title: Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity.
abstract: A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities.
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101.
authors.forename: Z
authors.initials: Z
authors.lastname: Sheng
authors.forename: B S
authors.initials: BS
authors.lastname: Pan
authors.forename: T E
authors.initials: TE
authors.lastname: Miller
authors.forename: J D
authors.initials: JD
authors.lastname: Potter
mesh_terms: D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002645:Chickens; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D009218:Myosins / Q000037:antagonists & inhibitors / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014336:Troponin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019210:Troponin I
chemical_list: D000199:Actins; D011994:Recombinant Proteins; D014336:Troponin; D019210:Troponin I; D004533:Egtazic Acid; D004247:DNA; D004492:Edetic Acid; D009218:Myosins; D002118:Calcium