High Concentrations of Magnesium for Formation of Thick-Filaments of Chicken Gizzard Myosin1

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Abstract
It has long been known that very high concentrations of magnesium are required for contraction of vertebrate smooth muscle (1–6). We thought of three possible reasons to account for the requirement of high concentrations of magnesium; 1) A high concentration of magnesium may be required for activation of myosin light-chain kinase. 2) It may be required for actin-activation of myosin-ATPase. 3) It may be required for formation of thick filaments. The three possible reasons were examined by studying superprecipitation of chicken gizzard actomyosin and its ATPase reaction. The following results were thus obtained. a) A MgCl 2 , concentration higher than 1 mM (in the presence of 1 mM ATP) was required for activity of myosin light-chain kinase. b) However, high concentrations of magnesium (>ca. 3 mM MgCl 2 , in the presence of 1 mM ATP) were also required for superprecipitation of acto-phosphorylated myosin. c) In the presence of 1 mM MgCl 2 , (and 1 mM ATP), superprecipitation of acto-phosphorylated myosin did not occur but actin-activation of phosphorylated myosin-ATPase did occur to a full extent. d) The ATPase activity of unphosphorylated myosin was about 1 nmol P1/min/mg of myosin at all the concentrations of magnesium we tested (1–20 mM MgCl 2 , ), but that of phosphorylated myosin increased from 3 to 9 nmol Pl/min/mg of myosin when magnesium concentration increased to higher than approximately 3 mM. e) Judging from the turbidity of the myosin suspensions, a magnesium concentration higher than approximately 3 mM (in the presence of 1 mM ATP) was also required for formation of thick filaments even when myosin was phosphorylated. It is therefore suggested that formation of thick filaments is responsible for the increase in the ATPase activity of phosphorylated myosin from 3 to 9 nmol P 1 /min/mg of myosin. These results did not support the first and second possible reasons, but they did support the third possible reason. It is therefore strongly suggested that high concentrations of magnesium are required for superprecipitation of gizzard acto-myosin because the formation of thick filaments is required for superprecipitation, and at least for “calcium-tension” in glycerinated fibers of chicken gizzard muscle.