Purification of the CaATPase of sarcoplasmic reticulum by affinity chromatography.

Coll, R J; Murphy, Alexander J.

doi:10.1016/s0021-9258(18)89885-4

Cited by 87 publications

(25 citation statements)

References 48 publications

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“…However, the excited-state lifetime of the FITC-CaATPase is 4.0 ns (Highsmith, 1986), so the increase in the rate of rotational Brownian motion of the CaATPase upon solubilization does not affect the polarization detected with FITC. The small -log CC12 , ] added difference between the polarization of the fully labeled detergent-solubilized CaATPase and that of the minimally labeled vesicular CaATPase, for which the probability of FITC-FITC neighbors is small, supports the conclusion that high concentrations of ¿12 $ monomerize the enzyme (Murphy et al, 1982; Coll & Murphy, 1984). The data in Figures 1 and 2 suggest that no cooperative binding of FITC is occurring and that C12E9 does not change the excited-state lifetime of the attached FITC.…”

Section: Resultsmentioning

confidence: 61%

“…pH 7.7, with 0.1 mM CaCl2 or 1 mM EGTA present, and at 0 °C, pH 7.7, with 0.1 mM CaCl2 present, in the labeling incubation mixture. Pick and others have shown that FITC binds at or near the ATPase binding site specifically and stoichiometrically (Pick & Bassilian, 1981;Highsmith, 1984;Coll & Murphy, 1984). Recently, its site of attachment has been assigned to Lys-514 (MacLennan et al, 1985).…”

Section: Resultsmentioning

confidence: 99%

“…Less clear is the role that aggregation of CaATPase monomers into oligomers may have in Ca2+ transport. The SR membrane is rich in Ca-ATPase, about 2:1 protein to lipid by weight (Stewart & MacLennan, 1974), with much of the protein being CaATPase (Meissner et al, 1973;Coll & Murphy, 1984;Barrabin et al, 1984), so there is little doubt that the CaATPase molecules are near one another. However, whether there are interactions between CaATPase monomers-either monomer-monomer binding in preferred spatial orientations or functional intermolecular interactions-has not been established.Several lines of physical evidence indicate that there is, at least, association between monomeric subunits.…”

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confidence: 99%

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Spatial organization of calcium ATPase molecules in sarcoplasmic reticulum vesicles

Highsmith¹,

Cohen²

1987

Biochemistry

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Fluorescence intensity, polarization, and (Ca2+-Mg2+)-ATPase (CaATPase) activity were measured for sarcoplasmic reticulum (SR) CaATPase with varying amounts of fluorescein isothiocyanate (FITC) attached at a specific site at or near the ATP binding site. The stoichiometry of attached FITC was proportional to the inhibition of ATPase activity, consistent with the independent labeling of one FITC site per CaATPase molecule. Polarization measurements on vesicular CaATPase indicated the occurrence of energy-transfer depolarization that increased as the fraction of binding sites labeled by FITC increased. Addition of the nonionic detergent dodecyl nonaoxyethylene alcohol (C12E9) eliminated the energy-transfer depolarization for all degrees of labeling with little direct effect on the attached FITC molecule. Fluorescence polarization measurements on sizing-column-purified FITC-labeled CaATPase in the presence of 30 mM C12E9 indicated that the sample consisted of homogeneous monomeric CaATPase. The attached FITC molecule was not sensitive to the bulk viscosity for either the vesicular or the detergent-solubilized CaATPase. The midpoints of the transition from vesicular to monomeric CaATPase as a function of increasing detergent concentration were determined from fluorescence polarization and light-scattering measurements. The dependence of these midpoints on the CaATPase concentration indicated a stoichiometry of 262 +/- 35 molecules of C12E9 per CaATPase in the detergent-protein complex. Both measurements gave the same result. The decrease of fluorescence polarization with increasing saturation of the FITC binding sites for vesicular and detergent-solubilized CaATPase was analyzed in terms of energy-transfer depolarization to determine the spatial arrangements of CaATPase molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 61%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Spatial organization of calcium ATPase molecules in sarcoplasmic reticulum vesicles

