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.

摘要: A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting 182-amino-acid protein with molecular mass of 21,162 daltons. translated is different from that reported by Wilkinson Grand (Wilkinson, J. M., Grand, R. A. (1978) Nature 271, 31-35) in Arg-153, Asp-154, Leu-155 must be inserted into their original sequence. Amino acid sequencing adult TnI confirmed this result. In order to investigate the role NH2 terminus its biological activity, we have expressed recombinant deletion mutant (TnId57), which lacks residues 1-57, bacterial expression system. Both wild type (WTnI) TnId57 inhibited acto-S1-ATPase activity inhibition could fully reversed C (TnC) presence Ca2+. Additionally both WTnI bound an actin affinity column. Thus, inhibitory binding Ca(2+)-dependent neutralization TnC were retained TnId57. chromatography used compare TnC. Using method, two types interaction between observed: 1) one metal independent (or structural) 2) dependent on Ca2+ or Mg2+ Ca(2+)-Mg2+ sites same experiments demonstrated 1 weakened, 2 lost. This method also revealed upon Ca(2+)-specific Taken together, these results suggest may constitute Ca(2+)-Mg(2+)-dependent site play, part, structural maintaining stability complex while COOH site-dependent as well previously Ca(2+)-sensitive activities.