scholarly journals Mechanism of inhibition of Ca2+-ATPase by myotoxin a

1995 ◽  
Vol 307 (2) ◽  
pp. 571-579 ◽  
Author(s):  
K J Baker ◽  
J M East ◽  
A G Lee
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Potent Inhibitor ◽  
Low Ph ◽  
High Ionic Strength ◽  
Nucleotide Binding Domain ◽  
Amphipathic Peptide ◽  
Mechanism Of Inhibition ◽  
Myotoxin A ◽  
Atpase Inhibition ◽  
Phosphorylation Domain

The peptide DCRQKWKCCKKGSG [myotoxin-(29-42)], corresponding to residues 29-42 of myotoxin a, inhibits the activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum, with a Kd value of 19.4 microM at pH 7.5, in 100 mM KCl. The peptide YKQCHKKGGHCFPKEK, corresponding to residues 1-16 of myotoxin a, is a less potent inhibitor. Inhibition by myotoxin-(29-42) is reduced at low pH and at high ionic strength, suggesting that charge interactions are important in binding to the ATPase. Inhibition of the ATPase has been shown to follow from a decrease in the rate of dephosphorylation, with no effect on the rate of phosphorylation of the ATPase or on the rate of the Ca2+ transport step (E1PCa2-->E2P). Binding of myotoxin-(29-42) decreased the affinity of the ATPase for Ca2+ and Mg2+, and increased the rate of dissociation of the outer Ca2+ ion from the ATPase. Unlike the amphipathic peptide melittin, it is suggested that myotoxin-(29-42) does not bind significantly to the lipid bilayer portion of the sarcoplasmic reticulum. Fluorescence quenching studies suggest that it could bind to the ATPase in the vicinity of Cys-344 in the phosphorylation domain and Lys-515 in the nucleotide binding domain. Inhibition of the ATPase is observed when the ATPase is reconstituted in monomeric form in sealed vesicles, suggesting that aggregation of the ATPase is not involved in inhibition.

Effect of Non-Solubilizing SDS Concentrations on High Affinity Ca2+ Binding and Steady State Phosphorylation by Inorganic Phosphate of the Sarcoplasmic Reticulum ATPase

1989 ◽  
Vol 44 (1-2) ◽  
pp. 139-152 ◽  
Author(s):  
Elisabeth Fassold ◽  
Wilhelm Hasselbach ◽  
Bernd Küchler
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Inorganic Phosphate ◽  
Atp Hydrolysis ◽  
Mutual Exclusion ◽  
Chemical Properties ◽  
Cooperative Interaction ◽  
Hydrophobic Force ◽  
High Affinity ◽  
Binding Domains ◽  
Phosphorylation Domain

Abstract In this investigation low, non-solubilizing concentrations of the strong anionic detergent SDS were used to perturbate the interaction of Ca2+ and Pi with their respective binding domains on the sarcoplasmic reticulum Ca-transport ATPase. Rising SDS concentrations produce a two-step decline of Ca2+-dependent ATP hydrolysis. At pH 6.15, SDS differently affects high affinity Ca2+ binding and phosphorylation by inorganic phosphate and releases the “mutual exclusion” of these two ligand binding steps. The degree of uncoupling is considerably more pronounced in the presence of 20% Me2SO. The reduction of Ca2+ binding by SDS is demonstrated to be a result of decreased affinity of one of the two specific high affinity binding sites and of perturbation of their cooperative interaction. Higher SDS partially restores the original high Ca2+ affinity but not the cooperativity of binding. Phosphorylation exhibits a higher SDS sensitivity than Ca2+ binding: Increasing SDS competitively inhibits and then completely abolishes phosphoenzyme formation. Thus. SDS binds to the phosphorylation domain, evidently involving the Lys352 residue of the ATPase molecule; this is accompanied by a more unspecific concentration-dependent SDS effect, probably mediated by hydrophobic force, which, finally, suppresses phosphorylation. Me2SO does neither qualitatively affect the SDS-dependent chemical properties of the vesicular material nor the SDS-dependent perturbation of the investigated reaction steps.

