scholarly journals MgATP-induced inhibition of the adenosine triphosphatase activity of submitochondrial particles

1981 ◽  
Vol 196 (2) ◽  
pp. 443-449 ◽  
Author(s):  
P N Lowe ◽  
R B Beechey
Keyword(s):  
Atpase Activity ◽  
Conformational Change ◽  
Atp Hydrolysis ◽  
Hydrolytic Activity ◽  
Inhibitor Protein ◽  
Endogenous Inhibitor ◽  
Submitochondrial Particles ◽  
Hydrolysis Products ◽  
Enzyme Species ◽  
Partial Release

1. The ATP-hydrolytic activity of ox heart submitochondrial particles can be increased from 2-3 mumol/min per mg of protein to 10-12 mumol/min per mg of protein by incubation in media containing 50 mM-Na2B4O7. This process appears to be due to the partial release of inhibitor protein from the particles. 2. The ATPase activity of submitochondrial particles can be inhibited by incubation with the substrate, MgATP. This inhibition is not due to the accumulation of the hydrolysis products, MgADP and Pi, but could involve the process of ATP hydrolysis. 3. The mechanism of MgATP-induced inhibition of ATPase activity is proposed to involve a conformational change in one of the intermediate enzyme species of the ATP-hydrolytic sequence. 4. MgATP inhibits the ATPase activity of control submitochondrial particles at a higher rate and to a greater extent than it does that of inhibitor-protein-depleted submitochondrial particles, suggesting that the conformational change involves the endogenous inhibitor protein.

scholarly journals Specific Molecular Chaperone Interactions and an ATP-dependent Conformational Change Are Required during Posttranslational Protein Translocation into the Yeast ER

1998 ◽  
Vol 9 (12) ◽  
pp. 3533-3545 ◽  
Author(s):  
Amie J. McClellan ◽  
James B. Endres ◽  
Joseph P. Vogel ◽  
Debra Palazzi ◽  
Mark D. Rose ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Conformational Change ◽  
Molecular Chaperone ◽  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Wild Type ◽  
J Domain ◽  
Chaperone Interactions ◽  
Dependent Interaction ◽  
Er Membrane

The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen. Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane. To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST–63Jp fusion protein containing the lumenal DnaJ region of Sec63p. We observed that BiP, but not Ssa1p, is able to associate with GST–63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold. In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP. To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP. We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST–63Jp. Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP. We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners.

scholarly journals Involvement of the endogenous inhibitor protein in the MgATP-induced inhibition of soluble mitochondrial adenosine triphosphatase activity

1981 ◽  
Vol 200 (3) ◽  
pp. 655-661 ◽  
Author(s):  
P N Lowe ◽  
R B Beechey
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Heart Mitochondria ◽  
Inhibitor Protein ◽  
Endogenous Inhibitor ◽  
Adenosine Triphosphatase Activity

Chloroform-released ATPase from ox heart mitochondria contains significant amounts of inhibitor protein. There is a correlation between processes that affect the interactions between the inhibitor protein and the ATPase molecule and the ability of MgATP to induce an inhibition of ATPase activity. Evidence is presented suggesting that the endogenous inhibitor protein is involved in the process of MgATP-induced inhibition of soluble ATPase activity.

scholarly journals The adenosine triphosphatase-inhibitor content of bovine heart submitochondrial particles. Influence of the inhibitor on adenosine triphosphate-dependent reactions

1977 ◽  
Vol 162 (2) ◽  
pp. 351-357 ◽  
Author(s):  
S J Ferguson ◽  
D A Harris ◽  
G K Radda
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Type I ◽  
Type Ii ◽  
Inhibitor Protein ◽  
Isolation Method ◽  
Atpase Inhibitor ◽  
Submitochondrial Particles ◽  
Bovine Heart ◽  
Tightly Bound Nucleotides

