Lucigenin as a substrate of microsomal NAD(P)H-oxidoreductases.

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Abstract

NADPH oxidation and cytochrome c reduction with and without lucigenin as well as NAD(P)H/lucigenin-dependent chemiluminescence of rat liver microsomes were studied. An increased rate of NADPH oxidation and cytochrome c reduction in the presence of lucigenin was related to one-electron lucigenin reduction by microsomal NADPH reductases. The apparent Michaelis constant values for lucigenin (Km appLuc) were 3.6 and 5.0 microM in normoxygenic (pO2 = 150 +/- 5 mm Hg) and 8.7 and 8.3 microM in hypoxygenic (pO2 = 45 +/- 4 mm Hg) media in the reactions of lucigenin-dependent NADPH oxidation and cytochrome c reduction, respectively. The maximal level of NADPH/lucigenin-dependent chemiluminescence was registered at lucigenin concentration close to the mean K Luc/m app in the lucigenin-reductase reaction. Increasing the lucigenin concentration from 5 to 100 microM was associated with a decrease in the chemiluminescence intensity; this could be due to the inactivation of cytochrome P450. In the presence of superoxide dismutase (SOD), the rate of lucigenin-dependent cytochrome c reduction and NADPH/lucigenin-dependent chemiluminescence were decreased by 10 and 30%, respectively. The addition of lucigenin to microsomes which contain the reduced hemoprotein--CO complex was followed by the disappearance of the differential absorption spectrum specific for the carboxy complex and by increase in chemiluminescence intensity versus the control (without carboxy complex). Thus, lucigenin-dependent chemiluminescence of microsomes may be due to some enzymes including lucigenin reductase (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase), generation of O2-. in the redox cycle of lucigenin radicals, dioxetane formation by (di)oxygenases, and catalytic action of the cytochrome P450 heme on dioxetane decomposition followed by light quantum emission. Thus, lucigenin cannot be used to measure the basal O2-. formation in tissue homogenates with high levels of NAD(P)H-oxidoreductases.

Original languageEnglish
Pages (from-to)25-32
Number of pages8
JournalBiochemistry. Biokhimiia
Volume64
Issue number1
Publication statusPublished - Jan 1999
Externally publishedYes

Fingerprint

NAD
Oxidoreductases
Substrates
Chemiluminescence
Luminescence
NADP
Cytochromes c
10,10'-dimethyl-9,9'-biacridinium
Microsomes
Oxidation
Cytochrome P-450 Enzyme System
Cytochrome-B(5) Reductase
NADPH-Ferrihemoprotein Reductase
Oxygenases
Liver Microsomes
Carbon Monoxide
Heme
Application programs
Liver
Superoxide Dismutase

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Lucigenin as a substrate of microsomal NAD(P)H-oxidoreductases. / Schepetkin, I. A.

In: Biochemistry. Biokhimiia, Vol. 64, No. 1, 01.1999, p. 25-32.

