scholarly journals Proton translocation of the bovine chromaffin-granule membrane

1978 ◽  
Vol 170 (3) ◽  
pp. 661-672 ◽  
Author(s):  
J H Phillips ◽  
V P Allison
Keyword(s):  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Potential Gradient ◽  
Protonmotive Force ◽  
Chromaffin Granule ◽  
Anion Transporter ◽  
Direct Measurements ◽  
Granule Membrane ◽  
Potential Gradients ◽  
Proton Movement

Bovine chromaffin granules were lysed and their membranes resealed to give osmotically sensitive ‘ghosts’. These swell in the presence of salts and MgATP. It is shown that this is due to proton entry accompanied by anions. The rate of swelling depends on the anion present, but swelling is not limited to media containing permeant anions. It is quite marked in solutions of sulphates, phosphates and acetates. It is not uncoupler-sensitive, suggesting that at least one component of swelling is due to coupled proton and anion entry (non-electrogenic proton translocation). Direct measurements of transmembrane pH and potential gradients generated in the presence of MgATP shows that these are rapidly established in sucrose media, and are rather little affected by the presence of salts. They contribute roughly equally to the total protonmotive force. The potential gradient is establihsed very rapidly, but the pH gradient is generated over several minutes. The gradients are not completely dissipated by uncoupler, and it is shown that, in media containing sulphate but no permeant anion, sulphate can be taken up by the ‘ghosts’. There thus appear to be two mechanisms of proton translocation across the membrane, both dependent on ATP hydrolysis: an electrogenic transfer of protons, and proton movement linked to an anion transporter of broad specificity.

scholarly journals Inhibition of adenosine triphosphatase, 5-hydroxytryptamine transport and proton-translocation activities of resealed chromaffin-granule ‘ghosts’

1980 ◽  
Vol 190 (2) ◽  
pp. 273-282 ◽  
Author(s):  
David K. Apps ◽  
James G. Pryde ◽  
Raul Sutton ◽  
John H. Phillips
Keyword(s):  
Molecular Weight ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Proton Translocation ◽  
Low Molecular Weight ◽  
Chromaffin Granule ◽  
Low Concentrations ◽  
Tributyltin Chloride ◽  
Granule Membrane ◽  
Chromaffin Granule Ghosts

1. Highly purified resealed chromaffin-granule ‘ghosts’ were assayed for ATPase and ATP-driven H+-translocation and 5-hydroxytryptamine-uptake activities, and for 5-hydroxytryptamine uptake driven by an imposed transmembrane H+-gradient. The effects of several inhibitors on these activities were studied. 2. Dicyclohexylcarbodi-imide inhibits all of these activities, but not in parallel; at low concentrations it decreases the permeability of the membrane to protons. 3. 4-Chloro-7-nitrobenzofuran (Nbf-Cl) and silicotungstate inhibit ATP-dependent activities, without effect on 5-hydroxytryptamine uptake driven by an imposed H+-gradient. 4. Tributyltin chloride inhibits all of the activities. 5. Treatment of the ‘ghosts’ with low concentrations of urea inhibits 5-hydroxytryptamine uptake and ATP-dependent generation of a transmembrane H+-gradient, without inhibiting ATPase activity. 6. Nbf-Cl and silicotungstate are without effect on the rate of leakage of 5-hydroxytryptamine from preloaded ‘ghosts’, whereas dicyclohexylcarbodi-imide and tributyltin chloride accelerate the rate of leakage. 7. Treatment of the membranes with 14C-labelled Nbf-Cl labels several proteins; membranes treated with dicyclohexyl[14C]carbodi-imide are labelled predominantly in a protein of low molecular weight, which may be analogous to the mitochondrial H+-conducting proteolipid. 8. It is concluded that Nbf-Cl and silicotungstate inhibit the H+-translocating ATPase of the granule membrane; that dicyclohexylcarbodi-imide inhibits the ATPase, and inhibits 5-hydroxytryptamine accumulation by accelerating leakage of the amine; and that the effects of tributyltin chloride are due to inhibition of the ATPase, and collapse of the transmembrane H+-gradient through OH−-anion exchange.

