Concanamycin A, the Specific Inhibitor of V-ATPases, Binds to the Vo Subunit c

Huss, Markus; Ingenhorst, Gudrun; König, Simone; Gaßel, Michael; Dröse, Stefan; Zeeck, Axel; Altendorf, Karlheinz; Wieczorek, Helmut

doi:10.1074/jbc.m207345200

Cited by 254 publications

(260 citation statements)

References 36 publications

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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 Assaysupporting

confidence: 53%

“…Our data is compatible only with a single binding site per monomeric enzyme. Since concanamycin A most likely binds to intramembranous subunits Whyteside et al 2005;Bowman et al 2006;Dixon et al 2008), the present result suggests that an interaction with a single subunit c is not sufficient for concanamycin A binding: it either binds to more than one subunits or it binds to either subunit a, c' or c" (Huss et al 2002), of which there are only one copies in the structure, although its interaction with the lobster c ring and other recent data Bowman et al 2006;Dixon et al 2008) argue against c' and c'' being the binding sites. The accuracy of our approach could be further improved by adding more points to the inhibitor titration curve.…”

Section: Discussionmentioning

confidence: 69%

“…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. Since these two inhibitors are known to be very potent and very selective inhibitors of V-ATPase (Bowman et al 1988;Farina and Gagliardi 1999;Huss et al 2002;Dixon et al 2008) ~60% of the total ATPase activity comes from V-ATPase in these vesicular preparations.…”

Section: Atpase Activity Assaymentioning

confidence: 99%

“…The macrolide antibiotics concanamycin A and bafilomycin are the most potent and most selective inhibitors of V-ATPase, with IC 50 values down to the nM region (Bowman et al 1988;Farina and Gagliardi 1999;Gagliardi et al 1999;Huss et al 2002;Dixon et al 2008). We have demonstrated recently that concanamycin A and its synthetic indole analogues (Dixon et al 2003) incorporate readily into membranes ) and interact with amino acid side chains of the 16kDa lobster (Nephrops norvegicus) protein and also with subunit c of yeast V-ATPase Dixon et al 2008).…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Atpase Activity Assaysupporting

confidence: 53%

Section: Discussionmentioning

confidence: 69%

Section: Atpase Activity Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2012

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“…This similarity in biological activity already suggested that 4, like other members of the archazolid family, should target V-ATPases. Indeed, by using established procedures it was shown that the purified V-ATPase holoenzyme from the midgut of the tobacco hornworm was inhibited with an IC 50 value of 1.2 mmol/mg enzyme [7,11,12]. Thus, 4 is much less inhibitory than 1 and 2 with an IC 50 value of 0.6 nmol/mg enzyme.…”

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confidence: 99%

The First Hydroxylated Archazolid from the Myxobacterium Cystobacter violaceus: Isolation, Structural Elucidation and V-ATPase Inhibition

et al. 2007

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The novel macrocyclic polyketide, 10-hydroxymethyl-archazolid-7-O-b -D-glucopyranoside (archazolid D), was obtained from the myxobacterium Cystobacter violaceus. The structure of this first hydroxylated archazolid was determined by spectroscopic analysis, in particular by HMBC, HMQC, and ROESY NMR investigations, and by degradation. This novel metabolite was evaluated for growth inhibition of murine connective tissue cells and V-ATPase inhibition in comparison to other known archazolids. This subunit forms an oligomer, building up a ring structure of six or more copies which transports protons across the membrane. Recently, the structure of the ring from a bacterial V-ATPase has been described at 2.1 Å resolution and showed that 10 copies form the c ring [4]. This increasing molecular understanding in combination with the very promising biological profile of the archazolids renders these macrolide antibiotics attractive structures for further development. Very recently, the full stereochemistry of the archazolids has been determined by extensive NMR studies and chemical derivatization [5], and was confirmed by total synthesis [6]. So far, however, only very limited SAR-data are available, relying on modifications of the hydroxyl groups at C-7, C-15 and C-1Ј [7,8]. Herein, we describe the isolation and structure elucidation of the first archazolid with a modified carbon backbone, the hydroxylated derivative 10-hydroxymethylarchazolid-7-O-b -D-glucopyranoside (archazolid D, 4), from fermentation broths of the myxobacterium Cystobacter violaceus and report on the V-ATPase inhibitory effect of this novel macrolide antibiotic in comparison to other known V-ATPase inhibitors.In a screening for novel and more efficient archazolid producing myxobacteria, we have recently identified C. violaceus strain Cb vi105 as the natural source of the first archazolid glycoside, archazolid-7-O-b -D-glucopyranoside (archazolid C, 3, Fig. 1), which was obtained as the major metabolite from fermentation broths together with the parent compound archazolid A. A detailed analysis of this

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Proton Translocating ATPases

Futai

Sun‐Wada

Wada

2006

Encyclopedia of Molecular Cell Biology and Molecular Medicine

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Concanamycin A, the Specific Inhibitor of V-ATPases, Binds to the Vo Subunit c

Abstract: Vacuolar-type ATPases (V-ATPases) 1 are complex, heteromultimeric proteins consisting of a peripheral, catalytic V 1 complex and a membrane bound, ion translocating V o complex.

Cited by 254 publications

References 36 publications

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

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

The First Hydroxylated Archazolid from the Myxobacterium Cystobacter violaceus: Isolation, Structural Elucidation and V-ATPase Inhibition

Proton Translocating ATPases

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