BINDING OF PHOSPHATE, ALUMINUM FLUORIDE, OR BERYLLIUM FLUORIDE TO F-ACTIN INHIBITS SEVERING BY GELSOLIN

Actin exhibits ATPase activity of unknown function that increases when monomers polymerize into filaments. Differences in the kinetics of AT P hydrolysis and the release of the hydrolysis products ADP and inorga nic phosphate suggest that phosphate-rich domains exist in newly polym erized filaments. We examined whether the enrichment of phosphate on f ilamentous ADP-actin might modulate the severing activity of gel-solin , a protein previously shown to bind differently to ATP and ADP actin monomers. Binding of phosphate, or the phosphate analogs aluminum fluo ride and beryllium fluoride, to actin filaments reduces their suscepti bility to severing by gelsolin. The concentration and pH dependence of inhibition suggest that HPO42- binding to actin filaments generates t his resistant state. We also provide evidence for two different bindin g sites for beryllium fluoride on actin. Actin has been postulated to contain two P-i binding sites. Our data suggest that they are sequenti ally occupied following ATP hydrolysis by HPO42- which is subsequently titrated to H2PO4-. We speculate that beryllium fluoride and aluminum fluoride bind to the HPO42- binding site. The cellular consequences o f this model of phosphate release are discussed.