Reconstitution of the sarcoplasmic reticulum Ca2+-ATPase: mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents

Lévy, Daniel; Gulik, Annette; Bluzat, Aline; Rigaud, Jean‐Louis

doi:10.1016/0005-2736(92)90415-i

Cited by 122 publications

(102 citation statements)

References 46 publications

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“…The level of accumulation of Ca# + was identical with that observed for vesicles reconstituted by adding the solubilized SR to a preparation of pre-formed large unilamellar vesicles, using the procedure of Levy et al [25] (results not shown). As reported by Levy et al [19], we found that addition of FCCP to make the reconstituted vesicles permeable to H + led to increased levels of accumulation of Ca# + (because the Ca# + -ATPase acts as a Ca# + \H + -ATPase), and that addition of valinomycin had no significant effect in the presence of FCCP, showing that no significant membrane potential built up in the presence of FCCP.…”

Section: Resultssupporting

confidence: 76%

“…Reconstituted vesicles were prepared by a modification of the method described by Levy et al [25]. Phospholipids (24 mg) were suspended in buffer (4n6 ml ; 10 mM Pipes\100 mM K # SO % , pH 7n1), containing 40 mM octyl-β--glucoside and sonicated in a bath sonicator (Ultrawave Model U100) for about 5 min.…”

Section: Methodsmentioning

confidence: 99%

“…The solubilized SR was mixed with the lipid sample to give a 1 : 40 ratio of protein : lipid (w\w). Detergent was removed by addition of four aliquots of washed SM # Bio-Beads, as described by Levy et al [25], to give the final preparation of sealed vesicles.…”

Section: Methodsmentioning

confidence: 99%

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Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

Dalton

Pilot

Mall

et al. 1999

Biochemical Journal

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Accumulation of Ca2+ by the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum has been measured in reconstituted, sealed vesicles as a function of lipid composition. Measurements were performed in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to eliminate any effects of H+ transport; in the presence of FCCP, addition of valinomycin had no effect on the level or rate of accumulation of Ca2+ showing that, in the presence of FCCP, no electrical potential built up across the membrane. Levels of accumulation were low when the phospholipid was dioleoylphosphatidylcholine (DOPC), even though DOPC supports high ATPase activity. Inclusion of 10 mol% anionic phospholipid [dioleoylphosphatidic acid (DOPA) or dioleoylphosphatidylserine (DOPS)] led to higher levels of accumulation of Ca2+, 10 mol% being the optimum concentration. Cardiolipin or phosphatidylinositol 4-phosphate were more effective than DOPA or DOPS in increasing accumulation of Ca2+. Effects of anionic phospholipids were seen in the presence of an ATP-regenerating system to remove ADP, and in the presence of phosphate within the reconstituted vesicles to precipitate calcium phosphate. Rates of passive leak of Ca2+ from the reconstituted vesicles were slow. The Ca2+-accumulation process was simulated assuming either simple passive leak of Ca2+ from the vesicles or assuming slippage on the ATPase, a process in which the phosphorylated intermediate of the ATPase releases bound Ca2+ on the cytoplasmic rather than the lumenal side of the membrane. The experimental data fitted to a slippage model, with anionic phospholipids decreasing the rate of slippage.

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Section: Resultssupporting

confidence: 76%

Section: Methodsmentioning

confidence: 99%

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Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

Dalton

Pilot

Mall

et al. 1999

Biochemical Journal

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“…To elucidate fully the structure and function of Ca# + -ATPase, and particularly the role of lipid-protein interactions in the conformational changes that accompany ion translocation, it is necessary to isolate the protein and separate it from other membrane constituents. The most successful approach so far involves the use of detergents for solubilization [3][4][5][6][7][8][9][10] and for reconstitution into defined lipids [11][12][13][14][15][16][17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