Mercury blocks Na-K-ATPase by a ligand-dependent and reversible mechanism

1992 ◽  
Vol 262 (5) ◽  
pp. F830-F836 ◽  
Author(s):  
B. M. Anner ◽  
M. Moosmayer ◽  
E. Imesch
Keyword(s):  
Metal Binding ◽  
Inhibitory Concentration ◽  
Potent Inhibitor ◽  
Inhibitory Receptor ◽  
Protein Inhibitor ◽  
Binding Interface ◽  
Cellular Expression ◽  
Metal Binding Domain ◽  
Cardioactive Steroids ◽  
Atpase Inhibition

An inhibitory receptor for cardioactive steroids such as digoxin and ouabain is located at the extracellular surface of the Na-K-adenosinetriphosphatase (ATPase) molecule. Besides cardioactive steroids, mercury is a potent inhibitor of the Na-K-ATPase activity. The half-maximal inhibitory concentration (IC50), determined within 30 min at 37 degrees C at 1 microgram protein/ml, was 200 nM, despite the presence of 1 mM EDTA; the IC50 decreased with increasing protein/inhibitor ratio, and it reached 2.7 microM at 0.1 mg protein/ml and 20 microM at 1 mg protein/ml. The IC50 for Na-K-ATPase inhibition by the diuretic compound mersalyl was 4 and 5 microM for the nondiuretic p-chloromercuribenzenesulfonic acid at 0.1 mg protein/ml. The IC50 for HgCl2 inhibition was modulated by the presence of EDTA as well as by the pump ligands Mg, Na, K, and ATP. The E2 conformation of the Na-K-ATPase molecule was more sensitive to HgCl2 than the E1 conformation. The mercury antidote 2,3-dimercapto-1-propanesulfonic acid was able to reactivate approximately 70% of the blocked enzyme. In conclusion, a metal-binding domain of the Na-K-ATPase molecule with particular high affinity for Hg(II) was described functionally in the present work. Therefore Na-K-ATPase belongs to the metal-binding proteins. Metals may modulate the cellular expression and activity of the system by interacting with its metal-binding interface.

scholarly journals The mechanism of inhibition of the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum by the cross-linker o-phthalaldehyde

1996 ◽  
Vol 317 (2) ◽  
pp. 439-445 ◽  
Author(s):  
Yamin M. KHAN ◽  
Anthony P. STARLING ◽  
J. Malcolm EAST ◽  
Anthony G. LEE
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Order Rate Constant ◽  
Molar Ratio ◽  
Slow Step ◽  
Phosphoryl Transfer ◽  
Back Reaction ◽  
Mechanism Of Inhibition ◽  
Fluorescence Change ◽  
Similar Conformation

Labelling the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum with o-phthalaldehyde (OPA) results in loss of ATPase activity at a 1:1 molar ratio of label to ATPase. The affinity of the ATPase for Ca2+ is unaffected, as is the E1/E2 equilibrium constant. The rate of dissociation of Ca2+ from the Ca2+-bound ATPase is also unaffected and Mg2+ increases the rate of dissociation, as for the unlabelled ATPase. Effects of Mg2+ on the fluorescence intensity of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin are also unaffected by labelling with OPA, consistent with the fluorescence change reporting on Mg2+ binding at the gating site on the ATPase. The affinity of the ATPase for ATP is reduced by labelling, as is the rate of phosphorylation. The rate of phosphorylation is independent of the concentration of ATP above 25 μM ATP, so that the slow step is the first-order rate constant for phosphorylation by bound ATP. The rate of the back reaction between phosphorylated ATPase and ADP is little affected, suggesting that the slow step in phosphorylation could be the slow conformation step before phosphoryl transfer. The rate of dephosphorylation of the phosphorylated ATPase is also decreased, suggesting that a similar conformation change could be involved in the dephosphorylation step. The rate of the Ca2+ transport step appears to be unaffected by labelling. The net result of these changes is that the labelled ATPase is present predominantly in a Ca2+-free, phosphorylated form at steady state in the presence of ATP.

Interaction of myotoxin a with the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum

1986 ◽  
Vol 246 (1) ◽  
pp. 90-97 ◽  
Author(s):  
Pompeo Volpe ◽  
Ernesto Damiani ◽  
Andreas Maurer ◽  
Anthony T. Tu
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Myotoxin A

Comparable Levels of Ca-ATPase Inhibition by Phospholamban in Slow-Twitch Skeletal and Cardiac Sarcoplasmic Reticulum†

Biochemistry ◽  
2002 ◽  
Vol 41 (44) ◽  
pp. 13289-13296 ◽  
Author(s):  
Deborah A. Ferrington ◽  
Qing Yao ◽  
Thomas C. Squier ◽  
Diana J. Bigelow
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Slow Twitch ◽  
Atpase Inhibition

