BINDING OF BIVALENT CATION AND NUCLEOTIDE TO G-ACTIN IN THE PRESENCE OF PROFILIN

The effect of profilin, a G-actin binding protein, on the mechanism of exchange of the tightly bound metal ion and nucleotide on G-actin, ha s been investigated, 1) In low ionic strength buffer, profilin increas es the rates of Ca2+ and Mg2+ dissociation from G-actin 250- and 50-fo ld, respectively, On the profilin-actin complex as well as on G-actin alone, nucleotide exchange is dependent on the concentration of divale nt metal ion and is kinetically limited, at low concentration of metal ion, by the dissociation of the metal ion, 2) Under physiological ion ic conditions, nucleotide exchange on G-actin is 1 order of magnitude faster than at low ionic strength. The rate of MgATP dissociation is i ncreased by profilin from 0.05 s(-1) to 2 s(-1), the rate of MgADP dis sociation is increased from 0.2 s(-1) to 24 s(-1). The dependences of the exchange rates on profilin concentration are consistent with a hig h affinity (5 x 10(6) to 10(7) M(-1)) of profilin for ATP G-actin, and a 20-fold lower affinity for ADP-G-actin, Profilin binding to actin l owers the affinity of metal-nucleotide by about 1 order of magnitude, These results restrain the possible roles of profilin in actin assembl y in vivo.