Self‐association of plasma membrane Ca<sup>2+</sup>‐ATPase by volume exclusion

Kosk‐Kosicka, Danuta; López, Marı́a M.; Fomitcheva, Ioulia; Lew, Virgilio L.

doi:10.1016/0014-5793(95)00870-f

Cited by 8 publications

(5 citation statements)

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“…We have previously demonstrated that dextran induces oligomerization of enzyme molecules at concentrations that are too low (beginning with 6 nM enzyme) for significant self-association under standard conditions (22). At present, we have shown that at insufficient Ca 2ϩ even dextran cannot force the enzyme to dimerize.…”

Section: Ca 2ϩ -Atpase Is a Dimer 9990mentioning

confidence: 81%

“…Dextran was used because its addition to the Ca 2ϩ -ATPase at 15 nM concentration (at which concentration the Ca 2ϩ -ATPase requires calmodulin for activation, suggesting that it is monomeric) induced self-association of the enzyme as judged by fluorescence resonance energy transfer experiments (22), and thus led to full, calmodulin-independent activity (Table II, set 4). Dextran had no effect on the 50 nM (dimeric) enzyme (Table II, Tables II and III), and centrifuged to equilibrium in the model E (A) or the XLA ultracentrifuge (B).…”

Section: Resultsmentioning

confidence: 99%

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The Active Species of Plasma Membrane Ca2+-ATPase Are a Dimer and a Monomer-Calmodulin Complex

Sackett

Kosk‐Kosicka

1996

Journal of Biological Chemistry

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We have previously shown that the Ca 2؉ -ATPase activity of the oligomeric enzyme is independent of calmodulin, in contrast to another active enzyme species, a presumable monomer, that is activated by calmodulin binding. Presently, we have succeeded in determining the molecular mass of the two active enzyme species by equilibrium ultracentrifugation. For the calmodulin-dependent species, the molecular mass is 170 ؎ 30 kDa, which is consistent with predominantly monomeric Ca 2؉ -ATPase with bound calmodulin. The molecular mass of calmodulin-independent oligomers is 260 ؎ 34 kDa, indicating that they are dimers. Results of experiments performed under different calcium and potassium concentrations and in the presence of dextran that causes molecular crowding verify a strict Ca 2؉ requirement of the dimerization process. We conclude that the active species of the Ca 2؉

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Section: Ca 2ϩ -Atpase Is a Dimer 9990mentioning

confidence: 81%

Section: Resultsmentioning

confidence: 99%

The Active Species of Plasma Membrane Ca2+-ATPase Are a Dimer and a Monomer-Calmodulin Complex

Sackett

Kosk‐Kosicka

1996

Journal of Biological Chemistry

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“…In the case of an ''excluded crowder'' with a large, negative preferential binding coefficient, the volume of exclusion is actually larger in the dissociated state than in the encounter complex or associated state. Thus, in stark contrast to neutral crowders, excluded crowders like sugars, polyols, and large, hydrophilic polymers favor association (Linder and Ralston, 1995;Kosk-Kosicka et al, 1995;Nichol et al, 1981).…”

Section: Figurementioning

confidence: 99%

Rational Design of Solution Additives for the Prevention of Protein Aggregation

Baynes

Trout

2004

Biophysical Journal

104

102

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We have developed a statistical-mechanical model of the effect of solution additives on protein association reactions. This model incorporates solvent radial distribution functions obtained from all-atom molecular dynamics simulations of particular proteins into simple models of protein interactions. In this way, the effects of additives can be computed along the entire association/dissociation reaction coordinate. We used the model to test our hypothesis that a class of large solution additives, which we term "neutral crowders," can slow protein association and dissociation by being preferentially excluded from protein-protein encounter complexes, in a manner analogous to osmotic stress. The magnitude of this proposed "gap effect" was probed for two simple model systems: the association of two spheres and the association of two planes. Our results suggest that for a protein of 20 A radius, an 8 A additive can increase the free energy barrier for association and dissociation by as much as 3-6 kcal/mol. Because the proposed gap effect is present only for reactions involving multiple molecules, it can be exploited to develop novel additives that affect protein association reactions although having little or no effect on unimolecular reactions such as protein folding. This idea has many potential applications in areas such as the stabilization of proteins against aggregation during folding and in pharmaceutical formulations.

