Relationship of phosphorylation and ADP-ribosylation using a synthetic peptide as a model substrate.
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 cAMP-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 ADP-ribosylated products of kemptide were purified by high-performance 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)ation was observed to occur on the second arginine. The minor product is di(ADP-ribosyl)ated. The Km and Vmax for mono(ADP-ribosyl)ation of kemptide are approximately 4.3 +/- 1.2 mM and 38.1 +/- 5.5 nmol min-1 mg-1, respectively. Phosphorylated seryl residue of kemptide suppresses ADP-ribosylation 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. Di(ADP-ribosyl)ated kemptide is not phosphorylated at all. These results suggest that a mere exposure of an arginyl residue in peptides is not a sufficient condition for effective ADP-ribosylation and that a relationship exists between ADP-ribosylation and phosphorylation.
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D002772:Cholera Toxin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D009842:Oligopeptides / Q000378:metabolism*; D010430:Pentosyltransferases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism
D009842:Oligopeptides; D000246:Adenosine Diphosphate Ribose; C037642:kemptide; D002772:Cholera Toxin; D036002:ADP Ribose Transferases; D010430:Pentosyltransferases; D011494:Protein Kinases