1. The activity of the ATPase (adenosine triphosphatase) of phosphorylating particles prepared by sonication of bovine heart mitochondria in the presence of MgCl2 and ATP is influenced by the isolation method for the mitochondria used in the preparation of particles. Type-I particles, made from mitochondria isolated in a medium lacking succinate, have a lower ATPase activity than to Type-II particles, which are prepared from mitochondria isolated in a medium containing succinate. 2. Centrifugation under appropriate energized conditions increases the ATPase activity of Type-I particles almost to that of the Type-II particles. The ATPase activity of Type-II particles was only slightly stimulated by this procedure. These data are interpreted as indicating a higher content of the ATPase-inhibitor protein in the Type-I particles. 3. A comparison was made of the ATP-driven enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and the exchange of the endogenous tightly bound nucleotides of the ATPase in Type-I and Type-II particles. The effect of exogenous inhibitor protein on both these reactions was also studied. 4. The time-scale on which the inhibitor protein can exchange between ATPase molecules is discussed.

Effect of denaturants on multisite and unisite ATP hydrolysis by bovine heart submitochondrial particles with and without inhibitor protein

2005 ◽  
Vol 439 (1) ◽  
pp. 129-137 ◽  
Author(s):  
Marietta Tuena de Gómez-Puyou ◽  
Lenin Domínguez-Ramírez ◽  
Gerardo Pérez-Hernández ◽  
Armando Gómez-Puyou
Keyword(s):  
Atp Hydrolysis ◽  
Inhibitor Protein ◽  
Submitochondrial Particles ◽  
Bovine Heart

scholarly journals Coupling of ATPase activity, microtubule binding and mechanics in the dynein motor domain

2018 ◽  
Author(s):  
Stefan Niekamp ◽  
Nicolas Coudray ◽  
Nan Zhang ◽  
Ronald D. Vale ◽  
Gira Bhabha
Keyword(s):  
Atpase Activity ◽  
Conformational Change ◽  
Conformational Changes ◽  
Large Scale ◽  
Atp Hydrolysis ◽  
Molecular Motor ◽  
Coiled Coil ◽  
Microtubule Binding ◽  
Dynein Motor ◽  
In The Wild

The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule-binding domain (MTBD) that is separated from the AAA ring by a ~135 Å coiled-coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule-independent hyperactive ATPase activity, and nucleotide-independent low affinity for microtubules. Cryo-electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide-dependent conformational changes occur normally in one half of the AAA ring, but are disrupted in the other half. The large-scale linker conformational change observed in the wild-type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.

ATPase activity of kidney mitochondria stimulated by sodium

1975 ◽  
Vol 228 (3) ◽  
pp. 815-820 ◽  
Author(s):  
MW Weiner
Keyword(s):  
Energy Metabolism ◽  
Atpase Activity ◽  
Ethacrynic Acid ◽  
Atp Hydrolysis ◽  
Intracellular Sodium ◽  
Kidney Cortex ◽  
Submitochondrial Particles ◽  
Kidney Mitochondria ◽  
Anionic Composition ◽  
Intracellular Energy

ATPase activity of intact mitochondria may be induced by the presence of sodium. Mitochondria obtained from kidney cortex or medulla demonstrate a higher sodium-stimulated ATPase than mitochondria prepared from liver. This difference is not due to a greater capacity of kidney mitochondria for ATP hydrolysis. Sodium-stimulated ATPase activity of mitochondria is not further enhanced by magnesium or potassium, nor is it inhibited by ouabain. Furthermore, the ATPase of submitochondrial particles is not enhanced by the presence of sodium. These results indicate that the ATPase activity of mitochondria which is stimulated by sodium is unrelated to the microsomal Na-K-ATPase. mitochondrial sodium-stimulated ATPase is affected by pH, anionic composition, furosemide, and ethacrynic acid. The demonstration that mitochondrial ATP hydrolysis can be activated by sodium suggests that intracellular sodium may directly influence intracellular energy metabolism.

