Effects of tryptic digestion on myosin subfragment- 1 and its actin-activated ATPase

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Abstract
Myosin [rabbit] subfragment 1 (S-1) was fluorescently labeled at its rapidly reacting thiol (SH1). Short exposure to trypsin cuts the S-1 H chain into 3 still-associated fragments (20K [kilodalton], 50K and 27K) which bind F-actin to the same extent as does the uncut labeled S-1, as indicated by time-resolved fluorescence anisotropy decay (4.degree. C, pH 7, in 0.15 M KCl and 5 mM MgCl2, .+-. 1 mM ADP). These results are in agreement with turbidity measurements on similar systems as reported by Mornet et al. 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. As the F-actin concentration is increased, the MgATPase activities for cut S-1 approach those for uncut S-1. In terms of an 8 spp. steady-state kinetics scheme involving actin binding to free S-1, S-1.cntdot.ATP, S-1.cntdot.ADP.cntdot.P and S-1.cntdot.ADP, actin affinity for the species S-1.cntdot.ADP.cntdot.P was 13.4 times greater for uncut S-1 than for cut S-1 [at 24.degree. C, pH 7.0, in 3 mM KCl, 1 mM ATP, 1 mM MgCl2 and 20 mM N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid].