Reactivity of mitochondrial sulfhydryl groups toward dithionitrobenzoic acid and bromobimanes under oligomycin-inhibited and uncoupling conditions

Freisleben, Hans-Joachim; Fuchs, Jürgen; Mainka, Luise; Zimmer, Guido

doi:10.1016/0003-9861(88)90239-1

Cited by 16 publications

(4 citation statements)

References 26 publications

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“…T-4,5-D also binds to SH residues of alcohol dehydrogenase (44) and guanine nucleotide-binding regulatory proteins (50) with resultant inhibition of their activities. Modifications of SH (cysteinyl) residues of mt respiratory enzyme complexes can evoke uncoupling (51,52) and loss of function (53). Thus, it is likely that covalent attachment of T-4,5-D to certain SH residues of mt respiratory enzyme complexes is responsible for uncoupling of respiration (Table 1) and irreversible inhibition of complex I (Table 2) and complex IV (Table 3).…”

Section: Discussionmentioning

confidence: 99%

Tryptamine-4,5-dione, a Putative Endotoxic Metabolite of the Superoxide-Mediated Oxidation of Serotonin, Is a Mitochondrial Toxin: Possible Implications in Neurodegenerative Brain Disorders

Jiang

Wrona

Dryhurst

1999

Chem. Res. Toxicol.

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The release and subsequent reuptake of 5-hydroxytryptamine (5-HT) and cytoplasmic superoxide (O2-*) generation have both been implicated as important factors associated with the degeneration of serotonergic neurons evoked by methamphetamine (MA) and cerebral ischemia-reperfusion (I-R). Such observations raise the possibility that tryptamine-4,5-dione (T-4,5-D), the major in vitro product of the O2-*-mediated oxidation of 5-HT, might be an endotoxicant that contributes to serotonergic neurodegeneration. When incubated with intact rat brain mitochondria, T-4,5-D (< or = 100 microM) uncouples respiration and inhibits state 3. Experiments with rat brain mitochondrial membrane preparations confirm that T-4,5-D evokes irreversible inhibition of NADH-coenzyme Q1 (CoQ1) reductase and cytochrome c oxidase (COX) apparently by covalently modifying key sulfhydryl (SH) residues at or close to the active sites of these respiratory enzyme complexes. Ascorbic acid blocks the inhibition of NADH-CoQ1 reductase by maintaining T-4,5-D predominantly as 4, 5-dihydroxytryptamine (4,5-DHT), thus preventing its reaction with SH residues. In contrast, ascorbic acid potentiates the irreversible inhibition of COX by T-4,5-D. This may be because the T-4,5-D-4, 5-DHT couple redox cycles in the presence of excess ascorbate and molecular oxygen to cogenerate O2-* and H2O2 that together react with trace levels of iron to form an oxo-iron complex that selectively damages COX. Thus, T-4,5-D might be an endotoxicant that, dependent on intraneuronal conditions, mediates irreversible damage to mitochondrial respiratory enzyme complexes and contributes to the serotonergic neurodegeneration evoked by MA and I-R.

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Section: Discussionmentioning

confidence: 99%

Tryptamine-4,5-dione, a Putative Endotoxic Metabolite of the Superoxide-Mediated Oxidation of Serotonin, Is a Mitochondrial Toxin: Possible Implications in Neurodegenerative Brain Disorders

Jiang

Wrona

Dryhurst

1999

Chem. Res. Toxicol.

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“…The commonly used bromobimanes are monobromobimane (mBBr), dibromobimane (dBBr), and monobromo(trimethylammonio)bimane (qBBr) (Figure ). However, mBBr is the most commonly used molecule because of its small size. − When compared to these bimanes, qBBr has advantages for detecting R-SH in DOM and in cell membranes due to its higher solubility in water and greater stability under ambient conditions. − The positive charge on qBBr also makes crossing the cell membrane difficult, and therefore makes qBBr ideal for detecting R-SH on the bacterial cell envelope …”

Section: Introductionmentioning

confidence: 99%

Estimation of Reactive Thiol Concentrations in Dissolved Organic Matter and Bacterial Cell Membranes in Aquatic Systems

Joe-Wong

Shoenfelt

Hauser

et al. 2012

Environ. Sci. Technol.

