scholarly journals The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

1970 ◽  
Vol 120 (1) ◽  
pp. 15-24 ◽  
Author(s):  
P. S. G. Goldfarb ◽  
R. Rodnight
Keyword(s):  
Potassium Chloride ◽  
Magnesium Chloride ◽  
Adenosine Triphosphatase ◽  
Time Course ◽  
Atp Hydrolysis ◽  
Protein Ratio ◽  
Low Concentrations ◽  
Hydrolysis Of ◽  
Emission Flame

1. The intrinsic Na+, K+, Mg2+ and Ca2+ contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na+ from 90±20 to 24±12, the bound K+ from 27±3 to 7±2, the bound Mg2+ from 20±2 to 3±1 and the bound calcium from 8±1 to <1nmol/mg of protein. 3. The activities of the Na++K++Mg2+-stimulated adenosine triphosphatase and the Na+-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5μm (ATP/protein ratio 12.5pmol/μg). 4. The Na+-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5μm-magnesium chloride and 2μm-potassium chloride. Addition of 2.5μm-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na+-dependent ATP hydrolysis was partly restored with 2.5μm-magnesium chloride; addition of K+ in the range 2–10μm-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0°C with 0.5nmol of K+/mg of protein so that the final added K+ in the reaction mixture was 0.1μm restored the Na+-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [42K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K+/mg of protein was linear over a period of 20min and was inhibited by Na+. Half-maximal inhibition of 42K+-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na+-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K+ and Mg2+ of the Na++K++Mg2+-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K+ from a solution of 0.5μm-potassium chloride.

scholarly journals Role of phospholipid in the intermediate steps of the sodium-plus-potassium ion-dependent adenosine triphosphatase reaction

1975 ◽  
Vol 146 (3) ◽  
pp. 729-738 ◽  
Author(s):  
K P Wheeler
Keyword(s):  
Phosphatase Activity ◽  
Adenosine Triphosphatase ◽  
Atp Hydrolysis ◽  
Reaction Sequence ◽  
Single Extraction ◽  
Background Value ◽  
Dependent Atpase ◽  
Membrane Atpase ◽  
Normal Enzyme

The phosphorylation and dephosphorylation steps of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) reaction have been compared in ‘normal’, lipid-depleted and ‘restored’ membrane ATPase preparations. Partial lipid depletion was achieved by a single extraction with Lubrol W, and ‘restoration’ by adding pure phosphatidylserine. γ-32-P-labelled ATP was used for phosphorylation. The main findings were as follows. (1) Partial lipid depletion decreased but did not prevent Na-+-dependent phosphorylation, although it virtually abolished both Na-+-dependent and (Na-++K-+)-dependent ATPase activities. (2) ‘Restoration’ with phosphatidylserine produced an increment in phosphorylation that was the same in the presence and absence of added Na-+. (3) K-+ decreased the extent of Na-+-dependent phosphorylation of the depleted enzyme without producing a corresponding release of Pi. (4) K-+ rapidly decreased the extent of phosphorylation of the ‘restored’ enzyme to near-background value, with a concomitant release of Pi. (5) Na-+-dependent ATP hydrolysis was not restored. (6) The turnover of the ‘restored’ enzyme seemed to be higher than that of the ‘normal’ enzyme. The reaction sequence is discussed in relation to these results and the fact that the depleted enzyme retained about 50% of K-+-dependent phosphatase activity.

scholarly journals Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase.

1984 ◽  
Vol 99 (2) ◽  
pp. 734-741 ◽  
Author(s):  
W A Braell ◽  
D M Schlossman ◽  
S L Schmid ◽  
J E Rothman
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Low Ph ◽  
Coated Vesicles ◽  
Magnesium Concentration ◽  
Cross Linking ◽  
Dependent Atpase ◽  
Event Triggering ◽  
Hydrolysis Of

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.

scholarly journals The effect of chemical agents on the turnover of the bound phosphate associated with the sodium-and-potassium ion-stimulated adenosine triphosphatase in ox brain microsomes

1970 ◽  
Vol 120 (1) ◽  
pp. 1-13 ◽  
Author(s):  
R. Rodnight
Keyword(s):  
Sodium Chloride ◽  
Atp Hydrolysis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Protein Ratio ◽  
Chemical Agents ◽  
Rapid Fall ◽  
Triton X ◽  
Hydrolysis Of ◽  
Sodium And Potassium

