Actin exhibits ATPase activity of unknown function that increases when monomers polymerize into filaments. Differences in the kinetics of ATP hydrolysis and the release of the hydrolysis products ADP and inorganic phosphate suggest that phosphate-rich domains exist in newly polymerized filaments. We examined whether the enrichment of phosphate on filamentous ADP-actin might modulate the severing activity of gelsolin, a protein previously shown to bind differently to ATP and ADP actin monomers. Binding of phosphate, or the phosphate analogs aluminum fluoride and beryllium fluoride, to actin filaments reduces their susceptibility to severing by gelsolin. The concentration and pH dependence of inhibition suggest that HPO binding to actin filaments generates this resistant state. We also provide evidence for two different binding sites for beryllium fluoride on actin. Actin has been postulated to contain two P binding sites. Our data suggest that they are sequentially occupied following ATP hydrolysis by HPO which is subsequently titrated to HPO. We speculate that beryllium fluoride and aluminum fluoride bind to the HPO binding site. The cellular consequences of this model of phosphate release are discussed.

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