Characterization of peptides released from rabbit skeletal muscle troponin-T by μ-calpain under conditions of low temperature and high ionic strength

Abstract

Rabbit skeletal muscle troponin-T (200 μg ml −1) was incubated with μ-calpain (2 μl ml −1) under conditions of low temperature and high ionic strength for 180 min at 4°C and the peptides released analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Troponin-T was hydrolyzed rapidly with the simultaneous appearance of eight peptides with masses of less than 14 up to 26 kDa. Two peptides produced by 10 min of incubation were electroblotted and analysis of these peptides by N-terminal sequencing and mass spectrometry showed that the principal cleavage sites of μ-calpain on troponin-T were at Thr 45–Ala 46, Leu 69–Met 70, Glu 220–Lys 221 and Asn 231–Val 232. The peptides present in insufficient quantity for electroblotting were isolated by reverse-phase high performance liquid chromatography (RP-HPLC). Cleavage sites were also identified at Met 151–Gly 152, Asn 188–Ile 189, Lys 223–Arg 224, Arg 233–Ala 234 and Ala 240–Lys 241. In general, μ-calpain cleaved bonds containing one hydrophobic amino acid residue and mainly towards the C-terminus of troponin-T.