Two genetically expressed troponin T fragments representing alpha and beta isoforms exhibit functional differences.
The carboxyl-terminal isoforms of troponin T (TnT), alpha and beta, differing in the sequence of a region near the COOH terminus, arise from alternative splicing of a primary transcript of the TnT gene and are expressed in a tissue-specific and developmentally regulated manner (Medford, R. M., Nguyen, H. T., Destree, A. T., Summers, E., and Nadal-Ginard, B. (1984) Cell 38, 409-421). To date, the beta isoform has not been studied directly at the protein level. To explore the potential functional differences between the alpha and beta sequences, we isolated two rabbit skeletal TnT cDNA clones: a full-length cDNA for a beta isoform and a partial-length cDNA for an alpha isoform. Two restriction fragments derived from the cDNA clones were used to direct overexpression, in Escherichia coli, of two TnT fragments, T2p-alpha and T2p-beta, each containing the last 108 amino acid residues of the alpha or beta isoform of TnT. Using purified T2p-alpha and T2p-beta along with fluorescent derivatives of troponin C (TnC) and alpha alpha-tropomyosin (Tm), we showed that T2p-alpha bound more strongly to TnC than did T2p-beta both in the presence and absence of Ca2+, and exhibited a higher affinity for Tm than did T2p-beta. More interestingly, the Ca2+ affinities of the Ca(2+)-specific regulatory sites of TnC in the T2p-alpha. TnC complex were found to be 3-fold higher than in T2p-beta.TnC complex. These results support the hypothesis that the sequence divergence between the alpha and beta isoforms of TnT may have functional significance in possibly contributing to the determination of the Ca2+ sensitivity of muscle fibers.
Pan
B S
BS
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101.
Potter
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D020107:Troponin T
D011994:Recombinant Proteins; D014336:Troponin; D020107:Troponin T; D004247:DNA