Kinetic rates of tryptic digestion of bovine cardiac myofibrils. An improved measurement of cross-bridge dissociation.
The rates of tryptic digestion of the 50/20-kDa junction in myosin in cardiac myofibrils were determined under various solvent conditions. This cleavage reaction is slow in the rigor solvent and proceeds at a fast rate in the presence of MgATP. When the reaction solvent contains 50% ethylene glycol, the digestion of myosin in the presence of MgATP occurs at the same rate as in myofibrils relaxed by Mg adenyl-5'-yl imidodiphosphate (AMP-PNP). It is shown that with the help of two reference rates of digestion, for attached and dissociated myosin heads, the initial cleavage rates of myosin in the presence of nucleotides accurately measure the dissociation of cross-bridges from actin in myofibrils. Under physiological salt conditions and at 24 degrees C, MgADP, MgPPi, and MgAMP-PNP cause only small cross-bridge detachment (less than or equal to 15%) in cardiac myofibrils. The dissociation of myosin from actin is greatly increased by lowering the solvent temperature to 4 degrees C. Lowering the salt concentration of the solvent from 0.1 to 0.01 M NaCl has the most pronounced effect on the rates of myosin digestion in the presence of MgATP. In the low salt medium a substantial fraction of myosin heads (at least 30%) appears to be attached to actin in the presence of 5 mM MgATP.
D000205:Actomyosin / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D066298:In Vitro Techniques; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism*; D015879:Myosin Subfragments; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D014357:Trypsin / Q000494:pharmacology*
D015879:Myosin Subfragments; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D000205:Actomyosin; D014357:Trypsin; D009218:Myosins