Two genetically expressed troponin T fragments representing α and β isoforms exhibit functional differences

Bo Sheng Pan, James D. Potter

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

The carboxyl-terminal isoforms of troponin T (TnT), α and β, 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 β isoform has not been studied directly at the protein level. To explore the potential functional differences between the α and β sequences, we isolated two rabbit skeletal TnT cDNA clones: a full-length cDNA for a β isoform and a partial-length cDNA for an α isoform. Two restriction fragments derived from the cDNA clones were used to direct overexpression, in Escherichia coli, of two TnT fragments, T2p-α and T2p-β, each containing the last 108 amino acid residues of the α or β isoform of TnT. Using purified T2p-α and T2p-β along with fluorescent derivatives of troponin C (TnC) and αα-tropomyosin (Tm), we showed that T2p-α bound more strongly to TnC than did T2p-β both in the presence and absence of Ca2+, and exhibited a higher affinity for Tm than did T2p-β. More interestingly, the Ca2+ affinities of the Ca2+-specific regulatory sites of TnC in the T2p-α TnC complex were found to be 3-fold higher than in T2p-β·TnC complex. These results support the hypothesis that the sequence divergence between the α and β isoforms of TnT may have functional significance in possibly contributing to the determination of the Ca2+ sensitivity of muscle fibers.

Original languageEnglish
Pages (from-to)23052-23056
Number of pages5
JournalJournal of Biological Chemistry
Volume267
Issue number32
StatePublished - Nov 15 1992
Externally publishedYes

Fingerprint

Troponin T
Troponin C
Protein Isoforms
Complementary DNA
Tropomyosin
Troponin
Clone Cells
Alternative Splicing
Escherichia coli
Muscle
Genes
Tissue
Rabbits
Derivatives
Amino Acids
Muscles
Fibers
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Two genetically expressed troponin T fragments representing α and β isoforms exhibit functional differences. / Pan, Bo Sheng; Potter, James D.

In: Journal of Biological Chemistry, Vol. 267, No. 32, 15.11.1992, p. 23052-23056.

Research output: Contribution to journalArticle

@article{0b31ee81ba1945d7b227e1fda716274f,
title = "Two genetically expressed troponin T fragments representing α and β isoforms exhibit functional differences",
abstract = "The carboxyl-terminal isoforms of troponin T (TnT), α and β, 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 β isoform has not been studied directly at the protein level. To explore the potential functional differences between the α and β sequences, we isolated two rabbit skeletal TnT cDNA clones: a full-length cDNA for a β isoform and a partial-length cDNA for an α isoform. Two restriction fragments derived from the cDNA clones were used to direct overexpression, in Escherichia coli, of two TnT fragments, T2p-α and T2p-β, each containing the last 108 amino acid residues of the α or β isoform of TnT. Using purified T2p-α and T2p-β along with fluorescent derivatives of troponin C (TnC) and αα-tropomyosin (Tm), we showed that T2p-α bound more strongly to TnC than did T2p-β both in the presence and absence of Ca2+, and exhibited a higher affinity for Tm than did T2p-β. More interestingly, the Ca2+ affinities of the Ca2+-specific regulatory sites of TnC in the T2p-α TnC complex were found to be 3-fold higher than in T2p-β·TnC complex. These results support the hypothesis that the sequence divergence between the α and β isoforms of TnT may have functional significance in possibly contributing to the determination of the Ca2+ sensitivity of muscle fibers.",
author = "Pan, {Bo Sheng} and Potter, {James D.}",
year = "1992",
month = "11",
day = "15",
language = "English",
volume = "267",
pages = "23052--23056",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "32",

}

TY - JOUR

T1 - Two genetically expressed troponin T fragments representing α and β isoforms exhibit functional differences

AU - Pan, Bo Sheng

AU - Potter, James D.

PY - 1992/11/15

Y1 - 1992/11/15

N2 - The carboxyl-terminal isoforms of troponin T (TnT), α and β, 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 β isoform has not been studied directly at the protein level. To explore the potential functional differences between the α and β sequences, we isolated two rabbit skeletal TnT cDNA clones: a full-length cDNA for a β isoform and a partial-length cDNA for an α isoform. Two restriction fragments derived from the cDNA clones were used to direct overexpression, in Escherichia coli, of two TnT fragments, T2p-α and T2p-β, each containing the last 108 amino acid residues of the α or β isoform of TnT. Using purified T2p-α and T2p-β along with fluorescent derivatives of troponin C (TnC) and αα-tropomyosin (Tm), we showed that T2p-α bound more strongly to TnC than did T2p-β both in the presence and absence of Ca2+, and exhibited a higher affinity for Tm than did T2p-β. More interestingly, the Ca2+ affinities of the Ca2+-specific regulatory sites of TnC in the T2p-α TnC complex were found to be 3-fold higher than in T2p-β·TnC complex. These results support the hypothesis that the sequence divergence between the α and β isoforms of TnT may have functional significance in possibly contributing to the determination of the Ca2+ sensitivity of muscle fibers.

AB - The carboxyl-terminal isoforms of troponin T (TnT), α and β, 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 β isoform has not been studied directly at the protein level. To explore the potential functional differences between the α and β sequences, we isolated two rabbit skeletal TnT cDNA clones: a full-length cDNA for a β isoform and a partial-length cDNA for an α isoform. Two restriction fragments derived from the cDNA clones were used to direct overexpression, in Escherichia coli, of two TnT fragments, T2p-α and T2p-β, each containing the last 108 amino acid residues of the α or β isoform of TnT. Using purified T2p-α and T2p-β along with fluorescent derivatives of troponin C (TnC) and αα-tropomyosin (Tm), we showed that T2p-α bound more strongly to TnC than did T2p-β both in the presence and absence of Ca2+, and exhibited a higher affinity for Tm than did T2p-β. More interestingly, the Ca2+ affinities of the Ca2+-specific regulatory sites of TnC in the T2p-α TnC complex were found to be 3-fold higher than in T2p-β·TnC complex. These results support the hypothesis that the sequence divergence between the α and β isoforms of TnT may have functional significance in possibly contributing to the determination of the Ca2+ sensitivity of muscle fibers.

UR - http://www.scopus.com/inward/record.url?scp=0026496306&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026496306&partnerID=8YFLogxK

M3 - Article

VL - 267

SP - 23052

EP - 23056

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 32

ER -