1. The effect of chemical agents on the turnover of the Na+-dependent bound phosphate and the simultaneous Na+-dependent hydrolysis of ATP by a membrane preparation from ox brain was studied at an ATP/protein ratio of 12.5pmol/μg. 2. The agents were added immediately after phosphorylation of the preparation in a medium containing 50mm-sodium chloride and 2.5μm-[γ-32P]ATP. 3. Concentrations of sodium chloride above 150mm, calcium chloride to 20mm and suramin to 1.4mm inhibited both phosphorylation and dephosphorylation and concomitantly slowed ATP hydrolysis. At 125mm-sodium chloride dephosphorylation and hydrolysis were slightly slowed without affecting phosphorylation. 4. Ethanol to 1.6m concentration inhibited dephosphorylation without affecting phosphorylation; the bound phosphate was increased and ATP hydrolysis slowed. 5. Ouabain to 4mm concentration partially inhibited ATP hydrolysis and caused a transient (1–2s) rise in bound phosphate followed by a rapid fall to a lower plateau value, which eventually declined to zero by the time ATP hydrolysis was complete. 6. Of the detergents examined Lubrol W, Triton X-100 and sodium deoxycholate had no significant effect on turnover. Sodium dodecyl sulphate and sodium decyl sulphate to 3.5mm and 20mm respectively completely inhibited turnover and ATP hydrolysis and stabilized the bound phosphate.

Role of the Sarcoplasmic Reticulum Ca2+-ATPase on Heat Production and Thermogenesis

2001 ◽  
Vol 21 (2) ◽  
pp. 113-137 ◽  
Author(s):  
Leopoldo de Meis
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atp Hydrolysis ◽  
Surrounding Medium ◽  
Chemical Energy ◽  
The Past ◽  
Sarcoplasmic Reticulum Membrane ◽  
Membrane Bound ◽  
Hydrolysis Of ◽  
Heat Released

The sarcoplasmic reticulum of skeletal muscle retains a membrane bound Ca2+-ATPase which is able to interconvert different forms of energy. A part of the chemical energy released during ATP hydrolysis is converted into heat and in the bibliography it is assumed that the amount of heat produced during the hydrolysis of an ATP molecule is always the same, as if the energy released during ATP cleavage were divided in two non-interchangeable parts: one would be converted into heat, and the other used for Ca2+ transport. Data obtained in our laboratory during the past three years indicate that the amount of heat released during the hydrolysis of ATP may vary between 7 and 32 kcal/mol depending on whether or not a transmembrane Ca2+ gradient is formed across the sarcoplasmic reticulum membrane. Drugs such as heparin and dimethyl sulfoxide are able to modify the fraction of the chemical energy released during ATP hydrolysis which is used for Ca2+ transport and the fraction which is dissipated in the surrounding medium as heat.

scholarly journals Glutathione-Dependent Conversion ofN-Ethylmaleimide to the Maleamic Acid by Escherichia coli: an Intracellular Detoxification Process

2000 ◽  
Vol 66 (4) ◽  
pp. 1393-1399 ◽  
Author(s):  
D. McLaggan ◽  
H. Rufino ◽  
M. Jaspars ◽  
I. R. Booth
Keyword(s):  
Escherichia Coli ◽  
Time Course ◽  
E Coli ◽  
Transient Activation ◽  
Low Concentrations ◽  
Maleamic Acid ◽  
High Concentrations ◽  
Hydrolysis Of ◽  
Strong Activator ◽  
Detoxification Process

ABSTRACT The electrophile N-ethylmaleimide (NEM) elicits rapid K+ efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K+ efflux systems. The fate of the ESG has not previously been investigated. In this report we demonstrate that NEM andN-phenylmaleimide (NPM) are rapidly detoxified by E. coli. The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released. N-Ethylmaleamic acid is not toxic to E. coli cells even at high concentrations. The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm. The detoxification is independent of new protein synthesis and NAD+-dependent dehydrogenase activity and entirely dependent upon glutathione. The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K+ efflux systems.

The role of ATP in the reactions of type II DNA topoisomerases

2010 ◽  
Vol 38 (2) ◽  
pp. 438-442 ◽  
Author(s):  
Andrew D. Bates ◽  
Anthony Maxwell
Keyword(s):  
Atp Hydrolysis ◽  
Dna Gyrase ◽  
Dna Topology ◽  
Type Ii ◽  
Free Energies ◽  
Dna Topoisomerases ◽  
Type Ii Dna Topoisomerases ◽  
Topology Simplification ◽  
Hydrolysis Of

Type II DNA topoisomerases catalyse changes in DNA topology in reactions coupled to the hydrolysis of ATP. In the case of DNA gyrase, which can introduce supercoils into DNA, the requirement for free energy is clear. However, the non-supercoiling type II enzymes carry out reactions that are apparently energetically favourable, so their requirement for ATP hydrolysis is not so obvious. It has been shown that many of these enzymes (the type IIA family) can simplify the topology of their DNA substrates to a level beyond that expected at equilibrium. Although this seems to explain their usage of ATP, we show that the free energies involved in topology simplification are very small (<0.2% of that available from ATP) and we argue that topology simplification may simply be an evolutionary relic.

scholarly journals Mechanochemical coupling in the relaxation of rigor-wave sea urchin sperm flagella.

