The effect of magnesium ions on the binding of calcium ions to glycerinated rabbit psoas muscle fibers

Abstract

The effect of Mg 2+ on Ca 2+ bound to glycerinated rabbit psoas muscle was studied by means of a double-isotope technique. The troponin-C content of the fibers was analyzed by quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis and found to be 0.5 μmol/g fiber protein. In the absence of Mg 2+ the fibers bound a maximum of 3.5 μmol Ca 2+ /g protein. This value could be readily accounted for in terms of the four Ca 2+ -binding sites of troponin-C and the two divalent cation-binding sites of myosin. In the presence of 1 mM Mg 2+ the entire Ca 2+ titration curve was shifted downward with a maximum bound Ca 2+ of slightly more than 2 μmol/g, or 4 mol Ca 2+/mol troponin-C . Further increase in Mg 2+ concentration to 10 mM had little effect on Ca 2+ binding when the free Ca 2+ concentration was in the upper part of the physiological range ( 5 · 10 −7–5 · 10 −6 M ) but caused a marked reduction when the Ca 2+ concentration was low. The results are consistent with biochemical data showing two groups of ( Mg 2+/Ca 2+ )-binding sites. One group, located in myosin, has a high affinity for Mg 2+ while the other group, located on troponin-C, has a low affinity for Mg 2+ . If the free Mg 2+ concentration in muscle is in the range of 2–5 mM, as suggested by recent data, it can be inferred that the binding sites on myosin will never be occupied by Ca 2+ under physiological conditions.