Isolation and physico-chemical properties of a high molecular weight subfragment-2 of myosin

Abstract An intact subfragment-2 particle of length sufficient to span the two proteasesensitive regions of myosin was isolated following digestion of the native molecule by various proteolytic enzymes (chymotrypsin, trypsin and papain). The molecular weight of the S-2 † particle (long S-2) prepared by chymotryptic digestion was determined to be 100,000 by sedimentation equilibrium and 120,000 by sodium dodecyl sulfate-containing gel electrophoresis. This S-2 fragment occupies about half the length of the myosin rod. It is soluble in dilute salt solutions and self-associates under physiological ionic strength conditions as revealed by sedimentation equilibrium and viscosity measurements. Tryptic digestion of long S-2 yields a smaller, well-defined S-2 particle which shows no tendency to undergo self-association over the range pH 2 to 7. Thermal melting studies of long S-2 show a broad, monophasic melting curve ( t m = 45 °C) suggesting that the thermal stability is uniform with respect to length throughout the particle. Although low pH (pH 3·9) increases the melting temperature of this particle ( t m = 64 °C), no ligand studied in the present work including Mg ions, MgADP and MgATP affected its thermal stability. Under physiological ionic strength conditions and temperature, about 15 to 25% of the helical structure of long S-2 is melted, indicating that the S-2 rod may be flexible under these conditions.