Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca2+–ATPase as an example

2008 ◽  
Vol 3 (11) ◽  
pp. 1782-1795 ◽  
Author(s):  
Marc le Maire ◽  
Bertrand Arnou ◽  
Claus Olesen ◽  
Dominique Georgin ◽  
Christine Ebel ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Membrane Proteins ◽  
Analytical Ultracentrifugation ◽  
Gel Chromatography ◽  
Stokes Radius

scholarly journals Structural membrane proteins and loosely associated proteins of the sarcoplasmic reticulum

1974 ◽  
Vol 139 (3) ◽  
pp. 509-513 ◽  
Author(s):  
A. Margreth ◽  
U. Carraro ◽  
G. Salviati
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Membrane Proteins ◽  
Osmotic Shock ◽  
Protein Composition ◽  
Polyacrylamide Gels ◽  
Associated Proteins

The protein composition of sarcoplasmic-reticulum vesicles, either unpurified or after fractionation on sucrose gradients, and with or without previous osmotic shock and sonication, was investigated by electrophoresis in acid polyacrylamide gels. The pattern of release of loosely bound proteins is discussed with respect to their localization in the interior of the vesicles.

scholarly journals Evidence for a tubulin-containing lipid-protein structural complex in ciliary membranes.

1985 ◽  
Vol 100 (4) ◽  
pp. 1082-1090 ◽  
Author(s):  
R E Stephens
Keyword(s):  
Membrane Proteins ◽  
Critical Micelle Concentration ◽  
Analytical Ultracentrifugation ◽  
Lipid Fraction ◽  
Moderate Increase ◽  
Or Efficiency ◽  
Lipid Complex ◽  
Ciliary Membrane ◽  
Freeze Thaw ◽  
Membrane Tubulin

The proteins and lipids of the scallop gill ciliary membrane may be reassociated through several cycles of detergent solubilization, detergent removal, and freeze-thaw, without significant change in overall protein composition. Membrane proteins and lipids reassociate to form vesicles of uniform, discrete density classes under a variety of reassociation conditions involving detergent removal and concentration. Freed of the solubilizing detergent during equilibrium centrifugation, a protein-lipid complex equilibrates to a position on a sucrose density gradient characteristic of the original membrane density. When axonemal tubulin is solubilized by dialysis, mixed with 2:1 lecithin/cholesterol dissolved in Nonidet P-40, freed of detergent, and reconstituted by freeze-thaw, vesicles of a density essentially equal to pure lipid result. If the lipid fraction is derived through chloroform-methanol extraction of natural ciliary membranes, a moderate increase in density occurs upon reconstitution, but the protein is adsorbed and most is removed by a simple low ionic strength wash, in contrast to vesicles reconstituted from membrane proteins where even high salt extraction causes no loss of protein. The proteins of the ciliary membrane dissolve with constant composition, regardless of the type, concentration, or efficiency of detergent. Analytical ultracentrifugation demonstrates that monodisperse mixed micelles form at high detergent concentrations, but that membranes are dispersed to large sedimentable aggregates by Nonidet P-40 even at several times the critical micelle concentration, which suggests reasons for the efficacy of certain detergent for the production of ATP-reactivatable cell models. In extracts freed of detergent, structured polydisperse particles, but not membrane vesicles, are seen in negative staining; vesicles form upon concentration of the extract. Membrane tubulin is not in a form that will freely undergo electrophoresis, even in the presence of detergent above the critical micelle concentration. All chromatographic attempts to separate membrane tubulin from other membrane proteins have failed; lipid and protein are excluded together by gel filtration in the presence of high concentrations of detergent. These observations support the idea that a relatively stable lipid-protein complex exists in the ciliary membrane and that in this complex membrane tubulin is tightly associated with lipids and with a number of other proteins.

scholarly journals Cholesterol in sarcoplasmic reticulum and the physiological significance of membrane fluidity

1981 ◽  
Vol 196 (2) ◽  
pp. 505-511 ◽  
Author(s):  
A Johannsson ◽  
C A Keightley ◽  
G A Smith ◽  
J C Metcalfe
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Membrane Proteins ◽  
Atpase Activity ◽  
Membrane Fluidity ◽  
Operating Conditions ◽  
Phospholipid Bilayer ◽  
General Question ◽  
Rabbit Muscle ◽  
Large Decrease ◽  
Physiological Conditions

Vesicles of sarcoplasmic reticulum from rabbit muscle can be loaded with cholesterol to at least 20 mol% with respect to endogenous sarcoplasmic-reticulum phospholipid without effect on the ATPase activity at 32 degrees C. This applies both to sarcoplasmic-reticulum vesicles in which the ATPase activity is stably coupled to Ca2+ accumulation, and to sarcoplasmic-reticulum vesicles in which the sarcoplasmic-reticulum ATPase is activated severalfold by fully uncoupling the enzyme from net Ca2+ accumulation. Since the incorporation of cholesterol causes a large decrease in fluidity of sarcoplasmic-reticulum phospholipid bilayer, these results for sarcoplasmic reticulum raise the more general question of whether bilayer fluidity is important in modulating the function of membrane proteins under physiological conditions as is widely assumed, or whether the function of membrane proteins may be effectively buffered under normal operating conditions against changes in bilayer fluidity due to extraneous agents.

scholarly journals 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

1997 ◽  
Vol 325 (2) ◽  
pp. 533-542 ◽  
Author(s):  
Bannikuppe D. SHIVANNA ◽  
Elizabeth S. ROWE
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Membrane Proteins ◽  
Conformational Transition ◽  
Specific Activity ◽  
Structure And Function ◽  
Native Structure ◽  
Lipid Interactions ◽  
Lauryl Ether ◽  
And Function

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.

scholarly journals Local Synthesis of Sarcolemma and Sarcoplasmic Reticulum Membrane Proteins in Cardiac Myocytes

2021 ◽  
Vol 120 (3) ◽  
pp. 52a-53a
Author(s):  
Vladimir Bogdanov ◽  
andrew M. Soltisz ◽  
Marina Ivanova ◽  
Ivan Andreev ◽  
Galina Sakuta ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Membrane Proteins ◽  
Cardiac Myocytes ◽  
Local Synthesis ◽  
Sarcoplasmic Reticulum Membrane
Sign in / Sign up

Export Citation Format

Share Document