Steady-state magnesium ion-adenosine triphosphatase activities of myosin and its subfragment 1 derivative

Abstract

The Mg 2+-ATPase activity of myosin and its subfragment 1 (ATP phosphohydrolase, EC 3.6.1.3) always followed normal Michaelis-Menten kinetics for ATP concentrations less than 10 μM. The average K m values at pH 7.4 and 25°C are 0.33 ± 0.04 μM for myosin and 0.43 ± 0.11 μM for subfragment 1. At low salt concentration myosin yields a second hyperbolic increase in Mg 2+-ATPase activity as the ATP rises from 10.2 μM to 153 μM: V doubles with a K m of 11 ± 5 μM. This second low-salt-dependent increase in Mg 2+-ATPase activity occurred between pH 6.8 and pH 8.7. It was not affected by the presence of 0.10 M EGTA to remove Ca 2+ contamination. Solubilization of the catalytic sites by assaying myosin for ATPase activity in the presence of 0.60 M NaCl or by conversion of myosin to subfragment 1 eliminated the secondary hyperbolic increase. Subfragment 1 has a significantly different pH-activity curve from that of myosin. Subfragment 1 has an activity peak at pH 6.0, a rising activity as the pH goes from 8.7 to 9.8, and a deep activity valley between pH 6.8 and pH 8.4. Myosin has a very shallow trough of activity at pH 6.8 to 8.4, and in 1.0 mM ATP its activity drops as the pH decreases from 6.8 to 6.0. NaCl is a non-competitive inhibitor of the Mg 2+-ATPase activity of myosin and subfragment 1. Myosin has a greater affinity for NaCl ( K i = 0.101 ± 0.004 M) than does subfragment 1 ( K i = 0.194 ± 0.009 M).