The relationship between ADP-ribosylation and phosphorylation using a model substrate, the specificity requirements for cholera toxin-catalyzed ADP-ribosylation using peptide substrates, and the role of ADP-ribosylation reactions in skeletal muscle were studied;Cholera toxin preferentially ADP-ribosylates the first arginyl residue of kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) and some ADP-ribosylation was observed on the second arginyl residue. A di(ADP-ribosyl)ated product was also formed. Phosphorylated seryl residue of kemptide decreased the extent of ADP-ribosylation of the arginyl residues by cholera toxin and mono(ADP-ribosyl)ated kemptide was a poor substrate for the cAMP-dependent protein kinase. Di(ADP-ribosyl)ated kemptide was not phosphorylated at all. These results suggest a possible relationship between ADP-ribosylation and phosphorylation;Synthetic peptides containing two arginyl groups with sequences derived from the GTP-binding proteins, Gs[alpha], T[alpha], Gi[alpha], and Go[alpha], were ADP-ribosylated by cholera toxin preferentially at the second arginyl residue. The specificity seen in the intact proteins, Gs[alpha] and T[alpha], was not seen. Peptides from Gi[alpha] and Go[alpha] were equally ADP-ribosylated by cholera toxin even though the native proteins are not substrates. The specificity could not be induced by various solvents or by an endogenous ADP-ribosylation factor, ARF. These results suggest that the tertiary structure of the native protein is important in the specificity observed in intact proteins and that peptides with disordered structures are not specifically ADP-ribosylated;An inhibitor, meta-iodobenzylguanidine (MIBG), for arginine-specific mono(ADP-ribosyl)transferases reversibly inhibited both proliferation and differentiation of embryonic chick myoblasts grown in culture. Micromolar (30 [mu]M) concentrations of MIBG blocked fusion, differentiation-specific increase in phosphocreatine kinase activity, both DNA and protein accumulation, expression of muscle-specific protein, titin, and also decreased the total NAD levels. These finding suggest a possible role for mono(ADP-ribosyl)ation reactions in myogenesis.