Binding and regulatory properties of expressed functional domains of chicken gizzard smooth muscle caldesmon

J Biol Chem. 1993 May 25;268(15):10969-76.

Abstract

We expressed the following fragments of chicken gizzard caldesmon in the pMW 172/BL21 (DE3) system at 0.4-2.2 mg of pure protein/liter of culture: full-length smooth muscle caldesmon (CDh) (amino acids 1-756), nonmuscle caldesmon (CDl), amino acids 1-128 (N128), 1-578 (N578), 230-419, 606-756 (606C), and 658-756 (658C). CDh bound tropomyosin with a Kd of 1.5 microM; N578, 230-419, and 606C bound with affinities at least 2-5 fold lower; N128 bound weakly; and 658C did not bind. Only N128 and N578 bound to smooth muscle myosin, both about 10-fold weaker than CDh and CDl. Only 606C and 658C bound to actin-tropomyosin with affinities CDh = 606C > 658C. The binding to actin-tropomyosin was biphasic, whereas the binding to actin was monophasic, corresponding to the weak binding component found in the presence of tropomyosin. Calmodulin bound only to the C-terminal fragments with the same affinity as CDh. CDh, 606C, and 658C inhibited actin-tropomyosin-activated myosin ATPase, with maximal inhibition correlated with 1 caldesmon bound/14 actins, and inhibition was reversed by Ca(2+)-calmodulin. Thus, the actomyosin ATPase regulatory function, calmodulin binding, and most actin binding is located within the C-terminal 99 amino acids, whereas tropomyosin binding is distributed throughout the rest of the molecule.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Calmodulin-Binding Proteins / genetics*
  • Calmodulin-Binding Proteins / metabolism*
  • Chickens
  • Cloning, Molecular
  • DNA / genetics
  • Gene Library
  • Gizzard, Avian / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Muscle, Smooth / metabolism*
  • Mutagenesis, Site-Directed
  • Oligodeoxyribonucleotides
  • Peptide Fragments / metabolism
  • Protein Binding
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Spectrometry, Fluorescence
  • Tropomyosin / metabolism

Substances

  • Actins
  • Calmodulin-Binding Proteins
  • Oligodeoxyribonucleotides
  • Peptide Fragments
  • Recombinant Proteins
  • Tropomyosin
  • DNA