NUCLEOTIDE-FREE ACTIN - STABILIZATION BY SUCROSE AND NUCLEOTIDE-BINDING KINETICS

We prepared nucleotide-free actin in buffer containing 48% (w/v) sucrose. Sucrose inhibits the irreversible denaturation of actin that follows nucleotide dissociation [Kasai er al. (1965) Biochim. Biophys. Acta 94, 494-503]. Our conditions removed nucleotide from similar to 80% of the actin. Stabilization of nucleotide-free actin depends on the sucrose concentration. The CD ellipticity (x 10(3) deg cm(2) dmol(-1)) at 222 nm of nucleotide-free actin in 48% sucrose is -3.54. The ellipticity of denatured nucleotide-free actin in dilute buffer is -2.01 and that of native actin is -4.19. In 48% sucrose nucleotide-free actin has 1.12 and native actin has 0.5 solvent-exposed thiol residues. The conformation of native actin is recovered when ATP and Mg2+ are added. Our ability to generate stable nucleotide-free actin permitted us to study the kinetics of nucleotide binding to actin. The observed rate constant of the reaction is linearly dependent on the concentration of epsilon ATP, a fluorescent analog of ATP. The inverse of the association rate constant is proportional to the viscosity of the solvent with an intercept near the origin as expected for a diffusion-limited reaction. The second-order association rate constant for Mg2+-ATP and Ca2+-ATP binding to nucleotide-free actin in water at 22 degrees C is 5 x 10(6) M(-1) s(-1). The Smoluchowski collision rate constant for actin and ATP is calculated to be 6.5 x 10(9) M(-1) s(-1), which makes the ''orientation factor'' 7.7 x 10(-4). From the ratio of the dissociation and association rate constants, we calculate dissociation equilibrium constants of 1.2 x 10(-9) M for Mg2+-ATP-actin, 4.4 x 10(-9) M for Mg2+ epsilon ATP-actin, and 1.2 x 10(-10) M for Ca2+-ATP-actin.