Effects of Tryptic Digestion on Myosin Subfragment 1 and Its Actin-Activated Adenosinetriphosphatase

Myosin subfragment 1 (S-1) was fluorescently labeled at its rapidly reacting thiol (“SH₁”). Short exposure to trypsin cuts the S-1 heavy chain into three still-associated fragments (20K, 50K, and 27K) [Balint, M., Wolf, L., Tarcsafalvi, A., Gergely, J., & Sreter, F.A. (1978) Arch. Biochem. Biophys. 190, 793–799] which bind F-actin to the same extent as does the uncut labeled S-1, as indicated by time-resolved fluorescence anisotropy decay (at 4 °C, pH 7, in 0.15 M KCl and 5 mM MgCl₂, ±1 mM ADP). These results are thus in agreement with turbidity measurements on similar systems as reported by Mornet et al. [Mornet, D., Pantel, P., Audemard, E., & Kassab, R. (1979) Biochem. Biophys. Res. Commun. 89, 925–932]. The excited-state lifetime of the fluorescent label on cut S-1 is indistinguishable from that on normal S-1 (±ADP, ±F-actin). F-Actin activation of MgATPase of cut S-1 is lower than that for normal S-1 at moderate concentrations of F-actin, as reported by Mornet et al. (1979). But as the F-actin concentration is increased, the MgATPase activities for cut S-1 approach those for uncut S-1. In terms of an eight-species steady-state kinetics scheme involving actin binding to free S-1, S-1·ATP, S-1-ADP·P, and S-1·ADP, actin affinity for the species S-1·ADP·P was found to be 13.4 times greater for uncut S-1 than for cut S-1 [at 24 °C, pH 7.0, in 3 mM KCl, 1 mM ATP, 1 mM MgCl₂, and 20 mM N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid].