Effect of ATP removal and inorganic phosphate on length redistribution of sheared actin filament populations. Evidence for a mechanism of end-to-end annealing.

The effect of inorganic phosphate (Pi) on the depolymerization of F-actin has been measured. Pi inhibits disassembly of pyrene-labelled F-actin at steady-state induced either by dilution, or by shearing, suggesting that Pi decreases the off rate constant, k-, for dissociation. This effect of Pi is maximal at 20 mM, unlike the effect of Pi in reducing the critical concentration at the pointed end (maximal at 2 mM). This difference in concentration dependence for the two effects is interpreted as different affinities of Pi for the barbed and pointed ends, presumably as ADP-Pi-actin species. The contribution of ATP/ADP phase changes at filament ends (i.e. "dynamic instability") to length redistribution in sheared polymer steady-state actin filament populations was determined by (1) converting ATP to ADP in the system to prevent phase changes, or (2) adding 20 mM-Pi to the system to inhibit depolymerization. The observed absence of effect of these treatments on length redistribution excludes all mechanisms which involve phase change-driven disassembly or monomer exchange at filament ends, and appears to constrain the mechanism to one of end-to-end annealing under these conditions.