scholarly journals Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H+-ATPase Activity and H+-Transport

Biomolecules ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1891
Author(s):  
Nikita K. Lapshin ◽  
Michail S. Piotrovskii ◽  
Marina S. Trofimova
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Hydrolytic Activity ◽  
Significant Inhibition ◽  
Plasma Membrane Vesicles ◽  
Kinetics Parameters ◽  
Hydrolytic Activities ◽  
Lipid Environment

Plasma membrane H+-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called “raft” domains. Studies on H+-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and inhibitions of its activity. Here, using sealed isolated plasma membrane vesicles, we investigated the effects of sterol depletion in the presence of methyl-β-cyclodextrin (MβCD) on H+-ATPase activity. The rate of ATP-dependent ∆µH+ generation and the kinetic parameters of ATP hydrolysis were evaluated. We show that the relative sterols content in membrane vesicles decreased gradually after treatment with MβCD and reached approximately 40% of their initial level in 30 mM probe solution. However, changes in the hydrolytic and H+-transport activities of the enzyme were nonlinear. The extraction of up to 20% of the initial sterols was accompanied by strong stimulation of ATP-dependent H+-transport in comparison with the hydrolytic activity of enzymes. Further sterol depletion led to a significant inhibition of active proton transport with an increase in passive H+-leakage. The solubilization of control and sterol-depleted vesicles in the presence of dodecyl maltoside negated the differences in the kinetics parameters of ATP hydrolysis, and all samples demonstrated maximal hydrolytic activities. The mechanisms behind the sensitivity of ATP-dependent H+-transport to sterols in the lipid environment of plasma membrane H+-ATPase are discussed.

Downmodulation of mitochondrial F0F1ATP synthase by diazoxide in cardiac myoblasts: a dual effect of the drug

2007 ◽  
Vol 292 (2) ◽  
pp. H820-H829 ◽  
Author(s):  
Marina Comelli ◽  
Giuliana Metelli ◽  
Irene Mavelli
Keyword(s):  
Atpase Activity ◽  
Atp Synthase ◽  
Transmembrane Potential ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Mitochondrial Transmembrane Potential ◽  
Mitochondrial Atpase ◽  
Inhibitor Protein ◽  
Atp Content ◽  
Intact Cells

Similar to ischemic preconditioning, diazoxide was documented to elicit beneficial bioenergetic consequences linked to cardioprotection. Inhibition of ATPase activity of mitochondrial F0F1ATP synthase may have a role in such effect and may involve the natural inhibitor protein IF1. We recently documented, using purified enzyme and isolated mitochondrial membranes from beef heart, that diazoxide interacts with the F1sector of F0F1ATP synthase by promoting IF1binding and reversibly inhibiting ATP hydrolysis. Here we investigated the effects of diazoxide on the enzyme in cultured myoblasts. Specifically, embryonic heart-derived H9c2 cells were exposed to diazoxide and mitochondrial ATPase was assayed in conditions maintaining steady-state IF1binding (basal ATPase activity) or detaching bound IF1at alkaline pH. Mitochondrial transmembrane potential and uncoupling were also investigated, as well as ATP synthesis flux and ATP content. Diazoxide at a cardioprotective concentration (40 μM cell-associated concentration) transiently downmodulated basal ATPase activity, concomitant with mild mitochondria uncoupling and depolarization, without affecting ATP synthesis and ATP content. Alkaline stripping of IF1from F0F1ATP synthase was less in diazoxide-treated than in untreated cells. Pretreatment with glibenclamide prevented, together with mitochondria depolarization, inhibition of ATPase activity under basal but not under IF1-stripping conditions, indicating that diazoxide alters alkaline IF1release. Diazoxide inhibition of ATPase activity in IF1-stripping conditions was observed even when mitochondrial transmembrane potential was reduced by FCCP. The results suggest that diazoxide in a model of normoxic intact cells directly promotes binding of inhibitor protein IF1to F0F1ATP synthase and enhances IF1binding indirectly by mildly uncoupling and depolarizing mitochondria.

