Specificity and Target Proteins of Arginine-Specific Mono-ADP-Ribosylation in T-Tubules of Rabbit Skeletal Muscle

In order to specify that protein labeling is the result of mono-ADP ribosylation, a careful evaluation of the reaction conditions and products is necessary. To investigate the specificity and target proteins of the arginine-specific mono-ADP-ribosyltransferase (mADP-RT) in rabbit skeletal muscle T-tubules (TT) biotin- or digoxigenin-coupled NAD-derivatives were synthesized. They were used for the nonradioactive labeling of proteins and compared with radioactive mono-ADP-ribosylation. According to the results of our studies, they cannot be used as substrates to detect arginine-specific or pertussis toxin-dependent mono-ADP-ribosylation of target proteins in skeletal muscle. In contrast, radioactive NAD can be used to monitor these reactions. Under the appropriate reaction conditions, the radioactive [adenylate-14C]NAD and [32P]NAD were found to be solely consumed by the arginine-specific mADP-RT of skeletal muscle TT. The incorporation studies confirmed earlier data on the localization of the mADP-RT and its targets in TT. The T-tubular targets were purified in a single-step procedure using phenylboronate affinity chromatography. Of 18 target proteins delineated by autoradiography of electrophoretically separated T-tubular proteins, a 42-kDa protein was suggested to be the stimulatory G protein (Gsα). Mono-ADP-ribosylation of Gsα resulted in an inhibition of the T-tubular adenylate cyclase activity as proven by the suppression of this inhibition using novobiocin as a specific inhibitor of mADP-RT.