Isolation of a 240-kilodalton actin-binding protein from Dictyostelium discoideum.
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.
D000199:Actins / Q000378:metabolism; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D003285:Contractile Proteins / Q000276:immunology; D003432:Cross-Linking Reagents; D004023:Dictyostelium / Q000032:analysis*; D004120:Dimethyl Suberimidate; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D064448:Filamins; D018260:Gelsolin; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000276:immunology; D008970:Molecular Weight
D000199:Actins; D002352:Carrier Proteins; D003285:Contractile Proteins; D003432:Cross-Linking Reagents; D000939:Epitopes; D064448:Filamins; D018260:Gelsolin; D046911:Macromolecular Substances; D008840:Microfilament Proteins; C016953:brevin; D004120:Dimethyl Suberimidate