Temperature-dependence of local melting in the myosin subfragment-2 region of the rigor cross-bridge.
We have used alpha-chymotrypsin as an enzyme-probe to detect local melting in the subfragment-2 region of the cross-bridges of rigor myofibrils and glycerinated psoas fibers. The kinetics of proteolysis and the sites of cleavage were determined at various temperatures over the range 5 to 40 degrees C by following the decay of the myosin heavy chain and the rates of appearance of light meromyosin fragments, using electrophoresis on sodium dodecyl sulfate-containing polyacrylamide gels. Cleavage occurs primarily at the 72,000 Mr and 64,000 Mr (per polypeptide chain from the C terminus of myosin) sites within the light meromyosin-heavy meromyosin hinge domain of the subfragment-2 region, under all experimental conditions. At pH 8.2 to 8.3 and at low divalent metal ion (0.1 mM), where the actin-bound cross-bridges are thought to be released from the thick filament surface, the intrinsic cleavage rate constant (k) increases markedly as the temperature is raised. This suggests substantial thermal destabilization of the released cross-bridge in the intact contractile apparatus. Addition of divalent metal ion (10 mM) lowers the cleavage rate and shifts the k versus temperature profile to higher temperatures. Normalized rate constants for chymotryptic cleavage within the subfragment-2 hinge region of released cross-bridges (pH 8.2, low divalent metal) of rigor fibers were markedly lower than activated fibers at all temperatures investigated (5 to 40 degrees C). Results show that conformational melting within the subfragment-2 hinge region is amplified on activation and is well above that observed when the actin-attached rigor bridge is passively released from the thick filament surface.
D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009119:Muscle Contraction; D009210:Myofibrils / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D013696:Temperature
D015879:Myosin Subfragments; D010446:Peptide Fragments; D002918:Chymotrypsin; D009218:Myosins