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

Rabbit skeletal muscle truponin-T (200 mug ml(-1)) was incubated with CI-ca lpain (3 mul ml(-1)) under conditions of low temperature and high ionic str ength for 180 min at 4 degreesC and the peptides released analyzed by sodiu m dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Troponin- T was hydrolyzed rapidly with the simultaneous appearance of right 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-termi nal sequencing and mass spectrometry showed that the principal cleavage sit es of mu -calpain on troponin-T were at Thr(45)-Ala(46), Leu(69)-Met(70), G lu(220)-Lys(221) and Asn(231)-Val(232). The peptides present in insufficien t quantity for electroblotting were isolated by reverse-phase high performa nce liquid chromatography (RP-HPLC), Cleavage sires 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, mu -calpain cleaved bonds containing one hydrophobic amino acid residue and mainly towards the C-terminus of tropon in-T. (C) 2001 Published by Elsevier Science Ltd.