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“…6 was designed to determine whether the monomers in the presence of VA were able to re‐associate. We have taken advantage of our discovery that dextran elicits self‐association of enzyme monomers [19]. In the presence of 3 m m isoflurane, the FRET of 8 n m enzyme decreased from 28 ± 6% to 4 ± 4% (compare Fig.…”

Section: Resultsmentioning

confidence: 99%

Effects of volatile anesthetic on the Ca²⁺‐ATPase activation by dimerization

López

Zelent

Kosk‐Kosicka

2000

European Journal of Biochemistry

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The phenomenological distance-dependent quenching (DDQ) model was employed to investigate the character of the interaction between volatile anesthetics (VAs) and the plasma membrane Ca 21 -ATPase (PMCA). The simultaneous analysis of the frequency-domain and steady-state data of tryptophan (Trp) fluorescence quenching by a VA points to a specific character of the apparent quenching effect of the VA, possibly arising from a significant contribution of static quenching. The apparent contributions of both static and dynamic quenching may be due to VA binding in the PMCA, which results in the modification of the conformational substates of the enzyme. To characterize further the molecular consequences of VA binding, we investigated its effects on the process of PMCA activation by self-association. VA shifted the equilibrium from enzyme dimers to monomers, as monitored by the loss of fluorescence energy transfer. The shift was apparently due to the VA-induced decrease in the affinity of PMCA molecules for self-association. Addition of a large molecular mass dextran to increase the proximity between enzyme monomers induced re-association of the VA-impaired PMCA, while the Ca 21 -ATPase activity was not recovered. The results are congruent with a dual VA effect on PMCA, a shift in the monomer/dimer equilibrium, and an inactivation of both monomers and dimers. -induced conformational change that PMCA undergoes in the initial step of its enzymatic cycle and the decrease in the intrinsic Trp fluorescence [4,5]. We have proposed that, upon binding to PMCA, the VAs disturb the sequence of conformational changes the enzyme normally undergoes in the process of catalytic activation, thus resulting in the inhibition of Ca 21 -ATPase activity [4,6]. Recently, we have shown that quenching of the total PMCA-intrinsic Trp fluorescence by halothane is dose-dependent, is saturable, and can be fitted to a binding curve [7].In this study we used two approaches to investigate the VA±PMCA interactions and their effects on enzyme activation. First, we employed the exponential distance-dependent quenching (DDQ) model (with parameters bimolecular rate constant of quenching k a , diffusion D and distances a and r e ) to analyze the Trp fluorescence quenching of PMCA by VA, which results in the loss of Ca 21 -ATPase activity. The DDQ model was developed to explain experimental frequency-domain and steady-state intensity decay data for which not only collisional but also through-space quenching was expected [8]. In using the DDQ model for our system we expanded our earlier spectroscopic studies [4] in which several characteristics of steady-state and preliminary frequency-domain data suggested that the PMCA±VA interaction is not just the result of mere collisional contacts. The frequency-domain data have been analyzed in more detail (see Materials and methods). Steadystate and frequency-domain Trp fluorescence intensity decays of the dimeric enzyme were measured under comparable experimental conditions at up to 5 mm isoflurane concentrations. S...

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Self‐association of plasma membrane Ca²⁺‐ATPase by volume exclusion

Cited by 8 publications

References 20 publications

The Active Species of Plasma Membrane Ca2+-ATPase Are a Dimer and a Monomer-Calmodulin Complex

The Active Species of Plasma Membrane Ca2+-ATPase Are a Dimer and a Monomer-Calmodulin Complex

Rational Design of Solution Additives for the Prevention of Protein Aggregation

Effects of volatile anesthetic on the Ca²⁺‐ATPase activation by dimerization

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Self‐association of plasma membrane Ca2+‐ATPase by volume exclusion

Cited by 8 publications

References 20 publications

The Active Species of Plasma Membrane Ca2+-ATPase Are a Dimer and a Monomer-Calmodulin Complex

The Active Species of Plasma Membrane Ca2+-ATPase Are a Dimer and a Monomer-Calmodulin Complex

Rational Design of Solution Additives for the Prevention of Protein Aggregation

Effects of volatile anesthetic on the Ca2+‐ATPase activation by dimerization

Contact Info

Product

Resources

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Self‐association of plasma membrane Ca²⁺‐ATPase by volume exclusion

Effects of volatile anesthetic on the Ca²⁺‐ATPase activation by dimerization