Interdependence of profilin, cation, and nucleotide binding to vertebrate non-muscle actin

The interaction of profilin and non-muscle beta,gamma -actin prepared from bovine spleen has been investigated under physiologic ionic conditions. Pro filin binding to actin decreases the affinity of actin for MgADP and MgATP by about 65- and 13-fold, respectively. Kinetic measurements indicate that profilin binding to actin weakens the affinity of actin for nucleotides pri marily due to an increased nucleotide dissociation rate constant, but the n ucleotide association rate constant is also increased about 2-fold. Removal of the actin-bound nucleotide and divalent cation produces the labile inte rmediate species in the nucleotide exchange reaction, nucleotide free actin (NF-actin), and increases the affinity of actin for profilin about 10-fold . Profilin binds NF-actin with high affinity, K-D = 0.013 muM, and slows th e observed denaturation rate of NF-actin. Addition of ATP to NF-actin weake ns the affinity for profilin and addition of Mg2+ to ATP-actin further weak ens the affinity for profilin. The high-affinity Mg2+ of actin regulates bi nding of both nucleotide and profilin to actin and is important for actin i nterdomain coupling. The data suggest that profilin binding to actin weaken s nucleotide binding to actin by disrupting Mg2+ coordination in the actin central cleft.