Synthesis and use of radioactive photoactivatable NAD⁺ derivatives as probes for G-protein structure

This chapter describes synthesis and use of radioiodinated phenylazide nicotinamide adenine dinucleotide (NAD⁺) derivatives ([¹²⁵I]AIPP-NAD⁺]. The synthesis and use of 2-azido-[³²P]NAD⁺ is described briefly. The chapter describes the photocross-linking and label transfer using pertussis toxin 2-azido-[³²P] adenosine diphosphate (ADP)-ribosylated transducin. An important feature of 2-azido-[³²P]NAD⁺ is that the photoactive moiety and the radioactive atom are positioned on the same side of the phosphodiester bond. Thus, ADP-ribosylation of a G protein, followed by a photolytic reaction of the azide moiety with a neighboring polypeptide, can be used to transfer the molecule to the site of azide insertion by cleavage of the thioglycosidic linkage using mercuric acetate or cleavage of the phosphodiester bond using snake venom phosphodiesterase. Two control experiments were performed to determine the pertussis toxin and azide dependence of photocross-linking. The results showed that radiolabel incorporation into α₁ is dependent on pertussis toxin treatment and that photoincorporation of radiolabel into transducin subunits is dependent on prior treatment with pertussis toxin. Evidence to support the identity of the cross-linked polypeptides is provided by immunoblotting using antisera specific for either α_{t or} β and γ subunits.