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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

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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 NaBH3CN 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.

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Abbreviations

6AA:

6-aminohexanoic acid

CBB:

Coomassie Brilliant Blue G-250

1D BN-PAGE:

1D blue native electrophoresis in polyacrylamide gel

1D HRCN-PAGE:

1D high resolution clear native electrophoresis in polyacrylamide gel

DIG:

digitonin

DM:

dodecyl-β-D-maltoside

DOC:

sodium deoxycholate

DTT:

dithiothreitol

GPCRs:

G-protein-coupled receptors

M app :

apparent molecular mass

MβCD:

methyl-β-cyclodextrin

PVDF:

polyvinylidene difluoride

Rh:

rhodopsin

RhA:

aggregate of Rh

RO:

N-retinylopsin

ROS:

rod outer segments

RPE:

N-retinyl-phosphatidylethanolamine

TBS:

Tris-buffered saline

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Authors and Affiliations

  1. Sechenov Institute for Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, pr. M. Toreza 44, 194223, St. Petersburg, Russia

    S. A. Shukolyukov

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  1. S. A. Shukolyukov
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Correspondence to S. A. Shukolyukov.

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Published in Russian in Biokhimiya, 2010, Vol. 75, No. 8, pp. 1157–1165.

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Shukolyukov, S.A. 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. Biochemistry Moscow 75, 1045–1051 (2010). https://doi.org/10.1134/S0006297910080146

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  • DOI: https://doi.org/10.1134/S0006297910080146

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