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A comparative study of reactive--SH groups of cardiac and skeletal muscle myosins
Abstract:
Cardiac and skeletal muscle myosins have been treated by N-ethylmaleimide in presence or absence of Mg-ADP. The variations of Ca2+ and K+-ATPase activities and the incorporation of N-[14C]ethylmaleimide into the whole myosin molecule and into its separated subunits (heavy and light chains) have been measured with N-ethylmaleimide treatment for different lengths of time. The results reported here show the following: 1. The Ca2+-ATPase activity of cardiac myosin is activated by N-ethylmaleimide treatment to a lesser extent than that of skeletal myosin. 2. The K+-ATPase activity of both myosins is inhibited in the same quantitative way. 3. The cardiac light chain L1 contains one highly reactive thiol group which is absent from the skeletal light chains. 4. The labelling of three SH-groups localized in the heavy subunits of both myosins induced the same degree of inactivation. 5. The difference observed between the degree of inhibition of the Ca2+-ATPase activity for the two types of myosin with longer treatments appears to be due to differences in the reactivity of the fourth--SH group labelled on the heavy chains.
Insights
N-ethylmaleimide treatment differentially affects cardiac and skeletal muscle myosins. Cardiac myosin shows less Ca2+-ATPase activation, while both myosins exhibit similar K+-ATPase inhibition due to specific thiol group reactivity differences.
Area of Science:
- Biochemistry
- Muscle Physiology
- Protein Chemistry
Background:
- Myosin, a key motor protein, drives muscle contraction.
- Understanding myosin's functional mechanisms requires investigating its enzymatic activities and structural components.
- N-ethylmaleimide (NEM) is a chemical probe used to study protein thiol groups.
Purpose of the Study:
- To compare the effects of N-ethylmaleimide (NEM) on cardiac and skeletal muscle myosins.
- To elucidate the role of specific thiol groups in myosin's Ca2+-ATPase and K+-ATPase activities.
- To identify differences in NEM reactivity between cardiac and skeletal myosin subunits.
Main Methods:
- Treatment of cardiac and skeletal myosins with N-ethylmaleimide (NEM) in the presence or absence of Mg-ADP.
- Assay of Ca2+-ATPase and K+-ATPase activities.
- Quantification of NEM incorporation into myosin heavy and light chains using N-[14C]ethylmaleimide.
Main Results:
- Cardiac myosin Ca2+-ATPase activity showed less activation by NEM compared to skeletal myosin.
- K+-ATPase activity of both myosins was inhibited similarly by NEM.
- Cardiac light chain L1 possesses a unique reactive thiol group absent in skeletal myosins.
- Inactivation of three SH-groups in heavy subunits similarly affected both myosins.
- Differential reactivity of a fourth SH-group in heavy chains explained varying Ca2+-ATPase inhibition.
Conclusions:
- NEM reactivity and its impact on ATPase activity differ between cardiac and skeletal myosins.
- Specific thiol groups in myosin heavy and light chains play distinct roles in enzymatic function.
- Cardiac myosin's unique light chain L1 thiol group contributes to functional differences.