Research output: Contribution to journalArticle

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title = "Lucigenin as a substrate of microsomal NAD(P)H-oxidoreductases.",
abstract = "NADPH oxidation and cytochrome c reduction with and without lucigenin as well as NAD(P)H/lucigenin-dependent chemiluminescence of rat liver microsomes were studied. An increased rate of NADPH oxidation and cytochrome c reduction in the presence of lucigenin was related to one-electron lucigenin reduction by microsomal NADPH reductases. The apparent Michaelis constant values for lucigenin (Km appLuc) were 3.6 and 5.0 microM in normoxygenic (pO2 = 150 +/- 5 mm Hg) and 8.7 and 8.3 microM in hypoxygenic (pO2 = 45 +/- 4 mm Hg) media in the reactions of lucigenin-dependent NADPH oxidation and cytochrome c reduction, respectively. The maximal level of NADPH/lucigenin-dependent chemiluminescence was registered at lucigenin concentration close to the mean K Luc/m app in the lucigenin-reductase reaction. Increasing the lucigenin concentration from 5 to 100 microM was associated with a decrease in the chemiluminescence intensity; this could be due to the inactivation of cytochrome P450. In the presence of superoxide dismutase (SOD), the rate of lucigenin-dependent cytochrome c reduction and NADPH/lucigenin-dependent chemiluminescence were decreased by 10 and 30{\%}, respectively. The addition of lucigenin to microsomes which contain the reduced hemoprotein--CO complex was followed by the disappearance of the differential absorption spectrum specific for the carboxy complex and by increase in chemiluminescence intensity versus the control (without carboxy complex). Thus, lucigenin-dependent chemiluminescence of microsomes may be due to some enzymes including lucigenin reductase (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase), generation of O2-. in the redox cycle of lucigenin radicals, dioxetane formation by (di)oxygenases, and catalytic action of the cytochrome P450 heme on dioxetane decomposition followed by light quantum emission. Thus, lucigenin cannot be used to measure the basal O2-. formation in tissue homogenates with high levels of NAD(P)H-oxidoreductases.",
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N2 - NADPH oxidation and cytochrome c reduction with and without lucigenin as well as NAD(P)H/lucigenin-dependent chemiluminescence of rat liver microsomes were studied. An increased rate of NADPH oxidation and cytochrome c reduction in the presence of lucigenin was related to one-electron lucigenin reduction by microsomal NADPH reductases. The apparent Michaelis constant values for lucigenin (Km appLuc) were 3.6 and 5.0 microM in normoxygenic (pO2 = 150 +/- 5 mm Hg) and 8.7 and 8.3 microM in hypoxygenic (pO2 = 45 +/- 4 mm Hg) media in the reactions of lucigenin-dependent NADPH oxidation and cytochrome c reduction, respectively. The maximal level of NADPH/lucigenin-dependent chemiluminescence was registered at lucigenin concentration close to the mean K Luc/m app in the lucigenin-reductase reaction. Increasing the lucigenin concentration from 5 to 100 microM was associated with a decrease in the chemiluminescence intensity; this could be due to the inactivation of cytochrome P450. In the presence of superoxide dismutase (SOD), the rate of lucigenin-dependent cytochrome c reduction and NADPH/lucigenin-dependent chemiluminescence were decreased by 10 and 30%, respectively. The addition of lucigenin to microsomes which contain the reduced hemoprotein--CO complex was followed by the disappearance of the differential absorption spectrum specific for the carboxy complex and by increase in chemiluminescence intensity versus the control (without carboxy complex). Thus, lucigenin-dependent chemiluminescence of microsomes may be due to some enzymes including lucigenin reductase (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase), generation of O2-. in the redox cycle of lucigenin radicals, dioxetane formation by (di)oxygenases, and catalytic action of the cytochrome P450 heme on dioxetane decomposition followed by light quantum emission. Thus, lucigenin cannot be used to measure the basal O2-. formation in tissue homogenates with high levels of NAD(P)H-oxidoreductases.

AB - NADPH oxidation and cytochrome c reduction with and without lucigenin as well as NAD(P)H/lucigenin-dependent chemiluminescence of rat liver microsomes were studied. An increased rate of NADPH oxidation and cytochrome c reduction in the presence of lucigenin was related to one-electron lucigenin reduction by microsomal NADPH reductases. The apparent Michaelis constant values for lucigenin (Km appLuc) were 3.6 and 5.0 microM in normoxygenic (pO2 = 150 +/- 5 mm Hg) and 8.7 and 8.3 microM in hypoxygenic (pO2 = 45 +/- 4 mm Hg) media in the reactions of lucigenin-dependent NADPH oxidation and cytochrome c reduction, respectively. The maximal level of NADPH/lucigenin-dependent chemiluminescence was registered at lucigenin concentration close to the mean K Luc/m app in the lucigenin-reductase reaction. Increasing the lucigenin concentration from 5 to 100 microM was associated with a decrease in the chemiluminescence intensity; this could be due to the inactivation of cytochrome P450. In the presence of superoxide dismutase (SOD), the rate of lucigenin-dependent cytochrome c reduction and NADPH/lucigenin-dependent chemiluminescence were decreased by 10 and 30%, respectively. The addition of lucigenin to microsomes which contain the reduced hemoprotein--CO complex was followed by the disappearance of the differential absorption spectrum specific for the carboxy complex and by increase in chemiluminescence intensity versus the control (without carboxy complex). Thus, lucigenin-dependent chemiluminescence of microsomes may be due to some enzymes including lucigenin reductase (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase), generation of O2-. in the redox cycle of lucigenin radicals, dioxetane formation by (di)oxygenases, and catalytic action of the cytochrome P450 heme on dioxetane decomposition followed by light quantum emission. Thus, lucigenin cannot be used to measure the basal O2-. formation in tissue homogenates with high levels of NAD(P)H-oxidoreductases.

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