Attempts to induce asymmetrical reconstitution of the sarcoplasmic reticulum calcium transporting system

Brèthes, Daniel; Avéret, Nicole; Gulik-Krzywicki, T.; Chevallier, Jean

doi:10.1016/0003-9861(81)90175-2

Cited by 14 publications

(2 citation statements)

References 28 publications

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“…There has been a long history of reconstitution as a method for studying the functional properties of the SR Ca 2+ -ATPases (25)(26)(27). Reconstitution has proven to be an effective mimic of native SR membranes, where pure Ca 2+ -ATPase and lipids (EYPC and EYPA) are reincorporated into membrane vesicles at low lipid-to-protein ratios (13).…”

Section: Resultsmentioning

confidence: 99%

The Effects of Mutation on the Regulatory Properties of Phospholamban in Co-Reconstituted Membranes

et al. 2005

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Reconstitution into proteoliposomes is a powerful method for studying calcium transport in a chemically pure membrane environment. By use of this approach, we have studied the regulation of Ca(2+)-ATPase by phospholamban (PLB) as a function of calcium concentration and PLB mutation. Co-reconstitution of PLB and Ca(2+)-ATPase revealed the expected effects of PLB on the apparent calcium affinity of Ca(2+)-ATPase (K(Ca)) and unexpected effects of PLB on maximal activity (V(max)). Wild-type PLB, six loss-of-function mutants (L7A, R9E, I12A, N34A, I38A, L42A), and three gain-of-function mutants (N27A, L37A, and I40A) were evaluated for their effects on K(Ca) and V(max). With the loss-of-function mutants, their ability to shift K(Ca) correlated with their ability to increase V(max). A total loss-of-function mutant, N34A, had no effect on K(Ca) of the calcium pump and produced only a marginal increase in V(max). A near-wild-type mutant, I12A, significantly altered both K(Ca) and V(max) of the calcium pump. With the gain-of-function mutants, their ability to shift K(Ca) did not correlate with their ability to increase V(max). The "super-shifting" mutants N27A, L37A, and I40A produced a large shift in K(Ca) of the calcium pump; however, L37A decreased V(max), while N27A and I40A increased V(max). For wild-type PLB, phosphorylation completely reversed the effect on K(Ca), but had no effect on V(max). We conclude that PLB increases V(max) of Ca(2+)-ATPase, and that the magnitude of this effect is sensitive to mutation. The mutation sensitivity of PLB Asn(34) and Leu(37) identifies a region of the protein that is responsible for this regulatory property.

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Section: Resultsmentioning

confidence: 99%

The Effects of Mutation on the Regulatory Properties of Phospholamban in Co-Reconstituted Membranes

et al. 2005

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“…[19] also supports the existence of a regular arrangem ent of the ATPase in native membranes. The reduction of excimers is best explained by the fact that the ATPase m ole cules were symmetrically incorporated in the re constituted vesicles [30][31][32][33]. The reconstitution ex periment excluded that the small am ounts of pyrenemaleinimide bound to other proteins (Fig.…”

Section: Discussionmentioning

confidence: 99%

Fluorescence Studies on N-(3-Pyrene)Maleinimide-Labeled Sarcoplasmic Reticulum ATPase in Native and Solubilized Membranes

Lüdi

Hasselbach

1982

Zeitschrift Für Naturforschung C

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Fluorescence polarization and formation of excimers were studied in N-(3-pyrene)maleinimide-labeled sarcoplasmic reticulum vesicles.1. The polarization of pyrenemaleinimide labeled vesicles does not change with temperature and shows a pronounced decrease at labeling concentrations larger than 1 mol pyrenemaleinimide per 10 mol ATPase.2. Solubilization of the membrane with myristoylglycerophosphocholine renders the polariza tion temperature dependent, but does not affect the concentration dependent depolarization observed in native vesicles.3. The polarization of labeled vesicles is much smaller than to be expected from the tempera ture independent polarization indicating that the pyrenemaleinimide polarization did not monitor the rotation of the entire ATPase. Thus segmental motion occurs.4. Pyrene excimers are observed at label concentrations larger than 1 mol label per 2.5 mol ATPase.5. The amount of excimers was critically dependent on added detergents. From the fact that non-solubilizing amounts of myristoylglycerophosphocholine strongly reduced the amount of pyrene excimers it is concluded that in the native sarcoplasmic reticulum vesicles at least two ATPase molecules must be in close contact.

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Freeze-Etching Electron Microscopy: Recent Developments and Application to the Study of Biological Membranes and Their Components

Gulik-Krzywicki

1985

Physical Methods on Biological Membranes and Their Model Systems

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Attempts to induce asymmetrical reconstitution of the sarcoplasmic reticulum calcium transporting system

Cited by 14 publications

References 28 publications

The Effects of Mutation on the Regulatory Properties of Phospholamban in Co-Reconstituted Membranes

The Effects of Mutation on the Regulatory Properties of Phospholamban in Co-Reconstituted Membranes

Fluorescence Studies on N-(3-Pyrene)Maleinimide-Labeled Sarcoplasmic Reticulum ATPase in Native and Solubilized Membranes

Freeze-Etching Electron Microscopy: Recent Developments and Application to the Study of Biological Membranes and Their Components

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