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Two chymotrypsin-susceptible sites of myosin rod from chicken gizzard
Abstract:
The rod prepared from chicken gizzard myosin has been found to have two sites sensitive to limited digestion with chymotrypsin; these sites were located at a subfragment 2/light meromyosin junction (site 1), and at a site 10 kDa remote from either C-terminal or N-terminal of light meromyosin (site 2). The site 1 was more sensitive to the digestion than the site 2. The cleavage at site 2 of the light meromyosin yielded a 74-kDa fragment that was soluble in a low ionic strength solution, contrary to the insolubility of the parent light meromyosin in the same solution. Studies on the effects of MgCl2, ATP and pH on the susceptibilities of these sites to chymotrypsin have given following results. (a) Millimolar concentrations of MgCl2 protected site 1 and site 2 from the chymotryptic cleavage. (b) The cleavage at site 1 of myosin rod in the low salt solution free of Mg2+ at pH 7.0 and pH 8.5, was not affected by the presence of 5 mM ATP. However, MgCl2-induced protection of site 1 was relieved by addition of ATP. On the other hand, the cleavage at site 2 was stimulated by addition of ATP, irrespective of the presence or absence of MgCl2. (c) The alkaline condition of pH 8.5 was more favorable for the chymotryptic cleavages at both site 1 and site 2 than the neutral condition of pH 7.0. These results suggest that myosin rod contains two flexible regions, the structures of which are influenced by such an ambient factor as MgCl2, ATP or pH.
Insights
Chicken gizzard myosin rod has two chymotrypsin-sensitive sites. MgCl2, ATP, and pH influence these sites, suggesting flexible regions in the myosin rod structure.
Area of Science:
- Biochemistry
- Muscle protein structure
- Enzymatic digestion
Background:
- Myosin rod structure and function are crucial for muscle contraction.
- Limited proteolysis is a key tool for dissecting protein domains.
- Understanding myosin rod flexibility is essential for elucidating muscle mechanics.
Purpose of the Study:
- To identify and characterize chymotrypsin-sensitive sites on chicken gizzard myosin rod.
- To investigate the influence of MgCl2, ATP, and pH on these cleavage sites.
- To infer the presence of flexible regions within the myosin rod.
Main Methods:
- Limited digestion of chicken gizzard myosin rod with chymotrypsin.
- Analysis of cleavage products and fragment solubility.
- Investigating the effects of MgCl2, ATP, and pH on chymotryptic cleavage susceptibility.
Main Results:
- Two chymotrypsin-sensitive sites were identified: one at the subfragment 2/light meromyosin junction (site 1) and another 10 kDa from the light meromyosin termini (site 2).
- Site 1 was more sensitive to digestion than site 2.
- Cleavage at site 2 produced a soluble 74-kDa fragment, unlike the insoluble parent light meromyosin.
- MgCl2 protected both sites, while ATP differentially affected cleavage: it relieved MgCl2-induced protection at site 1 but stimulated cleavage at site 2.
- Alkaline pH (8.5) favored cleavage at both sites compared to neutral pH (7.0).
Conclusions:
- Chicken gizzard myosin rod possesses two distinct flexible regions susceptible to chymotryptic digestion.
- The structural integrity and accessibility of these flexible regions are modulated by environmental factors like MgCl2, ATP, and pH.
- These findings contribute to a deeper understanding of myosin rod dynamics and its role in muscle function.