Enzyme kinetics and substrate stabilization of detergent-solubilized and membraneous (Ca2+ + Mg2+)-activated ATPase from sarcoplasmic reticulum. Effect of protein-protein interactions.

Møller, Jesper; Lind, Kirsten E.; Andersen, Johannes Lund

doi:10.1016/s0021-9258(19)85969-0

Cited by 242 publications

(80 citation statements)

References 35 publications

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“…Multiple studies since the 1970s have provided evidence for the assembly of skeletal isoform SERCA1a in homooligomeric complexes (9)(10)(11)(12). The functional significance of this was unclear, but early studies suggested SERCA-SERCA interactions altered SERCA responsiveness to Mg (12) or ATP (9). Some recent functional studies correlated oligomerization/aggregation with decreased SERCA catalytic activity (13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer

Blackwell

Zak

Robia

2016

Biophysical Journal

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The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation.

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Section: Introductionmentioning

confidence: 99%

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer

Blackwell

Zak

Robia

2016

Biophysical Journal

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“…The architecture of the NKA regulatory complex Previous studies employing diverse experimental approaches have provided evidence of  subunit oligomerization in the NKA regulatory complex (Boldyrev, 2001;Blackwell et al, 2016;Song et al, 2014a;Singh, R. B., and Dhalla, 2010;Moller et al, 1980;Vanderkooi et al, 1977;Clarke & Kane, 2007). Here, we explored the binding affinity between NKA subunits and investigated the stoichiometry of the NKA regulatory complex.…”

Section: Discussionmentioning

confidence: 99%

Stoichiometry of the Sodium Pump-Phospholemman Regulatory Complex

Šeflová

Habibi

Yap

et al. 2021

Preprint

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The sodium-potassium ATPase (NKA) establishes ion gradients that facilitate many physiological processes. In the heart, NKA activity is regulated by its interaction with phospholemman (PLM, FXYD1). Here we used a novel fluorescence lifetime-based assay to investigate the structure, stoichiometry, and affinity of the NKA-PLM regulatory complex. We observed concentration dependent association of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit followed by lower affinity alpha-alpha and alpha-PLM interactions. The data provide the first evidence that the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits in intact cells. Docking and molecular dynamics simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with stoichiometry (alpha-beta-PLM)2.

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“…Many proteins depend on Ca 2+ and Mg 2+ for their function. Some proteins are sensitive to the specific free Ca 2+ concentration present, where too high concentrations are inactivating or desensitizing, 26,27 while complete absence of free Ca 2+ can lead to irreversible inactivation. 26,28 Polyanionic copolymers such as AASTY are chelators of Ca 2+ through their carboxylate anions.…”

Section: Acrylic Acid Content Governs Divalent Cation Sensitivitymentioning

confidence: 99%

“…Some proteins are sensitive to the specific free Ca 2+ concentration present, where too high concentrations are inactivating or desensitizing, 26,27 while complete absence of free Ca 2+ can lead to irreversible inactivation. 26,28 Polyanionic copolymers such as AASTY are chelators of Ca 2+ through their carboxylate anions. Consequently, the Ca 2+ concentration that permits native nanodisc formation and stability is not necessarily equivalent to the amount of free Ca 2+ ions required for a Ca 2+ dependent protein to function, as the copolymer and protein are competing for Ca 2+ ions.…”

Section: Acrylic Acid Content Governs Divalent Cation Sensitivitymentioning

confidence: 99%

“…Some proteins require a constant presence of calcium and magnesium to retain activity, so adding back the divalent cations after purification might not be sufficient. 26 Therefore, we next sought to characterize which CaCl 2 and MgCl 2 concentrations would be permissible during nanodisc formation with the four AASTY copolymers and SMA2000 (Figure 3B,C and Supp. Figure S5).…”

Section: Formation Of Nanodiscs In the Presence Of Divalent Cationsmentioning

confidence: 99%

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Characterization of divalent cation interactions with AASTY native nanodiscs

Timcenko

Autzen

2021

Preprint

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Amphiphilic copolymers show great promise in extracting membrane proteins directly from lipid bilayers into 'native nanodiscs'. However, many such copolymers are polyanionic and sensitive to divalent cations, limiting their applicability towards Ca2+ or Mg2+ dependent proteins. Here, we characterize the Ca2+ and Mg2+ sensitivity of poly(acrylic acid-co-styrene) (AASTY) copolymers using analytical UV and fluorescent size exclusion chromatography, enabling us to separate signals from nanodiscs, copolymers, and soluble aggregates. Determination of free Ca2+ ion concentrations in the presence of copolymer shows that divalent cation tolerance is dependent on not only specific characteristics of a copolymer, but also on its concentration. We see that high ionic strength protects against aggregation facilitated by divalent cations, which is prominent in nanodiscs isolated from excess free copolymer through dialysis. Overall, we conclude that the behavior of amphiphilic copolymers in the presence of divalent cations is more complex than precipitation beyond a specific cation concentration.

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Enzyme kinetics and substrate stabilization of detergent-solubilized and membraneous (Ca2+ + Mg2+)-activated ATPase from sarcoplasmic reticulum. Effect of protein-protein interactions.

Cited by 242 publications

References 35 publications

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer

Stoichiometry of the Sodium Pump-Phospholemman Regulatory Complex

Characterization of divalent cation interactions with AASTY native nanodiscs

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