Relationship between the binding of dicyclohexylcarbodiimide and the inhibition of H<sup>+</sup>‐translocation in submitochondrial particles

Kopecký, Jan; Glaser, Elzbieta; Norling, Birgitta; Ernster, Lars

doi:10.1016/0014-5793(81)80369-9

Cited by 21 publications

(8 citation statements)

References 32 publications

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“…Each monomer contains a specific glutamyl residue, whose y-carboxyl group is the binding-site for dicyclohexylcarbodiimide and is assumed to be involved in H+-translocation. As concluded in a previous paper [45] and further supported by the present data, two moles of dicyclohexylcarbodiimide per mole F, seem to be required to completely inhibit passive H + -translocation. It appears, therefore, that F,, in the absence of F,, contains two H+-channels, both of which are equally active in H+-translocation.…”

Section: Discussionsupporting

confidence: 92%

“…Similar reasoning concerning the titers for dicyclohexylcarbodiimide leads to the conclusion that one mole of dicyclohexylcarbodiimide is needed for the inhibition of H+-flow through complete F,F, sites measured as inhibition of the ATPase activity, whereas two moles of the inhibitor are required for the inhibition of H +-translocation through F,-lacking F, sites, measured as stimulation of the respiratory control ratio. These findings have been discussed in a previous paper [45].…”

Section: Figuresupporting

confidence: 71%

“…F,-depleted particles were prepared from the Sephadex-treated particles by treatment with urea [47]. EDTA particles containing dextran labelled with fluoresceine isothiocyanate were prepared by including 1 mg of the labelled dextran/mg protein in the medium during sonication of the mitochondria [45].…”

Section: Preparationsmentioning

confidence: 99%

“…Binding of dicyclohexylcarbodiimide to the particles were estimated by the use of ['4C]dicyclohexylcarbodiimide as described previously [45].…”

Section: Incubations With Oligomycin and Dicyclohexylcarbodiimidementioning

confidence: 99%

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Comparison of the Effects of Oligomycin and Dicyclohexylcarbodiimide on Mitochondrial ATPase and Related Reactions

Glaser¹,

Norling²,

Kopecký³

et al. 1982

Eur J Biochem

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Titration curves with oligomycin of the ATPase activity, NADH oxidase activity and passive H+‐flow through Fo in submitochondrial particles from beef heart are similar and indicate that one mole of oligomycin bound per one mole of Fo is needed for maximal inhibition of H +‐translocation coupled to ATP hydrolysis and H+‐translocation through Fo in the absence of F1. Titration curves of the above activities with dicyclohexylcarbodiimide indicate that one mole of dicyclohexylcarbodiimide per one mole of Fo is needed for inhibition of H+‐translocation coupled to ATP hydrolysis and two moles of dicyclohexylcarbodiimide are needed for inhibition of H+‐translocation through Fo lacking F1. Small amounts of oligomycin stimulate ATP‐dependent transhydrogenase activity and an ATP‐dependent anilinonaphthalene sulfonate response whereas dicyclohexylcarbodiimide does not have this effect. Respiratory chain‐driven (succinate oxidase‐supported) transhydrogenase and the anilinonaphthalene sulfonate response can however be stimulated by both inhibitors. All the above effects are observed after preincubation of the particles with oligomycin or dicyclohexylcarbodiiniide at 0°C for 12–18 h. Short‐time incubation with larger amounts of oligomycin or dicyclohexylcarbodiimide results in a preferential inhibition of H+‐flow through Fo devoid of F1 as compared to H+‐flow through complete FoF1 complexes. Under these conditions, dicyclohexylcarbodiimide also stimulates ATP‐dependent transhydrogenase activity. The effects of oligomycin and dicyclohexylcarbodiimide on the ATPase activity are not additive, indicating different binding sites of the two inhibitors.

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Section: Discussionsupporting

confidence: 92%

Section: Figuresupporting

confidence: 71%

Section: Preparationsmentioning

confidence: 99%

“…Binding of dicyclohexylcarbodiimide to the particles were estimated by the use of ['4C]dicyclohexylcarbodiimide as described previously [45].…”

Section: Incubations With Oligomycin and Dicyclohexylcarbodiimidementioning

confidence: 99%

See 2 more Smart Citations

Comparison of the Effects of Oligomycin and Dicyclohexylcarbodiimide on Mitochondrial ATPase and Related Reactions

Glaser¹,

Norling²,

Kopecký³

et al. 1982

Eur J Biochem

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“…These 'other' inhibitors removed only about 20% of the total ATPase activity, even at relatively high inhibitor concentration. Di-cyclohexyl-carbodiimide, which inhibits proton translocation also in the related F-ATPase (see, e.g., (Kopecky et al 1981;Kopecky et al 1982;Kopecky et al 1983;Hermolin and Fillingame 1989;Wada et al 2000)), was more potent, but the most potent inhibitors were, as expected, concanamycin A and bafilomycin, even when applied at the lowest concentrations (Bowman et al 1988;Drose et al 1993;Gagliardi et al 1999;Huss et al 2002;Dixon et al 2008). In this system, and under the present conditions, these inhibitors removed ~60% of the total ATPase activity, which varied to some extent from isolation to isolation.…”

Section: Atpase Activity Assaymentioning

confidence: 99%

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

et al. 2012

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The rate of rotation of the rotor of the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or the steady parts of enzyme, is estimated in native vacuolar membrane vesicles of Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are spontaneously formed after exposing purified yeast vacuoles to osmotic shock. The fraction of the total ATPase activity originating from V-ATPase is determined using the potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during 10 min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit to the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data is incompatible with models assuming more binding sites) to the inhibitor titration curve determines the concentration of the enzyme. Combining it with the known rotation:ATP stoichiometry of V-ATPase and the assayed concentration of inorganic phosphate liberated by V-ATPase leads to an average rate of ~9.53 Hz of the 360 degrees rotation, which, according to the time-dependence of the activity, extrapolates to ~14.14 Hz for the beginning of the reaction. These are low limit estimates. To our knowledge this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and it is not fixed on a solid support, instead it is functioning in its native membrane environment.Special Issue: Structure, function, folding and assembly of membrane proteins -Insight from Biophysics.

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The Enzymes and the Enzyme Complexes of the Mitochondrial Oxidative Phosphorylation System

Hatefi

Ragan

Galante

1985

The Enzymes of Biological Membranes

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Relationship between the binding of dicyclohexylcarbodiimide and the inhibition of H⁺‐translocation in submitochondrial particles

Cited by 21 publications

References 32 publications

Comparison of the Effects of Oligomycin and Dicyclohexylcarbodiimide on Mitochondrial ATPase and Related Reactions

Comparison of the Effects of Oligomycin and Dicyclohexylcarbodiimide on Mitochondrial ATPase and Related Reactions

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

The Enzymes and the Enzyme Complexes of the Mitochondrial Oxidative Phosphorylation System

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Relationship between the binding of dicyclohexylcarbodiimide and the inhibition of H+‐translocation in submitochondrial particles

Cited by 21 publications

References 32 publications

Comparison of the Effects of Oligomycin and Dicyclohexylcarbodiimide on Mitochondrial ATPase and Related Reactions

Comparison of the Effects of Oligomycin and Dicyclohexylcarbodiimide on Mitochondrial ATPase and Related Reactions

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

The Enzymes and the Enzyme Complexes of the Mitochondrial Oxidative Phosphorylation System

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Product

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Relationship between the binding of dicyclohexylcarbodiimide and the inhibition of H⁺‐translocation in submitochondrial particles