A Contribution of the Mitochondrial Adenosinetriphosphatase Inhibitor Protein to the Thermal Stability of the F0F1-ATPase Complex

1997 ◽  
Vol 52 (7-8) ◽  
pp. 459-465 ◽  
Author(s):  
Jorge Saad-Nehme ◽  
Andre L. Bezerra ◽  
Luz Alba M. Fornells ◽  
Jerson L. Silva ◽  
Jose R. Meyer-Fernandes
Keyword(s):  
Thermal Stability ◽  
Thermal Inactivation ◽  
Half Time ◽  
Inhibitor Protein ◽  
Endogenous Inhibitor ◽  
Atpase Inhibitor ◽  
Submitochondrial Particles ◽  
Complete Inactivation ◽  
Natural Inhibitor ◽  
Thermal Stability Of

Abstract A complete inactivation is observed after a 3 min pre-incubation at 70 °C with mitochondrial F0F1-ATPase complex depleted of the ATPase natural inhibitor protein (ammonium-Sephadex submitochondrial particles) and activated MgATP-submitochondrial par­ticles (particles that after a 4 h-pre-incubation at 42 °C released the endogenous inhibitor protein). However, latent MgATP-submitochondrial particles (particles containing the inhibi­tor protein) pre-incubated under the same conditions are totally inactivated only after 15 min of pre-incubation. When ammonium-Sephadex particles are reconstituted with 20 μg/ml of purified ATPase inhibitor protein there is an increase of 15-fold in the half-time for ther­mal inactivation (t0.5), showing that the inhibitor protein protects the mitochondrial F0F1-ATPase complex against thermal inactivation.

Interdomain regulation of the ATPase activity of the ABC transporter haemolysin B from Escherichia coli

2016 ◽  
Vol 473 (16) ◽  
pp. 2471-2483 ◽  
Author(s):  
Sven Reimann ◽  
Gereon Poschmann ◽  
Kerstin Kanonenberg ◽  
Kai Stühler ◽  
Sander H.J. Smits ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Biochemical Characterization ◽  
Atp Hydrolysis ◽  
Protein A ◽  
Maximum Velocity ◽  
Hydrolytic Activity ◽  
Secretion Systems ◽  
Hydrolytic Activities ◽  
Wide Range

Type 1 secretion systems (T1SS) transport a wide range of substrates across both membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC (ATP-binding cassette) transporter. The ABC transporter HlyB (haemolysin B) is part of a T1SS catalysing the export of the toxin HlyA in E. coli. HlyB consists of the canonical transmembrane and nucleotide-binding domains. Additionally, HlyB contains an N-terminal CLD (C39-peptidase-like domain) that interacts with the transport substrate, but its functional relevance is still not precisely defined. In the present paper, we describe the purification and biochemical characterization of detergent-solubilized HlyB in the presence of its transport substrate. Our results exhibit a positive co-operativity in ATP hydrolysis. We characterized further the influence of the CLD on kinetic parameters by using an HlyB variant lacking the CLD (HlyB∆CLD). The biochemical parameters of HlyB∆CLD revealed an increased basal maximum velocity but no change in substrate-binding affinity in comparison with full-length HlyB. We also assigned a distinct interaction of the CLD and a transport substrate (HlyA1), leading to an inhibition of HlyB hydrolytic activity at low HlyA1 concentrations. At higher HlyA1 concentrations, we observed a stimulation of the hydrolytic activities of both HlyB and HlyB∆CLD, which was completely independent of the interaction of HlyA1 with the CLD. Notably, all observed effects on ATPase activity, which were also analysed in detail by mass spectrometry, were independent of the HlyA1 secretion signal. These results assign an interdomain regulatory role for the CLD modulating the hydrolytic activity of HlyB.

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