Action of diethylstilbestrol on the NADH-dehydrogenase region of the respiratory chain

doi:10.1016/0003-9861(74)90137-4

Archives of Biochemistry and Biophysics

Volume 165, Issue 1, November 1974, Pages 21-33

https://doi.org/10.1016/0003-9861(74)90137-4 Get rights and content

Abstract

Diethylstilbestrol reversibly inhibits electron transfer in particle-bound mitochondrial NADH-dehydrogenase (Keilin-Hartree preparation) at a site located in the vicinity of, or superimposed on, the piericidin A- and rotenone-specific sites. At this site, diethylstilbestrol half-maximal inhibitory concentration varies from 0.2 μm (at 37 μm NADH) to 2.6 μm (at 180 μm or higher NADH concentrations). The postulated localization of the diethylstilbestrol site is supported by (a) the similar effects of the diethylstilbestrol, piericidin A, and rotenone on the kinetics of the NADH-induced changes of the 470–500-nm chromophore; (b) the lack of action of diethylstilbestrol on the NADH-ferricyanide reductase and the NADH-acetylpyridine-adenine dinucleotide transhydrogenase reactions; (c) the lack of action of diethylstilbestrol on the NADH-2,3-dimethoxy-2′-methyl-1,4-benzoquinone reaction at infinite concentration of UQ₀; (d) the similar inhibitions of the NADH-UQ₂ reaction and NADH-cytochrome b_k reaction; (e) the noncompetitive inhibition of the NADH-UQ₂ reaction; (f) the similar effects of diethylstilbestrol and rotenone on the kinetics of the NADH-UQ₀ reaction; and (g) the inhibition of the NADH-cytochrome b_k reaction, in contrast with the insensitivity of the succinate-cytochrome b_k reaction. The interaction of diethylstilbestrol with the NADH-dehydrogenase protein is supported by the summation of diethylstilbestrol, piericidin A, rotenone, and p-chloromercuribenzoate inhibitions. Diethylstilbestrol inhibition involves specific structural requirements as shown by the lesser (if any) inhibitory potency of hexestrol, diethylstilbestrol monomethyl ether, diethylstilbestrol dimethyl ether, and hexestrol dimethyl ether.

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effective inhibitor. The inhibition was maximum (78%) in the presence of Q1, when the higher relative electron flow through the rotenone-sensitive site was established, and minimum (48%) in the presence of Q0, when two quinone reduction sites were operative, one on the substrate side and the other on the oxygen side of the rotenone-sensitive site (21-23). Previous data (9) and the results presented in this paper point to ubisemiqui- none and ubiquinol as the main sources of mitochondrial H2O2, but the operation of other H2O2 generators is not excluded.
Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated $O_{2}^{-}$ at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H₂O₂ at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H₂O₂ was essentially produced by disproportionation of $O_{2}^{-}$ , which constitutes the precursor of H₂O₂. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of $O_{2}^{-}$ and 0.63 mol of H₂O₂/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O₂ to $O_{2}^{-}$ , since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H₂0₂ generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H₂O₂ generation. The efficiency of added quinones as peroxide generators decreased in the order Q₁ > Q₀ > Q₂ > Q₆ = Q₁₀, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymati- cally by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of $O_{2}^{-}$ and H₂O₂ during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q₀H₂), benzoquinol, duroquinol and menadiol generated $O_{2}^{-}$ with k₃ values of 0.1 to 1.4 M^-1 s^-1 and H₂O₂ with k₄ values of 0.009 to 4.3 m^-1·s^-1
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Classical genomic and non-genomic signaling pathways mediated by nuclear and cell membrane estrogen receptors are considered to contribute to estrogen-induced cell proliferation. Here we propose that mitochondrial signals to the nucleus regulate estrogen-induced progression of the cell cycle. The influence of estrogen on mitochondrial oxidative phosphorylation and mitochondrial gene transcription support the idea that mitochondria are significant targets of estrogen. Mitochondria are the major source of reactive oxygen species (ROS) in epithelial cells. Estrogen redox cycling within mitochondria also generates ROS. Antioxidants inhibit estrogen-induced cell growth. A-Raf, Akt, PKC, MEK, ERK, and transcription factors AP-1, NF-κB, and CREB are targets of both estrogen and ROS. We provide four lines of evidence in support of our hypothesis that estrogen-induced mitochondrial ROS stimulate redox sensor kinase A-Raf, Akt or PKC, which, in turn, activate transcription factors NF-κB, CREB, or AP-1 via the MEK/ERK pathway. Thus, estrogen-induced mitochondrial ROS leading to the activation of cell cycle genes containing AP-1, NF-κB, or CREB response elements are involved in the progression of the cell cycle of the estrogen-dependent cells. Our novel concept will contribute to the development of new targets in the prevention and control of estrogen-induced disease including cancer.
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In order to investigate the possibility that fusicoccin(FC)-induced electrogenic H⁺ extrusion and K⁺ uptake in higher plants depend on the activity of a plasmalemma ATPase, we have studied the effect of two non-mitochondrial membrane-bound ATPase inhibitors, diethylstilbestrol (DES) and vanadate, on the transport of H⁺ and K⁺ in maize root segments.
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The effects of diethylstilbestrol (DES) and oligomycin, inhibitors of ion absorption in animal cells, on the energetics of ion absorption in oat roots were compared. Both compounds inhibited K⁺ and Cl⁻ absorption in excised roots and oxidative phosphorylation in isolated mitochondria. DES inhibited a plasma membrane ATPase that is involved with ion absorption but did not inhibit the mitochondrial ATPase, whereas, oligomycin inhibited the mitochondrial ATPase but inhibited the plasma membrane ATPase only slightly.
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Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O₂⁻ at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H₂O₂ at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H₂O₂ was essentially produced by disproportionation of O₂⁻, which constitutes the precursor of H₂O₂. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O₂⁻ and 0.63 mol of H₂O₂/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O₂ to O₂⁻, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H₂O₂ generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H₂O₂ generation. The efficiency of added quinones as peroxide generators decreased in the order Q₁ > Q₀ > Q₂ > Q₆ = Q₁₀, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymatically by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O₂⁻ and H₂O₂ during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q₀H₂), benzoquinol, duroquinol and menadiol generated O₂⁻ with k₃ values of 0.1 to 1.4 m⁻ · s⁻¹ and H₂O₂ with k₄ values of 0.009 to 4.3 m⁻¹ · s⁻¹.

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^☆: Aided by grants from Consejo Nacional de Investigaciones Científicas y Técnicas, The Jane Coffin. Childs Fund for Medical Research, and the United States Air Force Office under Grant AFOSR-68-1395, monitored by the Air Force Office of Scientific Research, Office of Aerospace Research.

²: Member of Investigator Career, Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina.

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Action of diethylstilbestrol on the NADH-dehydrogenase region of the respiratory chain☆

Abstract

Biochim. Biophys. Res. Commun

Biochim. Biophys. Acta

Arch. Biochem. Biophys

Arch. Biochem. Biophys

Arch. Biochem. Biophys

Arch. Biochem. Biophys

Arch. Biochem. Biophys

J. Biol. Chem

J. Chromatogr

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

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J. Mol. Biol

Arch. Biochem. Biophys

J. Biol. Chem

Fed. Eur. Biochem. Soc

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Biochim. Biophys. Res. Commun

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