Highsmith¹,

Cohen²

1987

Biochemistry

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“…In the simulations, an importantparameteris [EPImax.,themaximum observable level of phosphorylation. This is generally unknown, and studies have suggested that in many SR preparations active ATPase may constitute only 50% of the total protein (Gafni & Boyer, 1984;Coll & Murphy, 1984). As discussed previously (Martin & Tanford, 1981;de Meis et al, 1982;Inesi et al, 1984), this makes less certain the determination of the equilibrium constant for phosphorylation.…”

Section: Discussionmentioning

confidence: 98%

A model for the phosphorylation of the Ca2+ + Mg2+-activated ATPase by phosphate

Froud¹,

Lee²

1986

Biochemical Journal

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We have shown that changes in fluorescence intensity for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate following the addition of Ca2+ can give the ratio of the two conformations (E1 and E2) of the ATPase. We show that the fluorescence response to Ca2+ is unaffected by Mg2+, phosphate or K+, implying that these ions bind equally well to the E1 and E2 conformations. A model is presented for phosphorylation of the ATPase by phosphate as a function of pH, Mg2+, K+ and Ca2+.

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“…SR vesicles were prepared from rabbit fast-twitch muscle (40), then resuspended to 25 mg/mL total protein in SR buffer (30 mM 3-(n-morpholino) propanesulfonic acid (MOPS), 0.3 M sucrose, pH 7.0). SERCA was purified by reactive red chromatography (41), omitting reducing agents. For spin labeling, SERCA was diluted to 10 mg/mL with SR buffer, to which 300 mM MSL was added from a 50 mM stock in dimethylformamide.…”

Section: Serca Purification and Spin Labelingmentioning

confidence: 99%

Protein-Protein Interactions in Calcium Transport Regulation Probed by Saturation Transfer Electron Paramagnetic Resonance

James¹,

McCaffrey²,

Torgersen

et al. 2012

Biophysical Journal

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We have used electron paramagnetic resonance (EPR) to probe the homo- and heterooligomeric interactions of reconstituted sarcoplasmic reticulum Ca-ATPase (SERCA) and its regulator phospholamban (PLB). SERCA is responsible for restoring calcium to the sarcoplasmic reticulum to allow muscle relaxation, whereas PLB inhibits cardiac SERCA unless phosphorylated at Ser(16). To determine whether changes in protein association play essential roles in regulation, we detected the microsecond rotational diffusion of both proteins using saturation transfer EPR. Peptide synthesis was used to create a fully functional and monomeric PLB mutant with a spin label rigidly coupled to the backbone of the transmembrane helix, while SERCA was reacted with a Cys-specific spin label. Saturation transfer EPR revealed that sufficiently high lipid/protein ratios minimized self-association for both proteins. Under these dilute conditions, labeled PLB was substantially immobilized after co-reconstitution with unlabeled SERCA, reflecting their association to form the regulatory complex. Ser(16) phosphorylation slightly increased this immobilization. Complementary measurements with labeled SERCA showed no change in mobility after co-reconstitution with unlabeled PLB, regardless of its phosphorylation state. We conclude that phosphorylating monomeric PLB can relieve SERCA inhibition without changes in the oligomeric states of these proteins, indicating a structural rearrangement within the heterodimeric regulatory complex.

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Purification of the CaATPase of sarcoplasmic reticulum by affinity chromatography.

Cited by 87 publications

References 48 publications

Spatial organization of calcium ATPase molecules in sarcoplasmic reticulum vesicles

Spatial organization of calcium ATPase molecules in sarcoplasmic reticulum vesicles

A model for the phosphorylation of the Ca2+ + Mg2+-activated ATPase by phosphate

Protein-Protein Interactions in Calcium Transport Regulation Probed by Saturation Transfer Electron Paramagnetic Resonance

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