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Biochemistry (Moscow)

, Volume 74, Issue 6, pp 599–604 | Cite as

Aggregation of frog rhodopsin to oligomers and their dissociation to monomer: Application of BN- and SDS-PAGE

  • S. A. ShukolyukovEmail author
Accelerated Publication

Abstract

After solubilization of frog rod outer segments (ROS) with mild detergents (digitonin, n-dodecyl-β-D-maltoside, Chaps, Triton X-100) and subsequent one-dimensional blue native polyacrylamide gel electrophoresis (1D BN-PAGE), the position of rhodopsin (Rh) on the gradient gel does not match the monomer with molecular weight of 40 kDa but appears self-associated into aggregate of Rh (RhA) with molecular mass varying in different detergents from 85 to 125 kDa. Short-term treatment (∼2 h) of the excised BN-PAGE strip containing RhA by denaturing detergent mixture (10% SDS + 1 mM dithiothreitol (DTT)) followed by 2D SDS-PAGE revealed dissociation of the RhA into opsin monomer and unidentified proteins. Long-term treatment (∼2 days) of RhA that included extraction, denaturation, concentration, and electrophoresis induced, along with dissociation of RhA into opsin monomer + unidentified proteins, also formation of opsin dimers, trimers, and higher oligomers owing to a secondary aggregation of opsin. Direct solubilization of the ROS by harsh SDS + DTT detergent mixture followed by 1D SDS-PAGE revealed only opsin monomer that upon heating disappeared, trans-forming into higher oligomers owing to secondary aggregation. The data show that degree of Rh oligomerization depends on specific conditions in which it stays. In the native state in the photoreceptor membrane as well as in mild detergents frog Rh exists mainly as dimers or higher oligomers. After solubilization with denaturing detergents, RhA can dissociate into monomers that then spontaneously self-associate into higher oligomers under the influence of various factors (for example, heating).

Key words

frog rhodopsin oligomerization dissociation BN-PAGE SDS-PAGE 

Abbreviations

6AA

6-aminohexanoic acid

BN-PAGE

blue-native polyacrylamide gel electrophoresis

CBB

Coomassie brilliant blue G-250

Chaps

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

DIG

digitonin

DM

dodecyl-β-D-maltoside

DOC

deoxycholate

DTT

dithiothreitol

GPCRs

G-protein-coupled receptors

1D- and 2D-PAGE

1- and 2-dimension polyacrylamide gel electrophoresis

Mapp

apparent molecular mass

MSF

methylsulfonyl fluoride

Rh

rhodopsin

RhA

rhodopsin aggregate

ROS

rod outer segments

TX

Triton X-100

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Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  1. 1.Sechenov Institute for Evolutionary Physiology and BiochemistryRussian Academy of SciencesSt. PetersburgRussia

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