An enzyme-probe study of motile domains in the subfragment-2 region of myosin.
The temperature-dependence of local melting within the subfragment-2 region of rabbit skeletal muscle myosin has been investigated using an enzyme-probe technique. Rate constants of fragmentation of two long subfragment-2 particles (61,000 Mr and 53,000 Mr per polypeptide chain) and a short subfragment-2 particle (34,000 Mr per polypeptide chain) by three different enzymes (alpha-chymotrypsin, trypsin and papain) have been determined over the temperature range 5 to 40 degrees C. We followed the time-course of digestion at specific sites at high (I = 0.50, pH 7.3) and low (physiological, I = 0.15, pH 7.3) ionic strengths by electrophoresis of the digestion products on sodium dodecyl sulfate-containing gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzymes by comparison with model substrates. Normalized rate constant versus temperature profiles for the three enzyme-probes are similar in showing that local melting in long subfragment-2 (61,000 Mr) occurs in two distinct stages as was observed earlier for the intact myosin rod. Over the temperature range 5 to 25 degrees C a restricted region at Mr = 53,000 to 50,000 from the N terminus of the rod (the light meromyosin/heavy meromyosin junction) shows the highest susceptibility to proteolytic cleavage. At temperatures above 25 degrees C local melting was detected by all three enzymes at several specific sites within the hinge domain (Mr = 53,000 to 34,000). Activation energies for cleavage at the susceptible sites were similar for the three enzyme probes. They suggest that this region of the myosin rod has significantly lower thermal stability than the flanking light meromyosin and short subfragment-2 segments. These results, together with other physico-chemical studies, point to the hinge domain of the myosin cross-bridge as an important functional element in the mechanism of force generation in muscle.
D000818:Animals; D002918:Chymotrypsin; D004586:Electrophoresis; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis; D015879:Myosin Subfragments; D009218:Myosins; D010206:Papain; D010446:Peptide Fragments; D010447:Peptide Hydrolases; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin
D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010446:Peptide Fragments; D012967:Sodium Dodecyl Sulfate; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin; D010206:Papain; D009218:Myosins