TY - JOUR
T1 - Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments
AU - Casella, J. F.
AU - Maack, D. J.
AU - Shin Lin, Lin
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1986
Y1 - 1986
N2 - 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 M(r) = 36,000 (α) and 32,000 (β), R(s) = 37 Ȧ, s(20,w) = 4.0 S, and a calculated native molecular weight of ~61,000. The protein was obtained in milligram quantities at >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 1) inhibit actin filament assembly nucleated by spectrin-band 4.1-actin complex in 0.4 mM Mg2+, 2) accelerate actin filament formation and increase the critical concentration of actin in 2-5 mM Mg2+, 75-100 mM KCl, and 3) 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.
AB - 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 M(r) = 36,000 (α) and 32,000 (β), R(s) = 37 Ȧ, s(20,w) = 4.0 S, and a calculated native molecular weight of ~61,000. The protein was obtained in milligram quantities at >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 1) inhibit actin filament assembly nucleated by spectrin-band 4.1-actin complex in 0.4 mM Mg2+, 2) accelerate actin filament formation and increase the critical concentration of actin in 2-5 mM Mg2+, 75-100 mM KCl, and 3) 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.
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M3 - Article
C2 - 3733738
AN - SCOPUS:0023034626
SN - 0021-9258
VL - 261
SP - 10915
EP - 10921
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 23
ER -