Tryptic digestion of rabbit skeletal myofibrils: an enzymatic probe of myosin cross-bridges.
Tryptic digestion of rabbit skeletal myofibrils under physiological ionic strength and pH conditions was used as a probe of cross-bridge interaction with actin in the presence of nucleotides and pyrophosphate. Under rigor conditions, digestion of myofibrils at 24 degrees C results in the formation of 25K, 110K [heavy meromyosin (HMM)], and light meromyosin (LMM) fragments as the main reaction products. Very little if any 50K peptide is generated in such digestions. In the presence of magnesium pyrophosphate, magnesium 5'-adenylyl imidodiphosphate (MgAMPPNP), and MgATP, the main cleavage proceeds at two positions, 25K and 75K from the N-terminal portion of myosin, yielding the 25K, 50K, and 150K species. The relative amounts of the 50K, 110K, and 150K peptides and the rates of myosin heavy-chain digestion in the presence of pyrophosphate and AMPPNP indicate partial dissociation of myosin from actin. Direct centrifugation measurements of the binding of HMM and subfragment 1 (S-1) to actin in myofibrils confirm that cross-bridges partition between attached and detached states in the presence of these ligands. In the presence of MgADP, HMM and S-1 remain attached to actin at 24 degrees C. However, tryptic digestion of myofibrils containing MgADP is consistent with the existence of a mixed population of attached and detached cross-bridges, suggesting that only one head on each myosin molecule is attached to actin. As shown by tryptic digestion of myofibrils and the measurements of HMM and S-1 binding to actin, nucleotide- and pyrophosphate-induced dissociation of cross-bridges is more pronounced at 4 than at 24 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism* / Q000648:ultrastructure*; D009210:Myofibrils / Q000378:metabolism / Q000648:ultrastructure*; D009218:Myosins / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D014357:Trypsin; D009218:Myosins