scholarly journals Glutamate uptake into synaptic vesicles of bovine cerebral cortex and electrochemical potential difference of proton across the membrane

1989 ◽  
Vol 258 (2) ◽  
pp. 499-504 ◽  
Author(s):  
J Shioi ◽  
S Naito ◽  
T Ueda
Keyword(s):  
Cerebral Cortex ◽  
Synaptic Vesicles ◽  
Driving Force ◽  
Significant Contribution ◽  
Atp Hydrolysis ◽  
Glutamate Uptake ◽  
Protonmotive Force ◽  
Chromaffin Granule ◽  
Carbonyl Cyanide ◽  
Disulphonic Acid

Measurements have been made of the ATP-dependent membrane potential (delta psi) and pH gradient (delta pH) across the membranes of the synaptic vesicles purified from bovine cerebral cortex, using the voltage-sensitive dye bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxanol and the delta pH-sensitive fluorescent dye 9-aminoacridine respectively. A pre-existing small delta pH (inside acidic) was detected in the synaptic vesicles, but no additional significant contribution by MgATP to delta pH was observed. In contrast, delta psi (inside positive) increased substantially upon addition of MgATP. This ATP-dependent delta psi was reduced by thiocyanate anion (SCN-), a delta psi dissipator, or carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonmotive-force dissipator. Correspondingly, a substantially larger glutamate uptake occurred in the presence of MgATP, which was inhibited by SCN- and FCCP. A nonhydrolysable analogue of ATP, adenosine 5′-[beta gamma-methylene]triphosphate, did not substitute for ATP in either delta psi generation or glutamate uptake. The results support the hypothesis that a H+-pumping ATPase generates a protonmotive force in the synaptic vesicles at the expense of ATP hydrolysis, and the protonmotive force thus formed provides a driving force for the vesicular glutamate uptake. The delta psi generation by ATP hydrolysis was not affected by orthovanadate, ouabain or oligomycin, but was inhibited by N-ethylmaleimide, quercetin, trimethyltin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid. These results indicate that the H+-pumping ATPase in the synaptic vesicle is similar to that in the chromaffin granule, platelet granule and lysosome.

Measuring surface potential components necessary for transmembrane current computation using microfabricated arrays

2005 ◽  
Vol 289 (6) ◽  
pp. H2468-H2477 ◽  
Author(s):  
J. James Wiley ◽  
Raymond E. Ideker ◽  
William M. Smith ◽  
Andrew E. Pollard
Keyword(s):  
Spatial Resolution ◽  
Current Density ◽  
Potential Gradient ◽  
Electrode Spacing ◽  
Back Side ◽  
Transmembrane Current ◽  
Fine Wire ◽  
Microfabricated Arrays ◽  
Potential Gradients ◽  
Unipolar Electrograms

