guinea pig. / Navarro, Javier; Piccolo, Daniel E.; Kupfer, David.

In: Archives of Biochemistry and Biophysics, Vol. 191, No. 1, 1978, p. 125-133.

Research output: Contribution to journalArticle

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abstract = "The incubation of [5,6-3H]prostaglandin E1 ([3H]PGE1) with guinea pig kidney cortex microsomes in the presence of NADPH in an atmosphere of air, resulted in chromatographically polar metabolites. The incubation products were treated with base which converted PGE1 derivatives into PGB1 derivatives, with a λmax = 278 nm and the products were analyzed by TLC and high pressure-liquid chromatography (HPLC). Based on UV absorption, mobility on TLC and retention time in HPLC, as compared with authentic compounds, it was concluded that the two polar UV-absorbing peaks in HPLC represented 19-hydroxy-PGB1 (19-OH-PGB1) and 20-hydroxy-PGB1 (20-OH-PGB1). Further identification of the metabolites was obtained by derivatizing the incubation products as methyl esters and t-butyldimethylsilyl ethers, followed by co-injection with similarly derivatized authentic compounds in HPLC and gas chromatography. Finally, the derivatized metabolites were identified by comparing their mass fragmentation with that of similarly derivatized authentic compounds. There was an absolute requirement for NADPH, and NADH did not significantly support the hydroxylation of PGE1. Inhibitors of microsomal monooxygenase (SKF 525A, metyrapone, and cytochrome c) inhibited the hydroxylation of PGE1 by kidney cortex microsomes. By contrast, carbon monoxide at a CO:O2 ratio of 5:1 did not inhibit the hydroxylation of PGE1, pointing to a low or lack of CO sensitivity of the hydroxylation of PGE1. The addition of PGE1 or laurate to guinea pig kidney cortex microsomes elicited Type I spectral changes. The spectral dissociation constant (Ks) for PGE1 was 2.4 × 10-4 m. The kinetic constants for 19- and 20-hydroxylations of PGE1 were determined. The KM values for the 19- and 20-hydroxylation pathways were found to be identical, being 3.3 × 10-4 m, suggesting that the same enzyme is involved in both hydroxylations; however, the Vmax values for 19-hydroxylation and 20-hydroxylation of PGE1 were 50 nmol/hr and 20.8 nmol/hr respectively. These results demonstrate that PGE1 is a substrate for the kidney cortex microsomal monooxygenase. The similarities and differences of the kidney monooxygenase in the guinea pig with that in the rat are discussed.",
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N2 - The incubation of [5,6-3H]prostaglandin E1 ([3H]PGE1) with guinea pig kidney cortex microsomes in the presence of NADPH in an atmosphere of air, resulted in chromatographically polar metabolites. The incubation products were treated with base which converted PGE1 derivatives into PGB1 derivatives, with a λmax = 278 nm and the products were analyzed by TLC and high pressure-liquid chromatography (HPLC). Based on UV absorption, mobility on TLC and retention time in HPLC, as compared with authentic compounds, it was concluded that the two polar UV-absorbing peaks in HPLC represented 19-hydroxy-PGB1 (19-OH-PGB1) and 20-hydroxy-PGB1 (20-OH-PGB1). Further identification of the metabolites was obtained by derivatizing the incubation products as methyl esters and t-butyldimethylsilyl ethers, followed by co-injection with similarly derivatized authentic compounds in HPLC and gas chromatography. Finally, the derivatized metabolites were identified by comparing their mass fragmentation with that of similarly derivatized authentic compounds. There was an absolute requirement for NADPH, and NADH did not significantly support the hydroxylation of PGE1. Inhibitors of microsomal monooxygenase (SKF 525A, metyrapone, and cytochrome c) inhibited the hydroxylation of PGE1 by kidney cortex microsomes. By contrast, carbon monoxide at a CO:O2 ratio of 5:1 did not inhibit the hydroxylation of PGE1, pointing to a low or lack of CO sensitivity of the hydroxylation of PGE1. The addition of PGE1 or laurate to guinea pig kidney cortex microsomes elicited Type I spectral changes. The spectral dissociation constant (Ks) for PGE1 was 2.4 × 10-4 m. The kinetic constants for 19- and 20-hydroxylations of PGE1 were determined. The KM values for the 19- and 20-hydroxylation pathways were found to be identical, being 3.3 × 10-4 m, suggesting that the same enzyme is involved in both hydroxylations; however, the Vmax values for 19-hydroxylation and 20-hydroxylation of PGE1 were 50 nmol/hr and 20.8 nmol/hr respectively. These results demonstrate that PGE1 is a substrate for the kidney cortex microsomal monooxygenase. The similarities and differences of the kidney monooxygenase in the guinea pig with that in the rat are discussed.

AB - The incubation of [5,6-3H]prostaglandin E1 ([3H]PGE1) with guinea pig kidney cortex microsomes in the presence of NADPH in an atmosphere of air, resulted in chromatographically polar metabolites. The incubation products were treated with base which converted PGE1 derivatives into PGB1 derivatives, with a λmax = 278 nm and the products were analyzed by TLC and high pressure-liquid chromatography (HPLC). Based on UV absorption, mobility on TLC and retention time in HPLC, as compared with authentic compounds, it was concluded that the two polar UV-absorbing peaks in HPLC represented 19-hydroxy-PGB1 (19-OH-PGB1) and 20-hydroxy-PGB1 (20-OH-PGB1). Further identification of the metabolites was obtained by derivatizing the incubation products as methyl esters and t-butyldimethylsilyl ethers, followed by co-injection with similarly derivatized authentic compounds in HPLC and gas chromatography. Finally, the derivatized metabolites were identified by comparing their mass fragmentation with that of similarly derivatized authentic compounds. There was an absolute requirement for NADPH, and NADH did not significantly support the hydroxylation of PGE1. Inhibitors of microsomal monooxygenase (SKF 525A, metyrapone, and cytochrome c) inhibited the hydroxylation of PGE1 by kidney cortex microsomes. By contrast, carbon monoxide at a CO:O2 ratio of 5:1 did not inhibit the hydroxylation of PGE1, pointing to a low or lack of CO sensitivity of the hydroxylation of PGE1. The addition of PGE1 or laurate to guinea pig kidney cortex microsomes elicited Type I spectral changes. The spectral dissociation constant (Ks) for PGE1 was 2.4 × 10-4 m. The kinetic constants for 19- and 20-hydroxylations of PGE1 were determined. The KM values for the 19- and 20-hydroxylation pathways were found to be identical, being 3.3 × 10-4 m, suggesting that the same enzyme is involved in both hydroxylations; however, the Vmax values for 19-hydroxylation and 20-hydroxylation of PGE1 were 50 nmol/hr and 20.8 nmol/hr respectively. These results demonstrate that PGE1 is a substrate for the kidney cortex microsomal monooxygenase. The similarities and differences of the kidney monooxygenase in the guinea pig with that in the rat are discussed.

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