Digestion of troponin C with trypsin in the presence and absence of Ca2+. Identification of cleavage points.
The rate of tryptic digestion of troponin C has been shown to be dependent on Ca2+ (Drabikowski et al., Biochim. Biophys. Acta 490, 216-224). We have characterized the tryptic peptides produced both in the presence and absence of Ca2+ using amino acid composition and end-group analyses. In the presence of Ca2+ trypsin cleaves TnC at Arg-8, Lys-84 and Lys-88, leading to the formation of two large peptides, one containing the two low-affinity sites (TR1C), the other, the two high-affinity Ca2+-binding sites (TR2C). In the absence of Ca2+ (1 mM EDTA), digestion proceeds much more rapidly and takes place first at Arg-100, followed by Arg-104, Arg-120, Lys-153, Arg-8 and others. The data suggest that the points of cleavage are determined by the Ca2+-dependent conformational states of TnC, particularly in the C-terminal half of the protein where the cation is known to induce secondary structure.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D009124:Muscle Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C; D014357:Trypsin / Q000378:metabolism*
D000596:Amino Acids; D009124:Muscle Proteins; D010446:Peptide Fragments; D014336:Troponin; D019209:Troponin C; D014357:Trypsin; D002118:Calcium