This study was designed to test the feasibility of using microfabricated electrodes to record surface potentials with sufficiently fine spatial resolution to measure the potential gradients necessary for improved computation of transmembrane current density. To assess that feasibility, we recorded unipolar electrograms from perfused rabbit right ventricular free wall epicardium ( n = 6) using electrode arrays that included 25-μm sensors fabricated onto a flexible substrate with 75-μm interelectrode spacing. Electrode spacing was therefore on the size scale of an individual myocyte. Signal conditioning adjacent to the sensors to control lead noise was achieved by routing traces from the electrodes to the back side of the substrate where buffer amplifiers were located. For comparison, recordings were also made using arrays built from chloridized silver wire electrodes of either 50-μm (fine wire) or 250-μm (coarse wire) diameters. Electrode separations were necessarily wider than with microfabricated arrays. Comparable signal-to-noise ratios (SNRs) of 21.2 ± 2.2, 32.5 ± 4.1, and 22.9 ± 0.7 for electrograms recorded using microfabricated sensors ( n = 78), fine wires ( n = 78), and coarse wires ( n = 78), respectively, were found. High SNRs were maintained in bipolar electrograms assembled using spatial combinations of the unipolar electrograms necessary for the potential gradient measurements and in second-difference electrograms assembled using spatial combinations of the bipolar electrograms necessary for surface Laplacian (SL) measurements. Simulations incorporating a bidomain representation of tissue structure and a two-dimensional network of guinea pig myocytes prescribed following the Luo and Rudy dynamic membrane equations were completed using 12.5-μm spatial resolution to assess contributions of electrode spacing to the potential gradient and SL measurements. In those simulations, increases in electrode separation from 12.5 to 75.0, 237.5, and 875.0 μm, which were separations comparable to the finest available with our microfabricated, fine wire, and coarse wire arrays, led to 10%, 42%, and 81% reductions in maximum potential gradients and 33%, 76%, and 96% reductions in peak-to-peak SLs. Maintenance of comparable SNRs for source electrograms was therefore important because microfabrication provides a highly attractive methods to achieve spatial resolutions necessary for improved computation of transmembrane current density.

CRISPR/Cas9-Mediated Gene Editing of Vacuolar ATPase Subunit D Mediates Phytohormone Biosynthesis and Virus Resistance in Rice

2021 ◽  
Author(s):  
Qinghua Lu ◽  
Xiangwen Luo ◽  
Xiao Yang ◽  
Tong Zhou ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Virus Resistance ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Resistance Breeding ◽  
Rice Stripe Virus ◽  
Proton Pumps ◽  
Wild Type ◽  
Knockout Mutant ◽  
Atpase Subunit ◽  
Vacuolar Atpases

Abstract Background: Vacuolar ATPases (v-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis; Previous research revealed Osv-ATPases mediates phytohormes levels and resistance in rice. Osv-ATPase subunit d (Osv-ATPase d) is part of an integral, membrane-embedded V0 complex of V-ATPases complex, whether Osv-ATPase d involves in phytohormes biosynthesis and resistance in rice remains unknown.Finding: The knockout mutant line (line 5) of Osv-ATPase d was generated using the CRISPR/Cas9 system, mutation of Osv-ATPase d did not show any detrimental effect on plant growth or yield productivity. Transcriptomic results showed Osv-ATPase d probably involved in mediating the biosynthesis of plant hormones and resistance in rice. Mutation of Osv-ATPase d significantly increased JA and ABA biosynthesis than wild type. Compared to wild type, mutation of Osv-ATPase d increased the resistance against Southern rice black-streaked dwarf virus (SRBSDV), however, decreased the resistance against Rice stripe virus (RSV) in rice. Conclusion: Taken together, our data reveal the Osv-ATPase d mediates phytohormone biosynthesis and virus resistance in rice, which can be selected as a potential target for resistance breeding in rice.

scholarly journals Structure of a bacterial ATP synthase

2018 ◽  
Author(s):  
Hui Guo ◽  
Toshiharu Suzuki ◽  
John L. Rubinstein
Keyword(s):  
Atp Synthase ◽  
Biochemical Analysis ◽  
Atp Hydrolysis ◽  
Genetic Manipulation ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Mitochondrial Complex ◽  
Rotational States ◽  
Membrane Region ◽  
Atp Synthases

AbstractATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed theBacillusPS3 ATP synthase inEschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunitεshows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

scholarly journals Determination of the proportion of sealed vesicles in a preparation of chromaffin granule membrane 'ghosts'

FEBS Letters ◽  
1982 ◽  
Vol 144 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Anne Hunter ◽  
Keith Waldron ◽  
David K. Apps
Keyword(s):  
Chromaffin Granule ◽  
Granule Membrane
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