“…Reconstitution of SR Ca# + -ATPase into defined lipids has been performed with several detergents, including cholate and deoxycholate [13][14][15], C "# E ) [15,20,22], Triton X-100 [15,23] and octylglucoside [15]. From these studies it is clear that the nature of the detergent used for reconstitution is a key factor in determining the properties of the reconstituted system.…”

Section: Introductionmentioning

confidence: 99%

Preservation of the native structure and function of Ca2+-ATPase from sarcoplasmic reticulum: Solubilization and reconstitution by new short-chain phospholipid detergent 1,2-diheptanoyl-sn-phosphatidylcholine

Shivanna

Rowe

1997

Biochemical Journal

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The properties of Ca2+-ATPase purified and reconstituted from rabbit skeletal sarcoplasmic reticulum (SR) has been studied in comparison with the preparations obtained by the commonly used detergent poly(oxyethylene)8-lauryl ether (C12E8) and the bile salt detergents cholate and deoxycholate. 1,2-Diheptanoyl-sn-phosphatidylcholine (DHPC) has been shown to be excellent for solubilizing a wide variety of membrane proteins [Kessi, Poiree, Wehrli, Bachofen, Semenza and Hauser (1994) Biochemistry 33, 10825–10836]. The DHPC method consistently gave higher yields of purified Ca2+-ATPase with a greater specific activity than the methods with C12E8, cholate, or deoxycholate. DHPC and C12E8 were superior to cholate and deoxycholate in active enzyme yields and specific activity. DHPC-solubilized Ca2+-ATPase purified on a density gradient retained the E1Ca–E1*Ca conformational transition, whereas the enzyme from the C12E8 purification did not retain this transition. The coupling of Ca2+ transported to ATP hydrolysed in the DHPC-purified enzyme was maximal and matched the values obtained with native SR, whereas the coupling was much lower for the C12E8-purified enzyme. The specific activity of Ca2+-ATPase reconstituted into dioleoylphosphatidylcholine vesicles with DHPC was up to 2-fold greater than that achieved with C12E8, and is comparable to that measured in the native SR. Finally, the dissociation of Ca2+-ATPase into monomers by DHPC preserved the ATPase activity, whereas similar dissociation by C12E8 gave only one-sixth the activity of that obtained with DHPC. These studies show that the Ca2+-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C12E8 in significant ways, making it a preparation suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of membrane proteins.

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Generation of proteoliposomes from subcellular fractions

et al. 1997

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Intracellular membranes are highly dynamic, yet they retain their identity and functional characteristics. Integral membrane proteins, which must confer this specific membrane identity, remain poorly characterized at the biochemical level, largely because detergent-mediated solubilization is required for purification and analysis, and several properties of integral membrane proteins can only be investigated when the molecule is properly embedded in a lipid bilayer. We present a method for the efficient reconstitution into proteoliposomes of integral membrane proteins from subcellular fractions. Integral membrane proteins were identified on high-resolution two-dimensional gels after selective extraction of soluble and peripheral membrane proteins; they accounted for 8% of the number of resolved polypeptides. A reconstitution procedure based on membrane solubilization with dodecyl-octaoxyethylene (C12E8) and subsequent detergent removal with BioBeads SM-2 resulted in the efficient reconstitution of several membrane proteins into proteoliposomes of uniform density. The generated proteoliposomes strongly resemble the starting membrane fraction in protein composition. This reconstitution allows the functional characterization of integral membrane proteins after enrichment and/or specific (immuno)depletion.

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Reconstitution of the sarcoplasmic reticulum Ca2+-ATPase: mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents

Cited by 122 publications

References 46 publications

Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

Anionic phospholipids decrease the rate of slippage on the Ca2+-ATPase of sarcoplasmic reticulum

Preservation of the native structure and function of Ca2+-ATPase from sarcoplasmic reticulum: Solubilization and reconstitution by new short-chain phospholipid detergent 1,2-diheptanoyl-sn-phosphatidylcholine

Generation of proteoliposomes from subcellular fractions

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