Affinity labeling of the guanine nucleotide binding site of transducin by pyridoxal 5'-phosphate.
Transducin (T), a guanine nucleotide binding regulatory protein composed of alpha-, beta-, and gamma-subunits, serves as an intermediary between rhodopsin and cGMP phosphodiesterase during signaling in the visual process. Pyridoxal 5'-phosphate (PLP), a reagent that has been used to modify enzymes that bind phosphorylated substrates, was probed here as an affinity label for T. PLP inhibited the guanine nucleotide binding activity of T in a concentration dependent manner, and was covalently incorporated into the protein in the presence of [3H]NaBH4. Approximately 1 mol of 3H was bound per mol of T. GTP and GTP analogs appreciably hindered the incorporation of 3H to T, suggesting that PLP specifically modified the protein active site. Interestingly, PLP modified both the alpha- and beta-subunits of T. Moreover, PLP in the presence of GDP behaved as a GTP analog, since this mixture was capable of dissociating T from T:photoactivated rhodopsin complexes.
Departamento de Química, Universidad Simón Bolivar, Caracas, Venezuela.
Matthias
M
Jaffé
José
J
Bubis
D000818:Animals; D001665:Binding Sites; D001881:Borates / Q000494:pharmacology; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005123:Eye / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism; D008024:Ligands; D008027:Light; D010710:Phosphates / Q000494:pharmacology; D017680:Potassium Compounds / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000528:radiation effects; D011730:Pyridoxal / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000378:metabolism* / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods*; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism*; D014316:Tritium
D001881:Borates; D004791:Enzyme Inhibitors; D008024:Ligands; D010710:Phosphates; D017680:Potassium Compounds; D014316:Tritium; D006153:Guanosine Diphosphate; D006165:Guanylyl Imidodiphosphate; D011730:Pyridoxal; D011732:Pyridoxal Phosphate; D006160:Guanosine Triphosphate; C013216:potassium phosphate; D015293:Transducin