Characterization of transducin from bovine retinal rod outer segments. The role of sulfhydryl groups.
The properties and functions of the sulfhydryl groups of transducin were examined by 5,5' -dithiobis-(2-nitrobenzoic acid) titration and N-ethylmaleimide modification. The T beta gamma subunit of transducin contained a total of six free sulfhydryl groups and two were reactive under native conditions. Both reactive sulfhydryl groups were located in the beta polypeptide. The functions of transducin were not affected by the modification of these two sulfhydryl groups. The T alpha subunit of transducin contained three accessible sulfhydryl groups under both native and denaturing conditions. When 1.3 sulfhydryl groups were covalently modified by N-ethylmaleimide, the GTPase activity, the guanosine 5' -(beta, gamma-imido)triphosphate (Gpp(NH)p) uptake, and the rhodopsin-binding property of transducin were inhibited. The binding of Gpp(NH)p to T alpha blocked two of the three sulfhydryl groups from chemical modification and increased the reactivity of the remaining one. Modification of this specific sulfhydryl group of T alpha -Gpp(NH)p inhibited the exchange of the bound Gpp(NH)p for GTP. However, the modified T alpha-Gpp(NH)p was able to activate cGMP phosphodiesterase in solution and on positively charged liposomes. These findings demonstrated that a conformational change of T alpha occurs upon the binding of Gpp(NH)p and a specific sulfhydryl group of T alpha plays an important role in the activation of transducin in retinal rod outer segments.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009579:Nitrobenzoates / Q000494:pharmacology*; D010786:Photoreceptor Cells / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000032:analysis; D015293:Transducin; D014316:Tritium
D000596:Amino Acids; D008565:Membrane Proteins; D009579:Nitrobenzoates; D013438:Sulfhydryl Compounds; D014316:Tritium; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D004228:Dithionitrobenzoic Acid; D015293:Transducin; D005033:Ethylmaleimide