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

Abstract

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 filatments 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 .times. 106 M-1 in 100 mM KCl and 2 mM MgCl2, and 35 .times. 106 M-1 in 2 mM MgCl2.