scholarly journals Effect of pH on the activity of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1990 ◽  
Vol 267 (2) ◽  
pp. 423-429 ◽  
Author(s):  
F Michelangeli ◽  
J Colyer ◽  
J M East ◽  
A G Lee
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Atpase Activity ◽  
Binding Sites ◽  
Ph Dependence ◽  
Effect Of Ph ◽  
High Ph ◽  
Atp Concentration ◽  
Slow Dissociation

A kinetic model for the Ca2(+) + Mg2(+)-activated ATPase of sarcoplasmic reticulum was presented in a previous paper [Stefanova, Napier, East & Lee (1987) Biochem. J. 245, 723-730]. Here, that model is modified to account for the pH-dependence of ATPase activity and for the effects of Mg2+ on activity at high pH. It is shown that effects of Mg2+ on measurements of ATPase activity as a function of ATP concentration at pH 8.0 and pH 8.5 are consistent with binding of Mg2+ to the Ca2(+)-binding sites on the phosphorylated ATPase, such binding inhibiting dephosphorylation of the ATPase. It is also shown that slow dissociation of Ca2+ from the phosphorylated ATPase is consistent with the previously published model.

scholarly journals A kinetic model for Ca2+ efflux mediated by the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1987 ◽  
Vol 245 (3) ◽  
pp. 713-721 ◽  
Author(s):  
J M McWhirter ◽  
G W Gould ◽  
J M East ◽  
A G Lee
Keyword(s):  
Experimental Data ◽  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Atpase Activity ◽  
Binding Site ◽  
Binding Sites ◽  
Adenine Nucleotides ◽  
Contraction Coupling ◽  
High Affinity

We present a model for Ca2+ efflux from vesicles of sarcoplasmic reticulum (SR). It is proposed that efflux is mediated by the Ca2+ + Mg2+-activated ATPase that is responsible for Ca2+ uptake in this system. In the normal ATPase cycle of the ATPase, phosphorylation of the ATPase is followed by a conformational change in which the Ca2+-binding sites change from being outward-facing and of high affinity to being inward-facing and of low affinity. To mediate Ca2+ efflux, it is proposed that the ATPase can adopt a conformation in which the Ca2+-binding sites are of low affinity but still outward-facing. It is shown that experimental data on the rates of Ca2+ efflux can be simulated in terms of this model, with Ca2+-binding-site affinities previously proposed to explain ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227]. Effects of Mg2+ and adenine nucleotides on efflux rates are explained. It is suggested that Ca2+ efflux from SR mediated by the ATPase could be important in excitation-contraction coupling in skeletal muscle.

scholarly journals A kinetic model for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1986 ◽  
Vol 237 (1) ◽  
pp. 217-227 ◽  
Author(s):  
G W Gould ◽  
J M East ◽  
R J Froud ◽  
J M McWhirter ◽  
H I Stefanova ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Binding Sites ◽  
Ph Dependence ◽  
High Affinity ◽  
Phosphorylated Form ◽  
Low Concentrations ◽  
Kinetics Of ◽  
Single Binding Site ◽  
Micromolar Range

The Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum exhibits complex kinetics of activation with respect to ATP. ATPase activity is pH-dependent, with similar pH-activity profiles at high and low concentrations of ATP. Low concentrations of Ca2+ in the micromolar range activate the ATPase, whereas activity is inhibited by Ca2+ at millimolar concentrations. The pH-dependence of this Ca2+ inhibition and the effect of the detergent C12E8 (dodecyl octaethylene glycol monoether) on Ca2+ inhibition are similar to those observed on activation by low concentrations of Ca2+. On the basis of these and other studies we present a kinetic model for the ATPase. The ATPase is postulated to exist in one of two conformations: a conformation (E1) of high affinity for Ca2+ and MgATP and a conformation (E2) of low affinity for Ca2+ and MgATP. Ca2+ binding to E2 and to the phosphorylated form E2P are equal. Proton binding at the Ca2+-binding sites in the E1 and E2 conformations explains the pH-dependence of Ca2+ effects. Binding of MgATP to the phosphorylated intermediate E1′PCa2 and to E2 modulate the rates of the transport step E1′PCa-E2′PCa2 and the return of the empty Ca2+ sites to the outside surface of the sarcoplasmic reticulum, as well as the rate of dephosphorylation of E2P. Only a single binding site for MgATP is postulated.

