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 basal Mg2+-dependent ATPase activity is not part of the (H++K+)-transporting ATPase reaction cycle

1990 ◽  
Vol 267 (3) ◽  
pp. 565-572 ◽  
Author(s):  
H T W M Van der Hijden ◽  
S Kramer-Schmitt ◽  
E Grell ◽  
J J H H M de Pont
Keyword(s):  
Gastric Mucosa ◽  
Atpase Activity ◽  
Hydrolytic Activity ◽  
Reaction Cycle ◽  
Dependent Atpase ◽  
Atpase Reaction ◽  
Phosphorylation Reaction ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Purified gastric (H(+)+K+)-transporting ATPase [(H(+)+K+)-ATPase] from the parietal cells always contains a certain amount of basal Mg2(+)-dependent ATPase (Mg2(+)-ATPase) activity. lin-Benzo-ATP (the prefix lin refers to the linear disposition of the pyrimidine, benzene and imidazole rings in the ‘stretched-out’ version of the adenine nucleus), an ATP analogue with a benzene ring formally inserted between the two rings composing the adenosine moiety, is an interesting substrate not only because of its fluorescent behaviour, but also because of its geometric properties. lin-Benzo-ATP was used in the present study to elucidate the possible role of the basal Mg2(+)-ATPase activity in the gastric (H(+)+K+)-ATPase preparation. With lin-benzo-ATP the enzyme can be phosphorylated such that a conventional phosphoenzyme intermediate is formed. The rate of the phosphorylation reaction, however, is so low that this reaction with subsequent dephosphorylation cannot account for the much higher rate of hydrolysis of lin-benzo-ATP by the enzyme. This apparent kinetic discrepancy indicates that lin-benzo-ATP is not a substrate for the (H(+)+K+)-ATPase reaction cycle. This idea was further supported by the finding that lin-benzo-ATP was unable to catalyse H+ uptake by gastric-mucosa vesicles. The breakdown of lin-benzo-ATP by the (H(+)+K+)-ATPase preparation must be due to a hydrolytic activity which is not involved in the ion-transporting reaction cycle of the (H(+)+K+)-ATPase itself. Comparison of the basal Mg2(+)-ATPase activity (with ATP as substrate) with the hydrolytic activity of (H(+)+K+)-ATPase using lin-benzo-ATP as substrate and the effect of the inhibitors omeprazole and SCH 28080 support the notion that lin-benzo-ATP is not hydrolysed by the (H(+)+K+)-ATPase, but by the basal Mg2(+)-ATPase, and that the activity of the latter enzyme is not involved in the (H(+)+K+)-transporting reaction cycle (according to the Albers-Post formalism) of (H(+)+K+)-ATPase.

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 The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

1991 ◽  
Vol 278 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T L Kirley
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Low Concentrations ◽  
Sds Page ◽  
Molecular Masses ◽  
Relationship Of ◽  
Peptide Core

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.

Functional role of ecto-ATPase activity in goldfish hepatocytes

1998 ◽  
Vol 274 (4) ◽  
pp. R1031-R1038 ◽  
Author(s):  
Pablo J. Schwarzbaum ◽  
Michael E. Frischmann ◽  
Gerhard Krumschnabel ◽  
Rolando C. Rossi ◽  
Wolfgang Wieser
Keyword(s):  
Atpase Activity ◽  
Functional Role ◽  
Maximal Activity ◽  
Hydrolysis Rate ◽  
Hyperbolic Function ◽  
Extracellular Release ◽  
Transport Atpases ◽  
Hydrolysis Of ◽  
Hepatocyte Suspension

Extracellular [γ-32P]ATP added to a suspension of goldfish hepatocytes can be hydrolyzed to ADP plus γ-32Pidue to the presence of an ecto-ATPase located in the plasma membrane. Ecto-ATPase activity was a hyperbolic function of ATP concentration ([ATP]), with apparent maximal activity of 8.3 ± 0.4 nmol Pi ⋅ (106cells)−1 ⋅ min−1and substrate concentration at which a half-maximal hydrolysis rate is obtained of 667 ± 123 μM. Ecto-ATPase activity was inhibited 70% by suramin but was insensitive to inhibitors of transport ATPases. Addition of 5 μM [α-32P]ATP to the hepatocyte suspension induced the extracellular release of α-32Pi[8.2 pmol ⋅ (106cells)−1 ⋅ min−1] and adenosine, suggesting the presence of other ectonucleotidase(s). Exposure of cell suspensions to 5 μM [2,8-3H]ATP resulted in uptake of [2,8-3H]adenosine at 7.9 pmol ⋅ (106cells)−1 ⋅ min−1. Addition of low micromolar [ATP] strongly increased cytosolic free Ca2+([Formula: see text]). This effect could be partially mimicked by adenosine 5′- O-(3-thiotriphosphate), a nonhydrolyzable analog of ATP. The blockage of both glycolysis and oxidative phosphorylation led to a sixfold increase of[Formula: see text] and an 80% decrease of intracellular ATP, but ecto-ATPase activity was insensitive to these metabolic changes. Ecto-ATPase activity represents the first step leading to the complete hydrolysis of extracellular ATP, which allows 1) termination of the action of ATP on specific purinoceptors and 2) the resulting adenosine to be taken up by the cells.

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.

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.

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 Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
FuJung Chang ◽  
Alberto Riera ◽  
Cecile Evrin ◽  
Jingchuan Sun ◽  
Huilin Li ◽  
...  
Keyword(s):  
Dna Replication ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
S Phase ◽  
G1 Phase ◽  
Helicase Loading ◽  
Mcm Helicase

To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant ‘Cdc6-E224Q’ promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo.

Actin filaments stimulate the Na(+)-K(+)-ATPase

1995 ◽  
Vol 269 (5) ◽  
pp. F637-F643 ◽  
Author(s):  
H. F. Cantiello
Keyword(s):  
Sequence Analysis ◽  
Atpase Activity ◽  
Actin Filaments ◽  
Functional Role ◽  
Atp Hydrolysis ◽  
Rat Kidney ◽  
Alpha Subunit ◽  
Actin Binding ◽  
Binding Domains

In this report, the functional role of actin on Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity was explored. The Na(+)- and K(+)-dependent, ouabain-sensitive ATP hydrolysis mediated by rat kidney Na(+)-K(+)-ATPase increased by 74% in the presence of previously unpolymerized actin (24 microM), whereas addition of polymerized actin was without effect. Addition of actin was associated instead with an increase in the affinity of the Na(+)-K(+)-ATPase for Na+ but not other enzymatic substates. A maximal stimulatory effect (296%) was observed either at an Na(+)-K(+)-ATPase:actin ratio of 1:50,000 or at lower ratios (1:625) by shifting from the E2 (K+ selective) to the E1 (Na+ selective) conformation of the enzyme. Immunoblotting of actin to the purified Na(+)-K(+)-ATPase suggested that this interaction may be linked to binding of actin to the enzyme, which was further supported by sequence analysis indicating putative actin-binding domains in the alpha-subunit of the enzyme. The interaction between actin and the Na(+)-K(+)-ATPase may imply a novel functional role of the cytoskeleton in the control of ion transport.

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.

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