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 (Gsalpha). Mono-ADP-ribosylation of Gsalpha 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.
Klebl
B M
BM
Fakultät für Biologie, Universität Konstanz, Konstanz, D-78457, Germany. Bert.Klebl@nrc.ca
Göpel
S O
SO
Pette
D
D
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D002250:Carbon Radioisotopes; D004076:Digoxigenin / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011817:Rabbits
C107266:8-(N-(N-biotinyl)-8-aminohexylamino)NAD; D002250:Carbon Radioisotopes; D009124:Muscle Proteins; C109216:N(6)-(((N-(N-biotinyl-(ethenoaminocapronyl))-(6-aminohexyl))-carbamoyl)methyl)NAD; D010761:Phosphorus Radioisotopes; C085701:digoxigenin-conjugated NAD; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D001710:Biotin; D001120:Arginine; D036002:ADP Ribose Transferases; D019205:GTP-Binding Protein alpha Subunits, Gs; D004076:Digoxigenin