Specificity of cholera toxin and mono(ADP-ribosyl)transferase activity in cultured myoblasts

Abstract

KemptIde (Leu-Arg-Arg-Ala-Ser-Leu-Gly), is a good substrate for cholera toxin in comparison with the angiotensin peptides. Because kemptide contains two potential ADP-ribosylation sites and, is also a good substrate for cAHP-dependent protein kinase, it was possible to gain some insight into factors influencing the specificity of cholera toxin and to study the relationship between phosphorylation and ADP-ribosylation. The ADPribosylated products of kemptide were purified by highperformance liquid chromatography and characterized by peptide sequence analysis, trypsin digestion, and fast-atom bombardment mass spectrometry. The major product is mono(ADP-ribosyl)ated preferentially on the first arginyl residue and some mono(ADP-ribosyl)atlon was observed to occur on the second arginine. The minor product is di(ADPribosyl)ated. The Km and Vmax for mono(ADP-ribosyl)ation of kemptide are approximately 7.7 ± 2.1 mM and 38.1 ± 5.5 nmoles min mg~ , respectively. Phosphorylated seryl residue of kemptide almost completely blocks ADPribosylation of the arginyl residues by cholera toxin. Mono(ADP-ribosyl)ated kemptide is a poor substrate for the cAMP-dependent protein kinase in comparison with kemptide.