1982 ◽  
Vol 92 (3) ◽  
pp. 733-741 ◽  
Author(s):  
S M Penningroth ◽  
A Cheung ◽  
K Olehnik ◽  
R Koslosky
Keyword(s):  
Sea Urchin ◽  
Atp Hydrolysis ◽  
Bend Angle ◽  
Flagellar Motility ◽  
Lytechinus Pictus ◽  
Mechanochemical Coupling ◽  
Mechanochemical Cycle ◽  
Hydrolysis Of

The relaxation (straightening) of flagellar rigor waves, which is known to be induced by micromolar ATP concentrations was investigated with respect to its dependence on the binding and hydrolysis of ATP. Flagellar rigor waves were formed by the dilution of demembranated, reactivated sea urchin (Lytechinus pictus) spermatozoa into ATP-free buffer. Relaxation in response to nucleotide was quantitated by measuring theta, the mean flagellar bend angle per sperm; this novel assay permitted determination of the rate of relaxation. It was found that (a) the rate of flagellar relaxation induced by 4 X 10(-6) M ATP was inhibited 80% by vanadate concentrations of 3 X 10(-6) M and above; (b) of 16 hydrolyzable and nonhydrolyzable nucleotide di-, tri-, and tetraphosphates tested, only three, each of which was hydrolyzed by the flagellar axonemal ATPase activity (ATP, dATP, and epsilon-ATP) were also capable of effecting relaxation; (c) several hundred ATP molecules were estimated to be hydrolyzed by each dynein of ATP hydrolysis, which defines the efficiency of ATP utilization, increased 30-fold as the ATP relaxation depends on ATP hydrolysis; (b) because it depends on ATP hydrolysis, flagellar relaxation is an inappropriate model system for investigating the role of ATP binding in the mechanochemical cycle of dynein; and (c) the efficiency of mechanochemical coupling in flagellar motility is an ATP-dependent phenomenon. A general model of relaxation is proposed based on active microtubule sliding.

The Role of Membrane Proteins and Phospholipids in the Interaction of Ribosomes with Endoplasmic Reticulum Membranes

1975 ◽  
Vol 53 (9) ◽  
pp. 1039-1045 ◽  
Author(s):  
Serge Jothy ◽  
Jean-Louis Bilodeau ◽  
Henry Simpkins
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Binding Sites ◽  
Molecular Weights ◽  
Low Concentrations ◽  
Phospholipid Headgroups ◽  
Hydrolysis Of

Hydrolysis of the membrane proteins and phospholipid headgroups of rat liver rough endoplasmic reticulum membranes showed that the ribosomal binding sites involve membrane proteins susceptible to low concentrations of trypsin, chymotrypsin, and papain. Three membrane proteins having molecular weights of 120 000, 93 000 and 36 000 are found to be altered by trypsin and chymotrypsin treatment. Also the polar headgroup of phosphatidylinositol appears to play a role in the binding process.

scholarly journals Elements in nucleotide sensing and hydrolysis of the AAA+ disaggregation machine ClpB: a structure-based mechanistic dissection of a molecular motor

2014 ◽  
Vol 70 (2) ◽  
pp. 582-595 ◽  
Author(s):  
Cathleen Zeymer ◽  
Thomas R. M. Barends ◽  
Nicolas D. Werbeck ◽  
Ilme Schlichting ◽  
Jochen Reinstein
Keyword(s):  
Atp Hydrolysis ◽  
Mutational Analysis ◽  
Molecular Motor ◽  
Nucleotide Binding ◽  
Side Chain ◽  
Side Chain Conformation ◽  
Proposed Model ◽  
Hydrolysis Of ◽  
Conserved Residue

ATPases of the AAA+ superfamily are large oligomeric molecular machines that remodel their substrates by converting the energy from ATP hydrolysis into mechanical force. This study focuses on the molecular chaperone ClpB, the bacterial homologue of Hsp104, which reactivates aggregated proteins under cellular stress conditions. Based on high-resolution crystal structures in different nucleotide states, mutational analysis and nucleotide-binding kinetics experiments, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2), one of the motor subunits of this AAA+ disaggregation machine, is dissected mechanistically. The results provide insights into nucleotide sensing, explaining how the conserved sensor 2 motif contributes to the discrimination between ADP and ATP binding. Furthermore, the role of a conserved active-site arginine (Arg621), which controls binding of the essential Mg2+ion, is described. Finally, a hypothesis is presented as to how the ATPase activity is regulated by a conformational switch that involves the essential Walker A lysine. In the proposed model, an unusual side-chain conformation of this highly conserved residue stabilizes a catalytically inactive state, thereby avoiding unnecessary ATP hydrolysis.

Sign in / Sign up

Export Citation Format

Share Document