Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments.
We describe herein the purification of a protein from skeletal muscle that binds to ("caps") the morphologically defined barbed end of actin filaments. This actin-capping protein appeared to be a heterodimer with chemically and immunologically distinct subunits of Mr = 36,000 (alpha) and 32,000 (beta), Rs = 37 A, s20,w = 4.0 S, and a calculated native molecular weight of approximately 61,000. The protein was obtained in milligram quantities at greater than 95% purity from acetone powder of chicken skeletal muscle by extraction in 0.6 M KI, precipitation with ammonium sulfate, sequential chromatographic steps on DEAE-cellulose, hydroxylapatite, and Sephacryl S-200, followed by preparative rate zonal sucrose density gradient centrifugation. In immunoblots of myofibrillar proteins, affinity-purified antibodies selectively recognized protein bands of the same molecular weight as the subunits of the capping protein to which they were made, indicating that the isolated capping protein is a native myofibrillar protein, and not a proteolytic digestion product of a larger muscle protein. A specific interaction of the capping protein with the barbed end of actin filaments was indicated by its ability to inhibit actin filament assembly nucleated by spectrin-band 4.1-actin complex in 0.4 mM Mg2+, accelerate actin filament formation and increase the critical concentration of actin in 2-5 mM Mg2+, 75-100 mM KCl, and inhibit the addition of actin monomers to the barbed end of heavy meromyosin-decorated actin filaments as determined by electron microscopy. All of these effects occurred at nanomolar concentrations of capping protein and micromolar concentrations of actin, suggesting a high affinity interaction.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D005260:Female; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009124:Muscle Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009132:Muscles / Q000378:metabolism*
D000199:Actins; D046911:Macromolecular Substances; D009124:Muscle Proteins; D008274:Magnesium