Comparative Studies on the ATP-Binding Sites in Ca2+-ATPase and (Na+ + K+)-ATPase by the Use of ATP-Analogues

1982 ◽  
Vol 37 (7-8) ◽  
pp. 692-705 ◽  
Keyword(s):  
Atpase Activity ◽  
Binding Sites ◽  
Sulfhydryl Groups ◽  
Atp Binding ◽  
Stable Form ◽  
Atp Binding Site ◽  
Atp Analogues ◽  
Transport Atpases ◽  
High Concentrations ◽  
Hydrolysis Of

Abstract The effects of ATP-analogues on Ca2+-ATPase and (Na+ + K+)-ATPase have been studied. The participation of sulfhydryl groups in the recognition of ATP by both transport ATPases is indicat ed by the fact, that the disulfide of thioinosine triphosphate inactivates both enzymes. The reactivity of rapidly and slowly reacting sulfhydryl groups in the ATP binding sites of both enzymes is altered by the presence of transport substrates. At least in (Na+ + K+)-ATPase Na+ and Mg2+ appear to alter the structure of the ATP binding site, which conclusion is fortified by the fact, that the photoinactivation of the enzyme by 3′-O-[3-(2-nitro-4-azidophenyl)-propionyl]-ATP need Mg2+. Chromium(III)ATP, a MgATP analogue, inactivated both transport ATPases by the formation of a stable chromo-phosphointermediate. In the case of Ca2+-ATPase this was concomited by the occlusion of Ca2+ in a stable form. No occlusion of Na+ was observable so far in the (Na++ K+)-ATPase. Contrary to the expectation of the Albers-Post-scheme the hydrolysis of the phosphointermediate formed from chromium(III)ATP was protected by K+, but activated by high concentrations of Na+. Consequently, despite of the inhibition of (Na+ + K+)-ATPase activity chromium(III)-ATP supported the Na+ - Na+ -exchange reaction in everted red bood cells.

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 Vibrio cholerae Strains with Mutations in an Atypical Type I Secretion System Accumulate RTX Toxin Intracellularly

2004 ◽  
Vol 186 (23) ◽  
pp. 8137-8143 ◽  
Author(s):  
Bethany Kay Boardman ◽  
Karla J. Fullner Satchell
Keyword(s):  
Binding Site ◽  
Vibrio Cholerae ◽  
Secretion System ◽  
Atp Binding ◽  
Type I ◽  
Atp Binding Site ◽  
Rtx Toxin ◽  
Component Type ◽  
Transport Atpases

ABSTRACT This study shows that the Vibrio cholerae RTX toxin is secreted by a four-component type I secretion system (TISS) encoded by rtxB, rtxD, rtxE, and tolC. ATP-binding site mutations in both RtxB and RtxE blocked secretion, demonstrating that this atypical TISS requires two transport ATPases that may function as a heterodimer.

scholarly journals A Variable Domain near the ATP-Binding Site in Drosophila Muscle Myosin Is Part of the Communication Pathway between the Nucleotide and Actin-binding Sites

2007 ◽  
Vol 368 (4) ◽  
pp. 1051-1066 ◽  
Author(s):  
Becky M. Miller ◽  
Marieke J. Bloemink ◽  
Miklós Nyitrai ◽  
Sanford I. Bernstein ◽  
Michael A. Geeves
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Variable Domain ◽  
Actin Binding ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Communication Pathway ◽  
Muscle Myosin

scholarly journals Effect of salinity on modulation by ATP, protein kinases and FXYD2 peptide of gill (Na+, K+)-ATPase activity in the swamp ghost crab Ucides cordatus (Brachyura, Ocypodidae)

2020 ◽  
Author(s):  
Francisco A. Leone ◽  
Malson N. Lucena ◽  
Leonardo M. Fabri ◽  
Daniela P. Garçon ◽  
Carlos F.L. Fontes ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Binding Site ◽  
Protein Kinases ◽  
Atp Binding ◽  
List Type ◽  
Inhibit Activity ◽  
Ucides Cordatus ◽  
Atp Binding Site ◽  
High Affinity ◽  
Endogenous Protein

ABSTRACTThe gill (Na+, K+)-ATPase is the main enzyme that underpins osmoregulatory ability in crustaceans that occupy biotopes like mangroves, characterized by salinity variation. We evaluated osmotic and ionic regulatory ability in the semi-terrestrial mangrove crab Ucides cordatus after 10-days acclimation to different salinities. We also analyzed modulation by exogenous FXYD2 peptide and by endogenous protein kinases A and C, and Ca2+- calmodulin-dependent kinase of (Na+, K+)-ATPase activity. Hemolymph osmolality was strongly hyper-/hypo-regulated in crabs acclimated at 2 to 35 ‰S. Cl- was well hyper-/hypo- regulated although Na+ much less so, becoming iso-natremic at high salinity. (Na+, K+)- ATPase activity was greatest in isosmotic crabs (26 ‰S), diminishing progressively from 18 and 8 ‰S (≈0.5 fold) to 2 ‰S (0.04-fold), and decreasing notably at 35 ‰S (0.07-fold). At low salinity, the (Na+, K+)-ATPase exhibited a low affinity ATP-binding site that showed Michaelis-Menten behavior. Above 18 ‰S, an additional, high affinity ATP-binding site, corresponding to 10-20% of total (Na+, K+)-ATPase activity appeared. Activity is stimulated by exogenous pig kidney FXYD2 peptide, while endogenous protein kinases A and C and Ca2+/calmodulin-dependent kinase all inhibit activity. This is the first demonstration of inhibitory phosphorylation of a crustacean (Na+, K+)-ATPase by Ca2+/calmodulin-dependent kinase. Curiously, hyper-osmoregulation in U. cordatus shows little dependence on gill (Na+, K+)-ATPase activity, suggesting a role for other ion transporters. These findings reveal that the salinity acclimation response in U. cordatus consists of a suite of osmoregulatory and enzymatic adjustments that maintain its osmotic homeostasis in a challenging, mangrove forest environment.Graphical abstractHighlightsGill (Na+, K+)-ATPase activity is greatest in isosmotic crabs, diminishing in lower and higher salinities.A high affinity ATP-binding site (10-20% of total activity) is exposed above 18 ‰S.Exogenous FXYD2 peptide stimulates activity; endogenous PKA, PKC and CaMK inhibit activity.First demonstration of inhibitory phosphorylation of crustacean (Na+, K+)-ATPase by CaMK.Hyper-osmoregulation shows little dependence on (Na+, K+)-ATPase activity.

