CAPPING OF THE BARBED ENDS OF ACTIN-FILAMENTS BY A HIGH-AFFINITY PROFILIN-ACTIN COMPLEX

Profilin, a ubiquitous 12 to 15-kDa protein, serves many functions, in cluding sequestering monomeric actin, accelerating nucleotide exchange on actin monomers, decreasing the critical concentration of the barbe d end of actin filaments, and promoting actin polymerization when barb ed ends are free. Most previous studies have focused on profilin itsel f rather than its complex with actin. A high-affinity profilin-actin c omplex (here called profilactin) can be isolated from a poly-(L)prolin e (PLP) column by sequential elution with 3 M and 7 M urea. Profilacti n inhibited the elongation rate of pyrenyl-G-actin from filament seeds in a concentration- and time-dependent manner. Much greater inhibitio n of elongation was observed with spectrin-F-actin than gelsolin-F-act in seeds, suggesting that the major effect of profilactin was due to c apping the barbed ends of actin filaments. Its dissociation constant f or binding to filament ends was 0.3 mu M; the on and off-rate constant s were estimated to be 1.7 x 10(3) M-1 s(-1) and 4.5 x 10(-4) s(-1), r espectively. Purified profilin (obtained by repetitive applications to a PLP column and assessed by silver-stained polyacylamide gels) did n ot slow the elongation rate of pyrenyl-G-actin from filament seeds. Ca pping protein could not be detected by Western blotting in the profila ctin preparation, but low concentrations of gelsolin did contaminate o ur preparation. However, prolonged incubation with either calcium or E GTA did not affect capping activity, implying that contaminating gelso lin-actin complexes were not primarily responsible for the observed ca pping activity. Reapplication of the profilactin preparation to PLP-co upled Sepharose removed both profilin and actin and concurrently elimi nated its capping activity. Profilactin that was reapplied to uncouple d Sepharose retained its capping activity. Phosphatidylinositol-4,5-bi sphosphate (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 regul ate actin filament growth. (C) 1997 Wiley-Liss, Inc.