The enhanced ATPase activity of glutathione-substituted actin provides a quantitative approach to filament stabilization.

[Cys374]glutathionyl-actin was prepared by isolation of the reaction product of G-actin with Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid], followed by reaction with glutathione. Filaments of this actin disulfide are susceptible to even weak shearing stress as exerted, for example, by heating to 37 degrees C. This treatment produces a 25-fold enhanced steady-state ATPase activity as compared to unsubstituted F-actin at room temperature. Monitoring the reduction of this enhanced ATPase activity is a reliable method for quantifying the effectiveness of filament-stabilizing agents and for determining their apparent dissociation constants. A detailed comparative study of filament-stabilizing agents was performed, and some hitherto unknown filament-protecting effects were revealed. Inorganic phosphate provides stabilization only to a maximum of 45% ATPase inhibition, but reaches this effect already at cytoplasmic Pi concentrations (approximately 4 mM). Arsenate seems to bind with similar affinity, but with distinctly less protective activity (maximum of 16%). High concentrations of alkali ions provide a more effective protection (maximum of 95%), Li+ being more efficient than Na+ and K+. Divalent cations (Ca2+, Mg2+) had a strong stabilizing effect on KCl-polymerized actin; we confirmed the presence of two distinct classes of binding sites for divalent metal ions with moderate and low affinities, apparent in a strong stabilizing effect on KCl-polymerized actin. The stabilizing effects of KCl and Pi are independent and additive. Correspondingly, at K2HPO4 concentrations greater than 4 mM, K+ ions contribute considerably to stabilization. In the presence of 100 mM KCl plus 4 mM Pi, conditions which mimic the physiological environment, filament protection is nearly as effective as with the mushroom toxin phalloidin. The strong stabilizing effect of phalloidin occurred at concentrations far below stoichiometric, suggesting a very high degree of cooperativity in its interaction with actin filaments.