Reconstitution of sarcoplasmic reticulum Ca2+-ATPase with excess lipid dispersion of the pump units.
Sarcoplasmic reticulum Ca2+-ATPase has been reconstituted with excess lipid (25-150 g egg phosphatidylcholine per g sarcoplasmic reticulum protein) by a procedure combining the use of a non-ionic detergent with cholate dialysis. The reconstituted vesicles were analyzed by sucrose density fractionation and freeze-fracture electron microscopy. At the lowest lipid to protein ratios some vesicles containing aggregated protein were observed. At a lipid to protein ratio of 150:1 (w/w) only 30-40% of the reconstituted protein sedimented through 7% (w/v) sucrose. The remainder of the latter preparation was characterized by a high Ca2+-uptake capacity and a coupling ratio of 1.6 mol Ca2+ transported per mol ATP hydrolyzed. Intramembranous particles in this preparation occurred isolated in the membrane. In most cases only one particle could be seen on a fracture face. Cross-linking with cupric phenanthroline indicated that protein-protein contacts were drastically reduced by reconstitution. It is concluded that aggregation of intramembranous particles is not required for optimal Ca2+-transport function. The dispersed preparation obtained by a combined reconstitution and sucrose density fractionation procedure is useful for further characterization of the Ca2+ pump.
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D005614:Freeze Fracturing; D007700:Kinetics; D008081:Liposomes; D008563:Membrane Lipids / Q000502:physiology*; D008854:Microscopy, Electron; D010713:Phosphatidylcholines / Q000494:pharmacology*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D000252:Calcium-Transporting ATPases