scholarly journals OppA, the Substrate-Binding Subunit of the Oligopeptide Permease, Is the Major Ecto-ATPase of Mycoplasma hominis

2004 ◽  
Vol 186 (4) ◽  
pp. 1021-1028 ◽  
Author(s):  
Miriam Hopfe ◽  
Birgit Henrich
Keyword(s):  
Atpase Activity ◽  
Binding Protein ◽  
Substrate Binding ◽  
Mycoplasma Hominis ◽  
Disulfonic Acid ◽  
Atp Binding ◽  
Atpase Inhibitor ◽  
Atp Binding Site ◽  
Oligopeptide Permease ◽  
Substrate Binding Protein

ABSTRACT Most ATPases, involved in energy-driven processes, act in the cytoplasm. However, external membrane-bound ATPases have also been described in parasites and eukaryotic cells. In Mycoplasma hominis, a bacterium lacking a cell wall, the surface-exposed substrate-binding protein OppA of an oligopeptide permease (Opp) contains an ATP binding P-loop structure in the C-terminal region. With ATP affinity chromatography and tryptic digestion in the presence or absence of ATP, the functionality of the Mg2+-dependent ATP binding site is demonstrated. In addition to ATP, ADP also could bind to OppA. The presence of an ATPase activity on the surface of M. hominis is indicated by the inactivation of ATP hydrolyzing activity of intact mycoplasma cells by the impermeable ATPase inhibitor 4′,4′-diisothiocyanostilbene-2′,2′-disulfonic acid and influenced by the ATP analog 5′-fluorosulfonyl-benzoyladenosine. Comparing equimolar amounts of OppA in intact mycoplasma cells and in the purified form indicated that more than 80% of the surface-localized ATPase activity is derived from OppA, implying that OppA is the main ATPase on the surface of mycoplasma cells. Together, these data present the first evidence that the cytoadhesive substrate binding protein OppA of the oligopeptide permease also functions as an ecto-ATPase in Mycoplasma hominis.

Fluorometric study on conformational changes in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase

1995 ◽  
Vol 15 (5) ◽  
pp. 317-326 ◽  
Author(s):  
Tohru Kanazawa ◽  
Hiroshi Suzuki ◽  
Takashi Daiho ◽  
Kazuo Yamasaki
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Site ◽  
Conformational Changes ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Tryptophan Fluorescence ◽  
Cytoplasmic Domain ◽  
Catalytic Cycle ◽  
Atp Binding ◽  
Atp Binding Site

Changes in the fluoresence of N-acetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca2+-ATPase without affecting the catalytic activity, as well as changes in the intrinsic tryptophan fluorescence were followed throughout the catalytic cycle by the steady-state measurements and the stopped-flow spectrofluorometry. EDANS-fluorescence changes reflect conformational changes near the ATP binding site in the cytoplasmic domain, while tryptophan-fluorescence changes most probably reflect conformational changes in or near the transmembrane domain in which the Ca2+ binding sites are located. Formation of the phosphoenzyme intermediates (EP) was also followed by the continuous flow-rapid quenching method. The kinetic analysis of EDANS-fluorescence changes and EP formation revealed that, when ATP is added to the calcium-activated enzyme, conformational changes in the ATP binding site occur in three successive reaction steps; conformational change in the calcium enzyme substrate complex, formation of ADP-sensitive EP, and transition of ADP-sensitive EP to ADP-insensitive EP. In contrast, the ATP-induced tryptophan-fluorescence changes occur only in the latter two steps. Thus, we conclude that conformational changes in the ATP binding site in the cytoplasmic domain are transmitted to the Ca2+-binding sites in the transmembrane domain in these latter two steps.

scholarly journals Adenosine nucleotide binding sites on Complex V from diverse organisms

2021 ◽  
Author(s):  
Abhinav Parashar ◽  
Kelath Murali Manoj
Keyword(s):  
Binding Sites ◽  
In Silico ◽  
Nucleotide Binding ◽  
Atp Binding ◽  
Atp Binding Site ◽  
In Silico Docking ◽  
Epsilon Subunit ◽  
Nucleotide Binding Sites ◽  
Alpha Beta ◽  
Adenosine Nucleotide

Using in silico docking approaches, we scan the various subunits of Complex V (FoF1ATPase) for putative adenosine nucleotide binding sites. We find that multiple generic ADP/ATP binding sites are present on the alpha-beta binding sites and a conserved ATP binding site is present on the epsilon subunit. These findings support the murburn model of Complex V.

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