title: Capping of the barbed ends of actin filaments by a high-affinity profilin-actin complex.
abstract: Profilin, a ubiquitous 12 to 15-kDa protein, serves many functions, including sequestering monomeric actin, accelerating nucleotide exchange on actin monomers, decreasing the critical concentration of the barbed end of actin filaments, and promoting actin polymerization when barbed ends are free. Most previous studies have focused on profilin itself rather than its complex with actin. A high-affinity profilin-actin complex (here called profilactin) can be isolated from a poly-(L)-proline (PLP) column by sequential elution with 3 M and 7 M urea. Profilactin inhibited the elongation rate of pyrenyl-G-actin from filament seeds in a concentration- and time-dependent manner. Much greater inhibition of elongation was observed with spectrin-F-actin than gelsolin-F-actin seeds, suggesting that the major effect of profilactin was due to capping the barbed ends of actin filaments. Its dissociation constant for binding to filament ends was 0.3 microM; the on- and off-rate constants were estimated to be 1.7 x 10(3) M-1 s-1 and 4.5 x 10(-4) s-1, respectively. Purified profilin (obtained by repetitive applications to a PLP column and assessed by silver-stained polyacylamide gels) did not slow the elongation rate of pyrenyl-G-actin from filament seeds. Capping protein could not be detected by Western blotting in the profilactin preparation, but low concentrations of gelsolin did contaminate our preparation. However, prolonged incubation with either calcium or EGTA did not affect capping activity, implying that contaminating gelsolin-actin complexes were not primarily responsible for the observed capping activity. Reapplication of the profilactin preparation to PLP-coupled Sepharose removed both profilin and actin and concurrently eliminated its capping activity. Profilactin that was reapplied to uncoupled Sepharose retained its capping activity. Phosphatidylinositol-4,5-bisphosphate (PIP2) was the most potent phosphoinositol in reducing the capping activity of profilactin. Dissociation of the tight profilactin complex may serve as a unique mechanism by which profilin helps regulate actin filament growth.
authors.affiliation: Department of Medicine, Cooper Hospital/University Medical Center, UMDNJ/Robert Wood Johnson Medical School, Camden, USA. dinubile@umdnj.edu
authors.forename: M J
authors.initials: MJ
authors.lastname: DiNubile
authors.forename: S
authors.initials: S
authors.lastname: Huang
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000648:ultrastructure; D008840:Microfilament Proteins / Q000737:chemistry* / Q000648:ultrastructure; D008863:Microspheres; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry; D051304:Profilins; D011506:Proteins / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D000199:Actins; D008840:Microfilament Proteins; D010455:Peptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D051304:Profilins; D011506:Proteins; C024782:profilactin; C011083:polyproline