Ouabain modulation of cellular calcium stores and signaling

2007 ◽  
Vol 293 (5) ◽  
pp. F1518-F1532 ◽  
Author(s):  
Aurélie Edwards ◽  
Thomas L. Pallone
Keyword(s):  
Mathematical Model ◽  
Sarcoplasmic Reticulum ◽  
Active Transport ◽  
Intracellular Localization ◽  
Calcium Stores ◽  
Model Simulations ◽  
Store Depletion ◽  
Intracellular Ca ◽  
Atpase Inhibition ◽  
Predominant Effect

Ouabain-like factors modulate intracellular Ca2+ concentrations and Ca2+ stores. Recently, a role for Na+-K+-ATPase Na+ transport inhibition as a pivotal event in ouabain signaling was questioned (Kaunitz JD. Am J Physiol Renal Physiol 290: F995–F996, 2006). In the present study, we used a mathematical model of Ca2+ trafficking in cytoplasm and subplasmalemmal microdomains to simulate the pathways through which ouabain can affect Ca2+ signaling: inhibition of active transport by Na+-K+-ATPase α1- and α2-isoforms, activation of inositol trisphosphate (IP3) production, and increased IP3 receptor (IP3R) conductance. A fundamental prediction is that Na+-K+-ATPase inhibition favors sarcoplasmic reticulum Ca2+ store loading, whereas Src-mediated increases in IP3 production and IP3R sensitization favor store depletion. The model predicts that α2-isoform inhibition generates a peak-and-plateau pattern of cytosolic Ca2+ concentration ([Ca2+]cyt) elevation, whereas α1-isoform inhibition yields a monophasic rise. The effects of ouabain-mediated increases in IP3 production or IP3R conductance on [Ca2+]cyt depend on their relative distributions between cellular microdomains and the bulk cytoplasm. Simulations suggest that the intracellular localization of IP3 production is a pivotal determinant of the changes in compartmental Ca2+ concentrations that can be induced by ouabain. As a consequence of sequestration of the ouabain-sensitive α2-isoform into microdomains, inhibition of the α2-isoform in rodents is not predicted to significantly affect cytosolic Na+ concentration. Model simulations support the hypothesis that ouabain can enhance agonist-evoked [Ca2+]cyt transients when its predominant effect is to inhibit α2-isoform Na+ transport and, thereby, increase Ca2+ loading into sarcoplasmic reticulum stores.

scholarly journals Effects on ATPase activity of monoclonal antibodies raised against (Ca2+ + Mg2+)-ATPase from rabbit skeletal muscle sarcoplasmic reticulum and their correlation with epitope location

1989 ◽  
Vol 262 (2) ◽  
pp. 439-447 ◽  
Author(s):  
J Colyer ◽  
A M Mata ◽  
A G Lee ◽  
J M East
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibodies ◽  
Sarcoplasmic Reticulum ◽  
Epitope Mapping ◽  
Structural Models ◽  
Rabbit Skeletal Muscle ◽  
Protein Fragments ◽  
Binding Domains ◽  
Inhibitory Antibodies ◽  
Phosphorylation Domain

A total of 28 monoclonal antibodies have been raised against the (Ca2+ + Mg2+)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum. Epitope mapping, using protein fragments generated by proteolysis, indicates that these antibodies include examples binding to at least four distinct epitopes on the A1 and B tryptic fragments of the ATPase. Competition data also show that the 28 antibodies are directed against at least five spatially distinct regions. Altogether, nine inhibitory antibodies were produced: six of these inhibitory antibodies mapped to the same spatial region, although they appear to bind to two distinct epitopes located within the hinge region and the nucleotide-binding domains of current structural models; one antibody bound to an epitope located within the phosphorylation domain and the stalk-transmembranous region designated M4S4 by Brandl, Green, Korczak & MacLennan [(1986) Cell 44, 597-607]. Two of the inhibitory antibodies recognized assembled epitopes exclusively and could not be mapped. Binding to four of the five identified spatial regions was without effect on activity. These data show that the inhibition of catalytic activity by monoclonal antibodies is achieved only by binding to defined regions of the ATPase and they may therefore provide useful probes of structure-function relationships.

Heat-induced Gelation of Myosins/Subfragments from Chicken Leg and Breast Muscles at High Ionic Strength and Low pH

1991 ◽  
Vol 56 (4) ◽  
pp. 884-890 ◽  
Author(s):  
IL-SHIN CHOE ◽  
JON-ICHIRO MORITA ◽  
KATSUHIRO YAMAMOTO ◽  
KUNIHIKO SAMEJIMA ◽  
TSUTOMU YASUI
Keyword(s):  
Ionic Strength ◽  
Low Ph ◽  
High Ionic Strength
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