Rhodopsin-egg phosphatidylcholine reconstitution by an octyl glucoside dilution procedure.
The transmembrane protein bovine rhodopsin was reconstituted with egg phosphatidylcholine (PC) by using a modified detergent dilution technique employing the nonionic detergent octyl-beta-D-glucoside (octyl glucoside). Using this technique, reconstituted membranes having molar phospholipid/protein ratios between 60:1 and 255:1 were prepared. This is in contrast to the results obtained when an octyl glucoside dialysis technique was employed (Jackson, M.L. and Litman, B.J. (1982) Biochemistry 21, 5601-5608). In the latter case, the highest molar phospholipid/protein ratio that could be obtained when reconstituting rhodopsin with egg PC was approximately 50:1. Reconstituted vesicles prepared by the octyl glucoside dilution technique were examined by negative stain and freeze-fracture electron microscopy, and it was found that the vesicles were unilamellar providing the molar PC/protein ratio was below about 200:1, whereas in preparations having ratios higher than this, a significant number of the vesicles were multilamellar. The mean vesicle diameter showed no trend based on the molar PC/protein ratio within the range of 82:1 to 186:1. The mean diameters of the preparations were between 520 and 850 A. Approximately equal numbers of protein particles were observed on the concave and convex fracture faces of the freeze-fracture micrographs of the reconstituted membranes which is indicative of a symmetric distribution of the protein across the bilayer.
D002499:Centrifugation, Density Gradient; D003956:Dialysis; D005614:Freeze Fracturing; D005960:Glucosides; D006027:Glycosides; D007201:Indicator Dilution Techniques; D008567:Membranes, Artificial; D008854:Microscopy, Electron; D010713:Phosphatidylcholines; D012168:Retinal Pigments; D012243:Rhodopsin; D013053:Spectrophotometry
D005960:Glucosides; D006027:Glycosides; D008567:Membranes, Artificial; D010713:Phosphatidylcholines; D012168:Retinal Pigments; C018619:octyl-beta-D-glucoside; D012243:Rhodopsin