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.
Highlights
NaN3, pH 7.0, followedbyultracentrifugationat 114,000 x g for1h.The 240-kDa proteinin this extract was separated from actin by chromatography on ATP-saturated DEAE-cellulose and further purified by chromatography on hydroxylapatite and Sephacryl s-300
In high salt (500 mM NaC1) the 240-kDa protein eluted upon gel filtration very near the elution position of thyroglobulin (Fig. 5 A ) and its Stokes' radius was calculated to be 8.6 nm
Properties of the 240-kDa Actin-binding Protein-A 240kDa actin-binding protein has been purified from a soluble extract of vegetative amoebae of D. discoideum
Summary
Purification of the 240-kDa Protein-Vegetative amoebae were homogenizedin alow salt/low calcium buffer in a motordriven glass-Teflon homogenizer. The elution conditions were optimized so that the240-kDa protein eluted before the myosin 11 During this step, many of the lower molecular weight contaminants were purified away from the 240-kDa protein (Fig. 2e) and much of the contaminating nucleic acid was removed. Fractions 55-63 contained the 240-kDa protein and were pooled (H)B ,.the hydroxylapatite column was equilibrated in Buffer 3. A sharp increase in low shear viscosity ml linear gradient of 0-350 mM NaPOl in Buffer 3 was applied a t C. of actin was again seen at low molar ratios of the 240-kDa. Fractions 106-126 contained the 240-kDa protein and were pooled (H)C,.the Sephacryl S-300 gel filtration column was equilibrated in Buffer 4. Fractions pooled from the hydroxylapatite column were concentrated by vacuum dialysis, clarified by ultracentrifugation a t protein dimer to actin (Fig. 311, opencircles). In Dictyostelium, actin has been estimated to com- suberimidate cross-linking of BSA, Dictyosteliurn myosin 11, Actin-2Db4i0cnt-dykioDnsgatelium
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
