Purification and characterization of an F-actin-bundling 55-kilodalton protein from HeLa cells.
An F-actin-bundling protein with Mr of 55,000 has been purified from HeLa cells by a simple method using its affinity to F-actin. Briefly, muscle actin was mixed with supernatants of HeLa cell homogenates, and the resultant actin gel was precipitated by low speed centrifugation. The 55-kDa protein in the actin gel was dissociated by depolymerization of F-actin and purified sequentially by chromatography on DEAE-cellulose and hydroxylapatite. The Stokes radius and sedimentation coefficient of the 55-kDa protein were 32 A and 4.35 (S20,w), respectively. These results suggest that the 55-kDa protein is a monomeric globular protein with a native molecular weight of 57,000. The globular form of the protein was confirmed by electron microscopy of rotary shadowed specimens. The binding of the protein to actin was saturated at an approximate stoichiometry of 4 actin monomers to one 55-kDa molecule. The protein made F-actin aggregate side-by-side into bundles as has been reported for other F-actin-bundling proteins such as fimbrin (Mr = 68,000) and fascin (Mr = 58,000). The 55-kDa protein is a new actin-binding protein based on biochemical, morphological, and immunological characterization. Skeletal muscle tropomyosin inhibited the actin-bundling activity of 55-kDa protein by competitive binding to actin, suggesting that the 55-kDa protein binding site on F-actin is in the vicinity of the tropomyosin-binding site.
D000199:Actins / Q000032:analysis; D000596:Amino Acids / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification*; D002848:Chromatography, DEAE-Cellulose; D006367:HeLa Cells / Q000032:analysis*; D006801:Humans; D007163:Immunosorbent Techniques; D008840:Microfilament Proteins / Q000302:isolation & purification*; D008854:Microscopy, Electron; D008970:Molecular Weight; D011189:Potassium Chloride / Q000494:pharmacology; D014335:Tropomyosin / Q000494:pharmacology
D000199:Actins; D000596:Amino Acids; D002352:Carrier Proteins; C034037:F-actin-binding proteins; D008840:Microfilament Proteins; D014335:Tropomyosin; D011189:Potassium Chloride