Qualitative analysis of skeletal myosin as substrate of Ca2+-activated neutral protease: comparison of filamentous and soluble, native, and L2-deficient myosin.
Ca2+ -activated neutral protease (CAF) was capable of degrading myosin over a 200-fold range of protease concentrations. CAF selected the heavy chain of myosin, although either prolonged exposure to or high concentrations of the protease degraded the L1, but not the L2 or L3, light chains of myosin. The following results indicated that during the first hour of digestion, under conditions where native myosin was the substrate, CAF selected for the "head" region of the myosin heavy chain: (a) large heavy chain fragments of identical molecular weight were produced from filamentous and from soluble myosin; (b) light meromyosin was not a substrate; (c) agents known to bind to the head of myosin (actin, MgATP, and L2) had both a qualitative and quantitative effect on degradation; and (d) similar cleavage sites could be demonstrated for myosin and for heavy meromyosin (HMM) despite the fact that HMM was a much poorer substrate than myosin. This observation is interpreted as an indication that the conformation of myosin heavy chain is altered in the preparation of HMM. The principal cleavage sites on the heavy chain of myosin were 20,000, 35,000 and 50,000 D from the N-terminus, producing large fragments with molecular weights of 180,000, 165,000, and 150,000 which comprised a "nicked" species of myosin. This nicked species retained both normal solubility properties and normal hydrolytic activities. For this reason, it is concluded that "nicked myosin" is an important pathophysiological species.
D000818:Animals; D002154:Calpain; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D015879:Myosin Subfragments; D009218:Myosins / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis*; D011817:Rabbits; D013379:Substrate Specificity
D015879:Myosin Subfragments; D010446:Peptide Fragments; D010450:Endopeptidases; D002154:Calpain; D009218:Myosins