Affinity labeling of the guanine nucleotide binding site of transducin by pyridoxal 5′-phosphate

被引:5
|
作者
Jaffé, M
Bubis, J
机构
[1] Univ Simon Bolivar, Dept Biol Celular, Caracas 1081A, Venezuela
[2] Univ Simon Bolivar, Dept Quim, Caracas 1081A, Venezuela
来源
JOURNAL OF PROTEIN CHEMISTRY | 2002年 / 21卷 / 05期
关键词
visual process; transducin; affinity labeling; G-protein-coupled signaling;
D O I
10.1023/A:1019942318202
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
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页码:339 / 348
页数:10
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