INSTABILITY OF F-ACTIN IN THE ABSENCE OF ATP - A SMALL AMOUNT OF MYOSIN DESTABILIZES F-ACTIN

The effects of the neutral salt concentration, pH, and coexistence of myosin on the denaturation of F-actin without ATP at low temperature were studied using the DNase I inhibition assay. The percent denaturation of F-actin gradually increased with a decrease in pH from 8.0 to 5.2, on incubation for 2 weeks in the presence of 50 mM KCl at 0-degrees-C. This change was much faster in 0.5 M KCl and more than 75% of the F-actin became denatured on incubation for 1 week at pH 5.2. The buffer composition was found to exert a strong influence on the denaturation of F-actin. That is, there was a tendency for the denaturation of F-actin at pH 6.0 to be faster in MES[2-(N-morpholino)ethanesulfonic acid]-NaOH buffer than in sodium phosphate buffer, the critical concentrations of actin in 0.5 M KCl being 0.31 mg/ml for MES-NaOH buffer and 0.15 mg/ml for sodium phosphate buffer. A sigmoidal relationship was found between the percent denaturation of F-actin and the KCl concentration added, the greatest change occurring at KCl concentrations between 0.25 and 0.75 M. The time courses of the denaturation of F-actin showed that the percent denaturation rose at first and that in time the rate of the increase decreased. In the case of pH 8.0 and 0.5 M KCl, it took about 1 week for the denaturation rate to begin to drop. The pH of 6.0 further promoted the instability of F-actin exposed to high KCl concentrations. When a constant concentration of F-actin (2 mg/ml) was mixed with various concentrations of heavy meromyosin (HMM), a small amount of HMM markedly accelerated the denaturation of F-actin. The maximum denaturation was observed at an HMM/actin weight ratio of 1: 100, at 0.5 M KCl and pH 8.0. A decrease in the KCl concentration to 50 mM made it shift toward a higher HMM/actin weight ratio. A large amount of HMM effectively protected F-actin against the denaturation by overcoming the destabilizing effect of a high salt concentration or low pH. A scheme for interpretation of the destabilizing effect of a small amount of HMM was proposed.