ISSN:
1432-1424
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Summary The extraction of rhodopsin-phospholipid complexes into lipid solvents is described. Two general procedures were developed: (i) the extraction of detergent-solubilized rhodopsin which involved: (a) solubilization of rhodopsin from bovine retinal rod disc membranes in detergent solutions; (b) partial removal of detergent from solubilized rhodopsin; (c) recombination with a defined lipid mixture by sonication of lipid and protein in salt media; and (d) sequential extraction into organic solvents. (ii) The direct transfer of rhodopsin from purified retinal rod disc membranes into apolar solvents which involved: (a) resuspension of membranes in hexane containing phospholipids; (b) sonication; (c) charge neutralization with salts; and (d) sequential extraction into organic solvents. The extraction yield was dependent on several parameters such as: phospholipid to rhodopsin ratio, sonication, type of solvent, and others. The composition of the extracts regarding the molar ratio of phospholipid, detergent, and rhodopsin varied according to the extraction protocol. The spectral characteristics of rhodopsin in the organic phase were similar to those of native rhodopsin in disc membranes. The capacity to regenerate the characteristic dark absorbance maximum at ∼485 nm after incubation of dark chromophore (9-cis retinal) with bleached rhodopsin was preserved to about 70%. The general applicability of these techniques in functional reconstitution studies of membrane systems is considered.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01869042
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