title: Labeling of the beta gamma subunit complex of transducin with an environmentally sensitive cysteine reagent. Use of fluorescence spectroscopy to monitor transducin subunit interactions.
abstract: 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.
authors.affiliation: Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853.
authors.forename: W J
authors.initials: WJ
authors.lastname: Phillips
authors.forename: R A
authors.initials: RA
authors.lastname: Cerione
mesh_terms: 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*
chemical_list: 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