Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments

James F Casella, D. J. Maack, Lin Shin Lin

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)10915-10921
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number23
StatePublished - 1986

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Actin Cytoskeleton
Purification
Muscle
Actins
Skeletal Muscle
Proteins
Actin Capping Proteins
Molecular Weight
Molecular weight
Myosin Subfragments
Spectrin
DEAE-Cellulose
Antibody Affinity
Density Gradient Centrifugation
Muscle Proteins
Protein Subunits
Ammonium Sulfate
Durapatite
Centrifugation
Acetone

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments. / Casella, James F; Maack, D. J.; Shin Lin, Lin.

In: Journal of Biological Chemistry, Vol. 261, No. 23, 1986, p. 10915-10921.

Research output: Contribution to journalArticle

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