Characterization of transducin from bovine retinal rod outer segments. Mechanism and effects of cholera toxin-catalyzed ADP-ribosylation.
Transducin, a guanine nucleotide-binding protein consisting of two subunits (T alpha and T beta gamma), mediates the signal coupling between rhodopsin and a membrane-bound cyclic GMP phosphodiesterase in retinal rod outer segments. The T alpha subunit is an activator of the phosphodiesterase, and the function of the T beta gamma subunit is to physically link T alpha with photolyzed rhodopsin. In this study, the mechanism of cholera toxin-catalyzed ADP-ribosylation of T alpha has been examined in a reconstituted system consisting of purified transducin and stripped rod outer segment membranes. Limited proteolysis of the labeled T alpha with trypsin indicated that the inserted ADP-ribose is located exclusively on a single proteolytic fragment with an apparent molecular weight of 23,000. Maximal incorporation of ADP-ribose was achieved when guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p) and T beta gamma were present at concentrations equal to that of T alpha and when rhodopsin was continuously irradiated with visible light in the 400-500 nm region. The stimulating effect of illumination was related to the direct interaction of the retinal chromophore with opsin. These findings strongly suggest that a transient protein complex consisting of T alpha X Gpp(NH)p, T beta gamma, and a photointermediate of rhodopsin is the required substrate for cholera toxin. Single turnover kinetic measurements demonstrated that the ADP-ribosylation of T alpha coincided with the appearance of a population of transducin molecules having a very slow rate of GTP hydrolysis. The hydrolysis rate of the bound GTP for this population was 1.1 X 10(-3)/s, which was 22-fold slower than the rate for the unmodified transducin.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002772:Cholera Toxin / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008027:Light; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D010786:Photoreceptor Cells / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013696:Temperature; D015293:Transducin
D006898:Hydroxylamines; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009702:Nucleoside Diphosphate Sugars; D000246:Adenosine Diphosphate Ribose; D019811:Hydroxylamine; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D002772:Cholera Toxin; D015293:Transducin