A new method of vesicle formation by salting-out and its application to the reconstitution of sarcoplasmic reticulum.
This paper describes a new method of forming membrane vesicles. It was found that the addition of salt such as KCl into a solution containing lipid (asolectin) and a non-ionic surfactant, Triton X-114, led to the formation of closed membrane vesicles. The vesicles were separated from Triton X-114 by hydrophobic interaction chromatography. Electron microscopy revealed that the mean diameter of the vesicles was 110 nm +/- 69 nm (S.D.). Measurement of osmotic volume change showed that the permeability of the vesicle was very low to salts, sugar (glucose) and amphoteric ion (glycine), but very high to glycerol, ethylene glycol and water. Vesicle formation by this 'salting-out' method is very useful for reconstitution of transport systems in biomembranes because of its advantages: completion within a short time; high yield; and the possibility of utilizing samples in non-ionic surfactant solution. When we applied the method to the reconstitution of sarcoplasmic reticulum, Ca2+-ATPase was incorporated into the reconstituted vesicles and was enzymatically active in the membrane.
D000818:Animals; D002458:Cell Fractionation; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D017830:Octoxynol; D009995:Osmosis; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000648:ultrastructure*
D011092:Polyethylene Glycols; D011189:Potassium Chloride; D017830:Octoxynol; C010615:Nonidet P-40