title: Proof of oligomeric state of frog rhodopsin: visualization of dimer and oligomers on gels after BN- and HRCN-PAGE using antibodies to rhodopsin and by retinylopsin fluorescence.
abstract: Staining by antibodies to rhodopsin (Rh) and fluorescence of N-retinylopsin (RO) have shown that digitonin (DIG)- , dodecyl-β-D-maltoside (DM)- , and sodium dodecyl sulfate (SDS)-solubilized frog Rh after BN- and HRCN-PAGE is situated in the gradient gel in the state of dimer with a slight content of higher oligomers (trimer, tetramer, etc.). With increasing detergent harshness (DIG < DM < SDS), the proportion of higher oligomers in extracts becomes more prominent. Formation of RO in rod outer segments (ROS) in the presence of 0.7 M NaBH(3)CN at pH 5.0 occurs only when Rh is simultaneously photolyzed during reduction. Dithiothreitol at the concentration of 0.005 M failed to induce RO production. Formation of a stable C-N bond between all-trans-retinal and opsin in RO is accompanied by decrease in the dimer share and increase in the share of the higher oligomers due to secondary dissociation-aggregation of solubilized opsin. The position of the Rh dimer in relation to the anode during both native electrophoreses is determined not only by its molecular mass, but probably also depends on unfolding degree (or form): the harsher the detergent, the closer to the anode the dimer is located. Treatment of ROS by agents modifying the cholesterol component of lipid membrane (MβCD, filipin III, nystatin, saponin) did not change the character of Rh oligomerization, thus showing that integrity of the cholesterol component of photoreceptor membrane is not a crucial factor for oligomerization of opsin. It is supposed that the dimer-oligomer "portrait" of frog Rh, which has been found by two methods of native electrophoresis in three detergents with different degree of harshness, corresponds to a physiological state of this protein in native photoreceptor membrane.
authors.affiliation: Sechenov Institute for Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, 194223, Russia. tinycapers@mail.ru
authors.forename: S A
authors.initials: SA
authors.lastname: Shukolyukov
mesh_terms: D000818:Animals; D000906:Antibodies; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides / Q000378:metabolism; D010786:Photoreceptor Cells / Q000378:metabolism; D011896:Rana temporaria; D012243:Rhodopsin / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism
chemical_list: D000906:Antibodies; D005960:Glucosides; C040358:dodecyl maltoside; D012243:Rhodopsin