Tropomyosin Fragments Cleaved at the Cysteinyl Residue1

Abstract

One cysteinyl residue in a polypeptide chain of rabbit skeletal α-tropomyosin subunit was titrated with PCMB in accord with the reported sequence. When α-tropomyosin was treated with 2-nitro-5-thiocyanobenzoic acid, the specific peptide bond between Lys 189 and Cys 190 was cleaved to yield two fragments with molecular weights of 25,000 and 11,000 daltons as determined by SDS-gel electrophoresis; these were designated as the N-chain (residues 1 to 189 in the intact chain) and the C-chain (residues 190 to 284). Each fragment was separated by a gel filtration method in the presence of urea. The apparent molecular weights of the two fragments in the absence of urea, estimated by a gel filtration technique, were about 110,000 and 40,000 daltons for the N- and C-chains, respectively, in 1 M NaCl at pH 7.0, indicating association of the individual chains after the removal of urea. Gel electrophoretic patterns of both chains after the removal of urea showed a single band for the N-chain and a double band for the C-chain; no complex formation between the N-chain and the C-chain could be observed. The N-chain showed 64% α-helical content and the C-chain 46+ in 20 mM phosphate buffer at pH 7.0 as determined by CD measurements. These aαhelical contents were increased by the addition of salts. An enhancement of the α-helical content was also observed when the chains were mixed together. Troponin had no significant binding capacity with either of the chains, while the mixture of both chains showed a clear binding with troponin in gel electrophoresis, suggesting that some cooperative interaction must exist between the N- and C-chain mixture and troponin. The binding was independent of Ca²⁺ in solution.