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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 [Schroer, E. & Wegner, A (1985) Eur. J. Biochem. 153, 515-5201, 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.In smooth muscle of chicken gizzard an actin polymerization-inhibiting protein has been reported to occur which inhibits polymerization of actin by binding to the barbed ends of actin filaments [l -31. The polymerizationinhibiting protein has the unusual property that it does not nucleate actin polymerization, i. e. actin monomers do not polymerize onto the polymerization-inhibiting protein. The polymerization-inhibiting protein copurifies with vinculin and initially vinculin was thought to be the polymerization-inhibiting protein [4]. Later it was demonstrated that the polymerization inhibition activity can be separated from vinculin by chromatography on carboxymethyl-cellulose or hydroxyapatite [l -3, 51. Furthermore the polymerization inhibition activity, which is eluted from a DEAE-cellulose column, does not coincide with vinculin [2].Purification of the polymerization-inhibiting protein is difficult because of the heterogeneity of this protein. SDS/ polyacrylamide gels of preparations of the polymerizationinhibiting proteins reveal numerous peptide bands. Polymerization inhibition activity can be eluted from SDS/polyacrylamide gels in the molecular mass range 20-80 kDa [2]. It is not known which bands of SDS/polyacrylamide gels represent polymerization-inhibiting peptides or contaminants. In this paper we purified a 21-kDa peptide polymerization-inhibiting peptide and we worked out methods for identifying the peptides observed on SDS/polyacrylamide gels as polymerization-inhibiting peptides.

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