Target proteins for arginine-specific mono(ADP-ribosyl) transferase in membrane fractions from chick skeletal muscle cells.
In a previous study, we found that a specific inhibitor of cellular arginine-specific mono(ADP-ribosyl) transferase, meta-iodobenzylguanidine (MIBG), reversibly inhibited both proliferation and differentiation of cultured embryonic chick primary muscle myoblasts. In addition, we observed that arginine-specific ADP-ribosyltransferase activity increased with muscle-cell differentiation in cultures. Therefore, muscle-cell cultures, especially the 96-h myotube cultures that contain the highest levels of ADP-ribosyltransferase, were used as a working system to determine the cellular protein substrates for arginine-specific ADP-ribosyltransferase. When membrane fractions extracted from 96-h chick myotubes were incubated with [32P]NAD at 30 degrees C for 30 min, only a few proteins were labeled. The labeling of two proteins of 36 and 56 kDa was inhibited by the presence of an arginine-specific mono(ADP-ribosyl) transferase inhibitor, MIBG, and by novobiocin. To prove that these proteins are indeed the targets for arginine-specific mono(ADP-ribosyl)ation, active recombinant muscle ADP-ribosyltransferase was incubated with membrane proteins under the same conditions. ADP-ribosylation of these two membrane proteins, as seen in the endogenous reactions, was also catalyzed by the added muscle transferase and was also inhibited by MIBG and novobiocin. By using antibody specific for desmin for immunoprecipitation and immunoblot analysis, we found that a 56-kDa protein associated with the membrane of myotubes is desmin. Our results showed that incorporation of isotope into this protein band from [32P]NAD is due to ADP-ribosylation of desmin.
Huang
H Y
HY
Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa, 50011, USA.
Zhou
H
H
Huiatt
T W
TW
Graves
D J
DJ
D019797:3-Iodobenzylguanidine; D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D000918:Antibody Specificity; D001345:Autoradiography; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002642:Chick Embryo; D003893:Desmin / Q000032:analysis / Q000276:immunology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007462:Iodobenzenes / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000201:enzymology*; D018482:Muscle, Skeletal / Q000166:cytology*; D009675:Novobiocin / Q000494:pharmacology; D010761:Phosphorus Radioisotopes
D003893:Desmin; D004791:Enzyme Inhibitors; D007462:Iodobenzenes; D008565:Membrane Proteins; D010761:Phosphorus Radioisotopes; D009675:Novobiocin; D019797:3-Iodobenzylguanidine; D036002:ADP Ribose Transferases