Labeling of the beta gamma subunit complex of transducin with an environmentally sensitive cysteine reagent. Use of fluorescence spectroscopy to monitor transducin subunit interactions.
In this study, we have examined the interactions of the beta gamma subunit complex of the retinal GTP-binding protein transducin (beta gamma T) with its alpha subunit (alpha T) using fluorescence spectroscopic approaches. The beta gamma T subunit complex was covalently labeled with 2-(4'-maleimidylanilino)napthalene-6-sulfonic acid (MIANS), an environmentally sensitive fluorescent cysteine reagent. The formation of the MIANS beta gamma T complexes (two to five MIANS adducts per beta gamma T) resulted in 2-3-fold enhancements in the MIANS fluorescence, and 20-25-nm blue shifts in the fluorescence emission maxima, relative to the emission for identical concentrations of MIANS-labeled MIANS complexes. The addition of alpha T.GDP to these MIANS beta gamma T complexes resulted in an additional enhancement in the MIANS fluorescence (typically ranging from 20 to 40%) and a 5-10-nm blue shift in the wavelength for maximum emission. These fluorescence changes were specifically elicited by the GDP-bound form of alpha T and were not observed upon the addition of purified alpha T.guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) complexes to the MIANS beta gamma T species. Conditions which resulted in the activation of the alpha T.GDP subunit (i.e. the addition of AlF4- or the addition of rhodopsin-containing vesicles and GTP gamma S) resulted in a reversal of the alpha T.GDP-induced enhancement of the MIANS beta gamma T fluorescence. Thus the MIANS beta gamma T fluorescence provided a spectroscopic monitor for transducin-subunit association and transducin-activation. Based on the results from studies using this spectroscopic read-out, it appears that the association of the alpha T.GDP species with the beta gamma T subunit complex to form the holotransducin molecule is rapid and does not limit the rate of the rhodopsin-stimulated activation of holotransducin. However, either the dissociation of the activated alpha T subunit from the beta gamma T complex, or a conformational change in beta gamma T which occurs as a result of the subunit dissociation event, appears to be slow relative to the G protein-subunit association event.
Phillips
W J
WJ
Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853.
Cerione
R A
RA
D000535:Aluminum / Q000494:pharmacology; D017607:Aluminum Compounds; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D003545:Cysteine; D005459:Fluorides; D005461:Fluorine / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism*
D017607:Aluminum Compounds; D000817:Anilino Naphthalenesulfonates; D046911:Macromolecular Substances; D013439:Sulfhydryl Reagents; D006153:Guanosine Diphosphate; C050992:tetrafluoroaluminate; D005461:Fluorine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D012243:Rhodopsin; D000535:Aluminum; D020558:GTP Phosphohydrolases; D015293:Transducin; D003545:Cysteine; D005459:Fluorides