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 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 Unique kinetics of nicotinic acid–adenine dinucleotide phosphate (NAADP) binding enhance the sensitivity of NAADP receptors for their ligand

2000 ◽  
Vol 352 (3) ◽  
pp. 725-729 ◽  
Author(s):  
Sandip PATEL ◽  
Grant C. CHURCHILL ◽  
Antony GALIONE
Keyword(s):  
Nicotinic Acid ◽  
Sea Urchin ◽  
Binding Sites ◽  
Cell Types ◽  
Unique Property ◽  
Single Class ◽  
High Affinity ◽  
Sea Urchin Egg ◽  
Low Concentrations ◽  
Kinetics Of

Nicotinic acidŐadenine dinucleotide phosphate (NAADP) is a novel and potent Ca2+-mobilizing agent in sea urchin eggs and other cell types. Little is known, however, concerning the properties of the putative intracellular NAADP receptor. In the present study we have characterized NAADP binding sites in sea urchin egg homogenates. [32P]NAADP bound to a single class of high-affinity sites that were reversibly inhibited by NaCl but insensitive to pH and Ca2+. Binding of [32P]NAADP was lost in preparations that did not mobilize Ca2+ in response to NAADP, indicating that [32P]NAADP probably binds to a receptor mediating Ca2+ mobilization. Addition of excess unlabelled NAADP, at various times after initiation of [32P]NAADP binding, did not result in displacement of bound [32P]NAADP. These data show that NAADP becomes irreversibly bound to its receptor immediately upon association. Accordingly, incubation of homogenates with low concentrations of NAADP resulted in maximal labelling of NAADP binding sites. This unique property renders NAADP receptors exquisitely sensitive to their ligand, thereby allowing detection of minute changes in NAADP levels.

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.

scholarly journals Characterization of ruthenium red-binding sites of the Ca2+-ATPase from sarcoplasmic reticulum and their interaction with Ca2+-binding sites

1992 ◽  
Vol 287 (3) ◽  
pp. 767-774 ◽  
Author(s):  
S Corbalan-Garcia ◽  
J A Teruel ◽  
J C Gomez-Fernandez
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Binding Sites ◽  
Ruthenium Red ◽  
Cation Binding ◽  
Binding Studies ◽  
High Affinity ◽  
Cation Binding Site ◽  
Kinetics Of ◽  
Fast Release

Sarcoplasmic reticulum Ca(2+)-ATPase has previously been shown to bind and dissociate two Ca2+ ions in a sequential mode. This behaviour is confirmed here by inducing sequential Ca2+ dissociation with Ruthenium Red. Ruthenium Red binds to sarcoplasmic reticulum vesicles (6 nmol/mg) with a Kd = 2 microM, producing biphasic kinetics of Ca2+ dissociation from the Ca(2+)-ATPase, decreasing the affinity for Ca2+ binding. Studies on the effect of Ca2+ on Ruthenium Red binding indicate that Ruthenium Red does not bind to the high-affinity Ca(2+)-binding sites, as suggested by the following observations: (i) micromolar concentrations of Ca2+ do not significantly alter Ruthenium Red binding to the sarcoplasmic reticulum; (ii) quenching of the fluorescence of fluorescein 5′-isothiocyanate (FITC) bound to Ca(2+)-ATPase by Ruthenium Red (resembling Ruthenium Red binding) is not prevented by micromolar concentrations of Ca2+; (iii) quenching of FITC fluorescence by Ca2+ binding to the high-affinity sites is achieved even though Ruthenium Red is bound to the Ca(2+)-ATPase; and (iv) micromolar Ca2+ concentrations prevent inhibition of the ATP-hydrolytic capability by dicyclohexylcarbodi-imide modification, but Ruthenium Red does not. However, micromolar concentrations of lanthanides (La3+ and Tb3+) and millimolar concentrations of bivalent cations (Ca2+ and Mg2+) inhibit Ruthenium Red binding as well as quenching of FITC-labelled Ca(2+)-ATPase fluorescence by Ruthenium Red. Studies of Ruthenium Red binding to tryptic fragments of Ca(2+)-ATPase, as demonstrated by ligand blotting, indicate that Ruthenium Red does not bind to the A1 subfragment. Our observations suggest that Ruthenium Red might bind to a cation-binding site in Ca(2+)-ATPase inducing fast release of the last bound Ca2+ by interactions between the sites.

