Degradation of TN-C component of troponin by trypsin.
The peptide pattern obtained during degradation of TN-C component of troponin by trypsin markedly depends on the concentration of calcium ions. In the presence of calcium higher than 0.1 mM a peptide with a mass of about 16 000 daltons is formed, followed by its splitting into two peptides (8500 and 7500 daltons), which accumulate during further digestion. When calcium is sequestred by EGTA the degradation of TN-C is much faster. The first products of digestion are the 11 000 and 7500 dalton peptides. The former one is degraded to 9700 dalton peptide and, subsequently, both peptides are split to small fragments. In the presence of calcium only three -NH2 groups per molecule of TN-C appear during digestion, whereas in its absence almost all peptide bonds available for trypsin are cleaved. The fragments of TN-C obtained in the presence of calcium are still able to interact with TN-I component as judged by disc-electrophoresis performed in the presence of calcium. During digestion in the presence of calcium only a slight decrease of tyrosyl intrinsic fluorescence occurs and the further decrease after removal of calcium bound to TN-C is fully reversible. Similarly, the content of alpha-helix decreases only slowly during digestion in the presence of calcium. All the results suggest that calcium stabilizes the structure of TN-C molecule, so that trypsin in the presence of calcium releases one small peptide from N-terminal not involved in the first "EF-hand" and the other from the region between pairs 1-2 and 3-4 of "EF-hands" in the nomenclature of Kretsinger (see Kretsinger and Barry [16]). The obtained fragments, containing almost exclusively pairs of "EF-hands", restore most of the properties of original TN-C molecule.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D008970:Molecular Weight; D009124:Muscle Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*
D009124:Muscle Proteins; D010455:Peptides; D014336:Troponin; D014357:Trypsin; D002118:Calcium