INDUCTION OF THE POLYMERIZATION OF ACTIN FROM THE ACTIN-THYMOSIN BETA-4 COMPLEX BY PHALLOIDIN, SKELETAL MYOSIN SUBFRAGMENT 1, CHICKEN INTESTINAL MYOSIN-I AND FREE ENDS OF FILAMENTOUS ACTIN

Thymosin beta 4 is able to form 1:1 complexes with monomeric (G) actin , thereby stabilizing the intracellular pool of unpolymerized actin. W e have searched for factors that are able to induce the polymerization of actin from the actin:thymosin beta 4 complex. Phalloidin, subfragm ent 1 isolated from rabbit skeletal muscle myosin and chicken intestin al myosin I are demonstrated to be able to polymerize the actin from t his complex in the presence of 1 mM MgCl2. Polymerization of actin was verified by the DNase I inhibition assay, by cosedimentation and from the fluorescence increase of pyrene-labelled actin. Actin filaments f ormed under the influence of subfragment 1 or phalloidin were visualiz ed under the electron microscope after negative staining. Polymerizati on of skeletal muscle actin from the complex with thymosin beta 4 by p halloidin is accompanied by the hydrolysis of the actin-bound ATP to A DP. Polymerization was also induced by sonicated F-actin which possess ed a high concentration of free filament ends. F-actin was severed by 0.01 M human cytoplasmic gelsolin, which is known to possess blocked ends. Free, slowly growing - ends were unable to induce polymerizatio n of actin from the thymosin beta 4 complex. However, when gelsolin on its own or in complex with two actin molecules was added to actin:thy mosin beta 4 under nucleating conditions, it was found to be able to p romote actin repolymerization provided that its concentration was clos e to the dissociation constant (K-d) of actin:thymosin beta 4. This K- d was found to be 0.4 mu M in the presence of 1 mM MgCl2 and the absen ce of KCl and, thus, close to the critical concentration of actin poly merization under these conditions. The source of actin did not influen ce its polymerization from the thymosin: beta 4 complex; rabbit skelet al muscle actin and porcine brain actin were polymerized with equal ef ficiency from their complexes with thymosin beta 4 by both phalloidin and myosin subfragment 1. Skeletal muscle, but not cytoplasmic actin, was found to be also polymerized in the presence of increased CaCl2 co ncentrations to values above 1 mM.