1976 Volume 80 Issue 6 Pages 1359-1369
F-Actin (FA) and pyruvate kinase (PK) [EC 2.7.1.40] were immobilized on PAB-cellulose. HMM-Subfragment-1 (S-1) was applied to a column of immobilized FA and PK, and eluted with 1-1.5 μM ATP and 1 mat PEP in 50mM KCl, 2mM MgCl2, and 10mM Tris-HCl at pH 7.8 and 4°. The size of the initial burst of Pi liberation of S-1 applied to the column was 0.5 mole/mole. The size of the initial burst of S-1 in early fractions from the column was 0.8-0.9 molelmole S-1. The burst size of S-1 decreased with increase in the fraction number, and S-1 in later fractions showed a burst size of 0.1-0.3 mole/mole. On the other hand, the rate of the ATPase [EC 3.6.1.3] reaction in the steady state was almost independent of the burst size, and increased slightly with increase in the fraction number.
The ATPase activity of S-1 with a burst size of less than 0.2 mole/mole was scarcely activated by FA. Usually, the dependence on the burst size of S-1 of its ATPase activity in the presence of FA was sigmoidal, and marked activation by FA was observed when the burst size was larger than 0.3-0.4 mole/mole. Similar results were obtained with S-1 fractions separated by the ultracentrifugation method described in our previous paper ((1976) J. Biochem. 79, 419-434).