scholarly journals Effect of Heptane Treatment on the Response of Sarcoplasmic Reticulum Preparations to Phosphate

1976 ◽  
Vol 29 (6) ◽  
pp. 459
Author(s):  
Douglas J Horgan
Keyword(s):  
Steady State ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Binding Sites ◽  
Calcium Uptake ◽  
Straight Line ◽  
Enzyme Intermediates ◽  
Stimulation Of ◽  
Linear Shape ◽  
Burk Plot

The calcium-stimulated (extra) ATPase and calcium uptake activities of sarcoplasmic reticulum (SR) preparations treated with aqueous heptane mixtures were compared with those of untreated SR, and with those of SR treated with aqueous ether. Both treatments altered the kinetic behaviour of the extra ATPase, the Lineweaver-Burk plot being changed from its normal non-linear shape to a straight line. Kinetic constants, Vma ., Km for ATP and KI for phosphate, were measured. The extra ATPase activity of heptane-treated SR was inhibited by phosphate as was that of ether-treated SR, to a lesser extent. The magnitude of this inhibition by phosphate was found to be considerably less than the degree of stimulation of the extra ATPase activity of untreated SR caused by phosphate through its calcium-precipitating action. The steady-state concentrations of the phosphoryl-enzyme intermediates were measured and together with the K m and K, values they indicate that the binding of ATP to heptane-treated SR is weaker than it is to untreated SR, and that phosphate is an efficient competitor for the binding sites.

scholarly journals Calcium-Binding Properties and ATPase Activities of Rat Liver Plasma Membranes

1974 ◽  
Vol 64 (1) ◽  
pp. 104-126 ◽  
Author(s):  
Anne-Marie Chambaut ◽  
Françoise Leray-Pecker ◽  
Gérard Feldmann ◽  
Jacques Hanoune
Keyword(s):  
Atpase Activity ◽  
Rat Liver ◽  
Binding Sites ◽  
Calcium Binding ◽  
Plasma Membranes ◽  
Atp Hydrolysis ◽  
Ph Dependence ◽  
Classical Energy ◽  
Liver Plasma Membranes ◽  
Specificity And Sensitivity

Plasma membranes from rat liver purified according to the procedure of Neville bind calcium ions by a concentration-dependent, saturable process with at least two classes of binding sites. The higher affinity sites bind 45 nmol calcium/mg membrane protein with a KD of 3 µM. Adrenalectomy increases the number of the higher affinity sites and the corresponding KD. Plasma membranes exhibit a (Na+-K+)-independent-Mg2+-ATPase activity which is not activated by calcium between 0.1 µM and 10 mM CaCl2. Calcium can, with less efficiency, substitute for magnesium as a cofactor for the (Na+-K+)-independent ATPase. Both Mg2+- and Ca2+-ATPase activities are identical with respect to pH dependence, nucleotide specificity and sensitivity to inhibitors. But when calcium is substituted for magnesium, there is no detectable membrane phosphorylation from [γ-32P] ATP as it is found in the presence of magnesium. The existence of high affinity binding sites for calcium in liver plasma membranes is compatible with a regulatory role of this ion in membrane enzymic mechanisms or in hormone actions. Plasma membranes obtained by the procedure of Neville are devoid of any Ca2+-activated-Mg2+-ATPase activity indicating the absence of the classical energy-dependent calcium ion transport. These results would suggest that the overall calcium-extruding activity of the liver cell is mediated by a mechanism involving no direct ATP hydrolysis at the membrane level.