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Organic thiols are highly reactive ligands and play an important role in the speciation of several metals and organic pollutants in the environment. Although small thiols can be isolated and their concentrations can be estimated using chromatographic and derivatization techniques, estimating concentrations of thiols associated with biomacromolecules and humic substances has been difficult. Here we present a fluorescence-spectroscopy-based method for estimating thiol concentrations in biomacromolecules and cell membranes using one of the soluble bromobimanes, monobromo(trimethylammonio)bimane (qBBr). The fluorescence of this molecule increases significantly when it binds to a thiol. The change in the sample fluorescence due to thiols reacting with qBBr is used to determine thiol concentration in a sample. Using this method, small thiols such as cysteine and glutathione can be detected in clean solutions down to ~50 nM without their separation and prior concentration. Thiols associated with dissolved organic matter (DOM) can be detected down to low micromolar concentration, depending on the DOM background fluorescence. The charge on qBBr prevents its rapid diffusion across cell membranes, so qBBr is ideal for estimating thiol concentration at the cell membrane-water interface. This method was successfully used to determine the thiol concentration on the cell envelope of intact Bacillus subtilis to nanomolar concentration without any special sample preparation. Among the chemical species tested for potential interferences (other reduced sulfides methionine and cystine, carboxylate, salt (MgCl(2))), carboxylates significantly influenced the absolute fluorescence signal of the thiol-qBBr complex. However, this does not affect the detection of thiols in heterogeneous mixtures using the presented method.

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“…For detection of binding of the spin-probe several parameters were used. Splitting of the low field peak h,, into the two peaks, h,,, and h+l,, corresponding to 'mobile' and immobilized fractions, respectively, reflects binding of the probe [18,19]. If their separation is low, binding can be described simply by an increase in the half-height width of the h,, peak ( W(!/Z),+,).…”

Section: Epr Spectroscopymentioning

confidence: 99%

“…If their separation is low, binding can be described simply by an increase in the half-height width of the h,, peak ( W(!/Z),+,). Another measure of binding is the ratio of heights of the middle-and high-field peak, /I&-' [19,20], a term necessary for calculation of the rotational correlation time [11,12], given as:…”

Section: Epr Spectroscopymentioning

confidence: 99%

Fatty acid binding site of the mitochondrial uncoupling protein

Ježek

Freisleben

1994

FEBS Letters

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Fatty acid binding site on isolated mitochondrial uncoupling protein (UcP) is demonstrated using EPR spectroscopy of S-DOXYL-stearic acid (5SASL), which also activated H+ transport in proteoliposomes containing UcP. In the presence of UcP the EPR spectrum showed reproducible broadening of the low field peak as well as an increase in h+lI/h+,M ratio, rotational correlation time and in order parameter. The half-height width of the low field peak was even doubled in the presence of another UcP ligand, GDP. Palmitic acid reversed the effect of 5-SASL and non-ionizable 5-DOXYL-decane did not exhibit it.

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Reactivity of mitochondrial sulfhydryl groups toward dithionitrobenzoic acid and bromobimanes under oligomycin-inhibited and uncoupling conditions

Cited by 16 publications

References 26 publications

Tryptamine-4,5-dione, a Putative Endotoxic Metabolite of the Superoxide-Mediated Oxidation of Serotonin, Is a Mitochondrial Toxin: Possible Implications in Neurodegenerative Brain Disorders

Tryptamine-4,5-dione, a Putative Endotoxic Metabolite of the Superoxide-Mediated Oxidation of Serotonin, Is a Mitochondrial Toxin: Possible Implications in Neurodegenerative Brain Disorders

Estimation of Reactive Thiol Concentrations in Dissolved Organic Matter and Bacterial Cell Membranes in Aquatic Systems

Fatty acid binding site of the mitochondrial uncoupling protein

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