Fluorescent labeling of signal-transducing G-proteins. Pertussis toxin-catalyzed etheno-ADP ribosylation of transducin.
Nicotinamide 1,N6-ethenoadenine dinucleotide (etheno-NAD, epsilon-NAD), a fluorescent analogue of NAD, was able to serve as a substrate for the bacterial toxin-catalyzed epsilon-ADP ribosylation of signal-transducing G-proteins. Pertussis toxin and transducin were used as a model system to characterize this reaction. Similar to ADP ribosylation using NAD as substrate, the epsilon-ADP ribosylation occurs at the carboxyl-terminal 5-kDa tryptic fragment of the T alpha subunit of transducin with the same labeling stoichiometry; however, the rate of labeling is slightly slower. epsilon-NAD competes with NAD as a substrate which suggests that the epsilon-ADP ribosylation occurs at Cys-347 of the T alpha subunit. The biochemical effects of epsilon-ADP ribosylation on transducin are similar to those of ADP ribosylation and include inhibition of the GTPase and [3H]Gpp(NH)p-binding activities. The epsilon-ADP-ribosylated transducin exhibits a fluorescent spectrum which resembles that of epsilon-ADP with an excitation maximum at 292 nm and an emission maximum of 413 nm. Removal of the amino-terminal peptide of epsilon-ADP-ribosylated T alpha with either Staphylococcus aureus V8 protease or trypsin results in a decrease in the emission intensity. This result suggests that the amino- and carboxyl-terminal peptides of the T alpha molecule may interact with each other as suggested previously (Hingorani, V. N., and Ho, Y.-K. (1987) FEBS Lett. 220, 15-22). epsilon-NAD should prove to be a useful fluorescent substrate for future studies of the ADP ribosylation reaction in biological systems.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002417:Cattle; D005456:Fluorescent Dyes; D019204:GTP-Binding Proteins / Q000502:physiology*; D007700:Kinetics; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism; D037342:Pertussis Toxin; D010786:Photoreceptor Cells / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D015293:Transducin / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D005456:Fluorescent Dyes; D010566:Virulence Factors, Bordetella; D009243:NAD; D000246:Adenosine Diphosphate Ribose; C011038:nicotinamide 1,N(6)-ethenoadenine dinucleotide; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D015293:Transducin