scholarly journals Effects of Mg2+, anions and cations on the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1987 ◽  
Vol 245 (3) ◽  
pp. 723-730 ◽  
Author(s):  
H I Stefanova ◽  
R M Napier ◽  
J M East ◽  
A G Lee
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Atpase Activity ◽  
Binding Site ◽  
Conformational Transition ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Phosphorylated Form ◽  
Low Concentrations ◽  
Anions And Cations

In a previous paper [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227] we presented a kinetic model for the activity of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum. Here we extend the model to account for the effects on ATPase activity of Mg2+, cations and anions. We find that Mg2+ concentrations in the millimolar range inhibit ATPase activity, which we attribute to competition between Mg2+ and MgATP for binding to the nucleotide-binding site on the E1 and E2 conformations of the ATPase and on the phosphorylated forms of the ATPase. Competition is also suggested between Mg2+ and MgADP for binding to the phosphorylated form of the ATPase. ATPase activity is increased by low concentrations of K+, Na+ and NH4+, but inhibited by higher concentrations. It is proposed that these effects follow from an increase in the rate of dephosphorylation but a decrease in the rate of the conformational transition E1′PCa2-E2′PCa2 with increasing cation concentration. Li+ and choline+ decrease ATPase activity. Anions also decrease ATPase activity, the effects of I- and SCN- being more marked than that of Cl-. These effects are attributed to binding at the nucleotide-binding site, with a decrease in binding affinity and an increase in ‘off’ rate constant for the nucleotide.

pH-dependent inhibitory effects of Ca2+, Mg2+, and K+ on Ca2+ efflux mediated by sarcoplasmic reticulum ATPase

1994 ◽  
Vol 266 (5) ◽  
pp. C1376-C1381 ◽  
Author(s):  
H. Wolosker ◽  
L. de Meis
Keyword(s):  
Binding Sites ◽  
Divalent Cations ◽  
Ph Dependence ◽  
Specific Inhibitor ◽  
Inhibitory Effects ◽  
Efflux Rate ◽  
Effect Of Ph ◽  
High Affinity ◽  
Neutral Ph ◽  
High Concentrations

The effect of pH on the rate of Ca2+ efflux mediated by the Ca2+ adenosinetriphosphatase (ATPase) was measured. The cations Ca2+, Mg2+, and K+ decrease the rate of Ca2+ efflux at pH 7.5 but not at pH 6.0. The effect of pH on the affinity to Ca2+ during Ca2+ efflux was found to be similar to the pH dependence of the high-affinity Ca2+ binding sites of the ATPase. The inhibitory activity of cations was significantly increased by a rise in pH, whereas acidification amplified the magnitude of an efflux component insensitive to cations. Acidification of the assay medium allows efflux of Ca2+ through the Ca2+ pump, even in the presence of high concentrations of monovalent and divalent cations. The efflux rate was severalfold increased by addition of the hydrophobic drugs trifluoperazine, dibucaine, and imipramine. At neutral pH, the Ca2+ efflux induced by trifluoperazine was antagonized by the cations Ca2+, Mg2+, and K+ and by thapsigargin, a highly specific inhibitor of the Ca2+ pump. In contrast to that observed at neutral pH, the cations did not antagonize the effect of trifluoperazine on Ca2+ efflux at acid pH. It is concluded that H+ produces functional alterations in ATPase domains involved in Ca2+ efflux.

Electron Probe Analysis of Muscle

1982 ◽  
Vol 40 ◽  
pp. 398-401
Author(s):  
A. V. Somlyo ◽  
H. Shuman ◽  
A. P. Somlyo
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Resting State ◽  
Binding Sites ◽  
Electron Probe ◽  
Frog Skeletal Muscle ◽  
Electron Probe Analysis ◽  
Probe Analysis

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

scholarly journals Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

1984 ◽  
Vol 99 (3) ◽  
pp. 1024-1033 ◽  
Author(s):  
D P Kiehart ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Antigenic Site ◽  
Actin Binding ◽  
Filamentous Form ◽  
Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

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