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 [H-3]NaBH4. Approximately 1 mol of H-3 was bound per mol of T. GTP and GTP analogs appreciably hindered the incorporation of H-3 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.