Characterization of the actin polymerization-inhibiting protein from chicken gizzard smooth muscle.

An actin polymerization-inhibiting protein, occurring in crude preparations of vinculin from chicken gizzard, has been found to be heterogeneous. The molecular masses of the polymerization-inhibiting peptides have been reported to range from 20 kDa to 80 kDa [Schröer, E. & Wegner, A (1985) Eur. J. Biochem. 153, 515-520]. In this paper, a 21-kDa peptide was isolated from the bulk of the other peptides by gel chromatography. The 21-kDa peptide was identified as a polymerization-inhibiting peptide by its ability to retard nucleated actin polymerization and to bind polymeric actin when it was blotted onto nitrocellulose. Antiserum raised to the 21-kDa peptide was found to react with almost all peptides of the blotted heterogeneous polymerization-inhibiting protein. The same peptides which reacted with antiserum cosedimented with polymeric actin. The major peptides of the blotted polymerization-inhibiting protein bound polymeric actin. The largest peptide which reacted with antiserum and cosedimented with polymeric actin had a molecular mass of 85 kDa. The results suggest that the preparation of polymerization-inhibiting protein contains mainly polymerization-inhibiting peptides and only some contaminants, and that all the polymerization-inhibiting peptides are proteolytic fragments stemming from a common precursor.

Purification and characterization of a protein from chicken gizzard, which inhibits actin polymerization.

An actin-polymerization-inhibiting protein, that occurs in crude preparations of vinculin from chicken gizzard, has been purified by DEAE-cellulose and carboxymethyl ion-exchange chromatography. According to sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis and to gel filtration the polymerization-inhibiting protein is heterogeneous and the molecular mass ranges from 20 kDa to 80 kDa. After treatment with acid the polymerization-inhibiting activity was found to migrate on a SDS/polyacrylamide gel as a single band of molecular mass about 32 kDa. The mechanism of the action of the polymerization-inhibiting protein on actin assembly was investigated by the effect on the kinetics of actin polymerization. The polymerization-inhibiting protein blocks elongation of actin filaments at substoichiometric ratios but does not nucleate actin filaments. The equilibrium constant for binding of the polymerization-inhibiting protein to the barbed end of an actin filament was estimated to be 2 X 10(6) M-1 in 100 mM KCl and 2 mM MgCl2, and 35 X 10(6) M-1 in 2 mM MgCl2.