INTERACTION AND POLYMERIZATION OF THE G-ACTIN MYOSIN HEAD COMPLEX - EFFECT OF DNASE-I

The properties of polymerization and interaction of the G-actin-myosin S1 complexes (formed with either the S1(A1) or the S1(A2) isoform) ha ve been studied by light-scattering and fluorescence measurements in t he absence and in the presence of DNase I. In the absence of DNase I, the G-actin-S1(A1) and G-actin-S1(A2) complexes were found to be chara cterized by different limiting concentrations (l.c.), defined as the c omplex concentrations above which the polymerization occurs spontaneou sly within 20 h at 20-degrees-C in a ''no salt'' buffer (l.c. = 0.42 a nd 8.8 muM for G-actin-S1(A1) and G-actin-S1(A2), respectively). The o ccurrence of a limiting concentration for either complex together with the kinetic properties of the polymerization led us to conclude that the G-actin-S1 polymerization occurs via a nucleation-elongation proce ss. Fluorescence titrations and proteolysis experiments revealed that G-actin interacts with S1 with a 1:1 stoichiometry (independently of t he presence of ATP) with dissociation constants, in the absence of nuc leotide, of 20 and 50 nM for the G-actin-S1(A1) and G-actin-S1(A2) com plexes, respectively. In the presence of at least a 1.5-fold excess of DNase I, the polymerization of the G-actin-SI complexes was blocked e ven at high protein concentration or in the presence of salts. In addi tion, the affinity of either S1 isoform to actin was reduced 4-5-fold by DNase I, while the stoichiometry of the G-actin-S1 complexes was no t changed. However, since the dissociation constants remain in the sub micromolar range, we could demonstrate the existence of ternary DNase I-G-actin-S1 complexes stable under polymerizing conditions. Finally, the study of the effect of nucleotides and of various salts on the G-a ctin-S1 interaction further showed significant differences between the G-actin-S1 and F-actin-S1 interactions.