Isolation of a 240-kilodalton actin-binding protein from Dictyostelium disoideum

R. S. Hock, John S. Condeelis

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

A high molecular weight actin-binding protein with subunit mass of 240 kilodaltons has been purified from vegetative amoebae of Dictyostelium discoideum. Briefly, a cell extract was prepared by homogenizing vegetative amoebae in 5 mM EGTA, 5 mM 1,4-piperazineethanesulfonic acid, 1 mM dithiothreitol, 0.02% NaN3, pH 7.0, followed by ultracentrifugation at 114,000 x g for 1 h. The 240-kDa protein in this extract was separated from actin by chromatography on ATP-saturated DEAE-cellulose and further purified by chromatography on hydroxylapatite and Sephacryl S-300. The 240-kDa protein increases the low shear viscosity of F-actin. Covalent cross-linking with dimethyl suberimidate demonstrates that the 240-kDa protein can form dimers in high salt (500 mM NaCl). Hydrodynamic studies in high salt demonstrate the presence of an asymmetric dimer (Stokes' radius = 8.6 nm, sedimentation coefficient = 12 S, native molecular weight = 434,000, and frictional ratio = 1.7). Rotary shadowing demonstrates that the monomer is a flexible rod of approximately 70 nm in length that can associate end to end to form a dimer of approximately 140 nm in length. The 240-kDa protein cross-reacts with antibodies to chicken gizzard filamin. The properties of the 240-kDa protein suggest that it is a member of the filamin class of actin-associated proteins.

Original languageEnglish (US)
Pages (from-to)394-400
Number of pages7
JournalJournal of Biological Chemistry
Volume262
Issue number1
StatePublished - 1987

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Microfilament Proteins
Dictyostelium
Filamins
Dimers
Actins
Proteins
Amoeba
Chromatography
Dimethyl Suberimidate
Salts
Molecular Weight
Molecular weight
Avian Gizzard
DEAE-Cellulose
Sodium Azide
Shear viscosity
Dithiothreitol
Egtazic Acid
Ultracentrifugation
Protein Subunits

ASJC Scopus subject areas

  • Biochemistry

Cite this

Isolation of a 240-kilodalton actin-binding protein from Dictyostelium disoideum. / Hock, R. S.; Condeelis, John S.

In: Journal of Biological Chemistry, Vol. 262, No. 1, 1987, p. 394-400.

Research output: Contribution to journalArticle

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