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 Kinetics of binding, endocytosis, and recycling of EGF receptor mutants

1992 ◽  
Vol 117 (1) ◽  
pp. 203-212 ◽  
Author(s):  
S Felder ◽  
J LaVin ◽  
A Ullrich ◽  
J Schlessinger
Keyword(s):  
Point Mutation ◽  
Phorbol Ester ◽  
Egf Receptor ◽  
Receptor Internalization ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Low Concentrations ◽  
Internalization Rate ◽  
Kinetics Of

This report describes analysis of factors which regulate the binding of EGF to EGF receptor, receptor internalization, and receptor recycling. Three different methods were used to inhibit high-affinity EGF binding as measured at equilibrium: treatment of cells with an active phorbol ester (PMA), binding of a mAb directed against the EGF receptor (mAb108), and truncation of most of the cytoplasmic domain of the receptor. These treatments reduced the rate at which low concentrations of EGF bound to cells, but did not affect the rate of EGF dissociation. We conclude that high-affinity EGF binding on living cells results from a difference in the apparent on rate of EGF binding. We then used these conditions and cell lines to test for the rate of EGF internalization at different concentrations of EGF. We demonstrate that internalization of the EGF receptor is stimulated roughly 50-fold at saturating concentrations of EGF, but is stimulated an additional two- to threefold at low concentrations (less than 1 nM). Four treatments reduce the rate of internalization of low concentrations of EGF to the rate seen at saturating EGF concentrations. Phorbol ester treatment and mAb108 binding to "wild type" receptor reduce this rate (and reduce high-affinity binding). Point mutation at Lys721 (kinase negative EGF receptor) and point mutation at Thr654 (removing a major site of protein kinase C phosphorylation) reduce the internalization rate, without affecting high-affinity binding. We suggest that while EGF stimulates endocytosis for all receptors, high-affinity receptors bind and are internalized more quickly than low-affinity receptors. Tyrosine kinase activity and the Thr654 region appear necessary for this response.

scholarly journals Galactoside-proton symport in a lacYun mutant of Escherichia coli investigated by analysis of transport progress curves

1987 ◽  
Vol 242 (2) ◽  
pp. 539-550 ◽  
Author(s):  
M G Page
Keyword(s):  
Binding Sites ◽  
Type Strain ◽  
Wild Type Strain ◽  
Initial Rate ◽  
Ph Dependence ◽  
Initial Period ◽  
Wild Type ◽  
Proton Symport ◽  
Internal Concentration ◽  
Kinetics Of

The kinetics of galactoside-proton symport catalysed by a wild-type strain and one carrying a mutation, previously reported to cause uncoupling of the symport reaction, have been examined. The mutation does not affect the stoichiometry during the initial period of uptake, when the internal concentration of galactoside is low, but it does result in much greater competition from the galactoside as it is accumulated. Simple methods for the analysis of the uptake progress curves have been developed and used to estimate the initial rate of uptake and affinity for internal galactoside. The maximum rate of uptake is decreased by a factor of 2 at most whereas the affinity for internal galactoside is increased up to 50-fold by the mutation. The pH-dependence of the galactoside efflux reaction is changed in a manner which suggests that the defect is in the interaction between proton-binding and galactoside-binding sites rather than in the structure of either site.

scholarly journals The effects of membrane potential and lanthanides on the conformation of the Ca2+-transport ATPase in sarcoplasmic reticulum

1986 ◽  
Vol 234 (2) ◽  
pp. 363-371 ◽  
Author(s):  
I Jona ◽  
A Martonosi
Keyword(s):  
Membrane Potential ◽  
Sarcoplasmic Reticulum ◽  
Fluorescence Intensity ◽  
Binding Sites ◽  
Fluorescein Isothiocyanate ◽  
Tryptophan Fluorescence ◽  
Lanthanide Ions ◽  
Positive Potential ◽  
High Affinity ◽  
Ion Substitution

The effects of Ca2+, lanthanide ions (Gd3+, La3+ and Pr3+) and membrane potential on the fluorescence of tryptophan and covalently bound fluorescein were analysed in native and fluorescein isothiocyanate (FITC)-labelled sarcoplasmic reticulum vesicles. The binding of Ca2+ and lanthanides to the Ca2+-ATPase increases the fluorescence intensity of tryptophan and decreases the fluorescence intensity of FITC; the dependence of these effects on cation concentration is consistent with the involvement of the high-affinity Ca2+-binding sites of the Ca2+-ATPase in the cation-induced fluorescence changes. The fluorescence of FITC-labelled sarcoplasmic reticulum vesicles is also influenced by membrane potential changes induced by ion substitution. Inside positive potential increases, while inside negative potential decreases, the fluorescence of bound FITC. Smaller potential-dependent changes in tryptophan fluorescence were also observed. The effects of Ca2+, lanthanides and membrane potential on the fluorescence of tryptophan and FITC are discussed in terms of the two major conformations of the Ca2+-ATPase (E1 and E2), that are assumed to alternate during Ca2+ transport. The observations support the suggestion [Dux, Taylor, Ting-Beall & Martonosi (1985) J. Biol. Chem. 260, 11730-11743] that the vanadate-induced crystals of Ca2+-ATPase represent the E2, while the Ca2+ and lanthanide-induced crystals the E1, conformation of the enzyme.

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