Calcium binding to rabbit skeletal myosin under physiological conditions

Area of Science:

• Biochemistry
• Muscle Physiology
• Molecular Biology

Background:

• Calcium (Ca2+) plays a crucial role in muscle contraction by interacting with regulatory proteins.
• Myosin, the motor protein of muscle, has binding sites for divalent cations like Mg2+ and Ca2+.
• Understanding the interplay between Mg2+ and Ca2+ binding to myosin is essential for elucidating muscle function.

Purpose of the Study:

• To investigate the effect of free Mg2+ concentration on Ca2+ binding to myosin.
• To determine the mechanism by which Mg2+ influences Ca2+ binding.
• To assess the impact of Ca2+ binding to myosin on actin-activated ATPase activity.

Main Methods:

• Equilibrium dialysis or similar techniques to measure Ca2+ binding to purified myosin.
• Varying free Mg2+ concentrations (e.g., 1.0 mM) and Ca2+ concentrations (e.g., 20 muM).
• Assays for actin-activated ATPase activity in the presence and absence of regulatory proteins.

Main Results:

• Myosin binds one Ca2+ per molecule at 1.0 mM free Mg2+ and 20 muM Ca2+.
• Mg2+ affects Ca2+ binding in a complex manner, not simple competition.
• Mg2+ (20-100 muM) induces positive cooperativity in high-affinity Ca2+ binding sites and shifts binding to higher Ca2+ concentrations.
• High-affinity Ca2+ binding is insensitive to ATP, ionic strength (0.1), and temperature.
• Ca2+ binding to myosin inhibited, rather than increased, actin-activated ATPase activity without regulatory proteins.

Conclusions:

• Free Mg2+ concentration significantly modulates Ca2+ binding to myosin in a manner distinct from simple competition.
• Mg2+ induces positive cooperativity in Ca2+ binding, influencing the protein's response to physiological Ca2+ levels during muscle contraction.
• Ca2+ binding to myosin, under these conditions, does not enhance but rather suppresses actin-myosin interaction as measured by ATPase activity, suggesting complex regulatory roles beyond direct activation.