Chemical modification of bovine transducin: effect of fluorescein 5'-isothiocyanate labeling on activities of the transducin alpha subunit.
Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residues of bovine transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells. The incorporation of FITC showed a stoichiometry of approximately 1 mol of FITC/mol of transducin. The labeling was specific for the T alpha subunit. There was no significant incorporation on the T beta gamma subunit. The modification had no effect on the transducin-rhodopsin interaction or on the binding of guanosine 5'-(beta, gamma-imidotriphosphate) [Gpp(NH)p] to transducin in the presence of photolyzed rhodopsin. The dissociation of the FITC-transducin-Gpp(NH)p complex from rhodopsin membrane remained unchanged. However, the intrinsic GTPase activity of T alpha and its ability to activate the cGMP phosphodiesterase were diminished by FITC modification. The rate of FITC labeling of the transducin-Gpp(NH)p complex was about 3-fold slower than that of transducin. Limited tryptic digestion and peptide mapping were used to localize the FITC labeling site. The majority of the FITC label was on the 23-kilodalton fragment, and a minor amount was on the 9-kilodalton fragment of the T alpha subunit. These results indicate that FITC labeling does not alter the activation of transducin by photolyzed rhodopsin but does affect the GTP hydrolytic activity as well as the GTP-induced conformational change of T alpha, which ultimately leads to the activation of cGMP phosphodiesterase.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D012374:Rod Cell Outer Segment / Q000378:metabolism; D013861:Thiocyanates / Q000494:pharmacology*; D015293:Transducin; D014357:Trypsin
D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010446:Peptide Fragments; D013861:Thiocyanates; D006165:Guanylyl Imidodiphosphate; D014357:Trypsin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D015293:Transducin; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine