−日中医学学会助成事業−

 Myosin II is one of the typical motor proteins and is classified as non-regulated, phosphorylatable and Ca-binding myosins. Physarum and scallop myosin II belongs to Ca-binding one. However, Ca²⁺ works as an inhibitor for Physarum myosin and as an activator for scallop myosin. This similarity in the subunits composition has raised the question of what subunit determines the inhibitory and stimulatory effects of Ca²⁺ Myosin II regulated by Ca-binding has not yet expressed as a recombinant protein. Here, we report the expression of physarum myosin II together with preliminary characterizations.

　

Key Words　motility assay, calcium, recombinant myosin II, actin

　

 Plasmodia of Physarum polycephalum shows vigorous cytoplasmic streaming by changing direction every few minutes. This oscillatory streaming is regulated by Ca²⁺ is thought to be driven by a conventional myosin. It has been known that the superprecipitation of actomyosin preparation or myosin B from the plasmodia to examine the effect of Ca²⁺. It superprecipitated without requiring Ca²⁺. When Ca²⁺ at μM level was present, the superprecipitation was inhibited. This calcium inhibition was quite the opposite of the superprecipitation of actomyosin from vertebrate muscles, and we expected that the inhibitory mode could be involved in the plant cytoplasmic streaming. With the finding of the diverse classes of unconventional myosin such as myosin I and V in vertebrate muscles, the inhibitory mode was shown to play a role in cell motility in both animal and plant kingdoms. In this case the myosins have calmodulin (CaM) as the light chains and are regulated by interaction of Ca²⁺ with CaM, which exerts an inhibitory effect on activity. Since of the findings of calcium inhibition in the plasmodia, efforts have been made to define the way in which Ca²⁺ regulates the actomyosin system, leading to the discovery that, while that Physarum myosin is the major site of action of Ca²⁺, actin-linked regulation through actin-binding protein is involved in the inhibition. Further biochemical studies on the myosin-linked regulation showed that Ca²⁺ binding to the Ca-binding light chain (CaLC) inhibits the activity of Physarum myosin Similar to Physarum myosin, mollusk scallop myosin belongs to the myosinII isoform family and the activity of scallop myosin is regulated by Ca ²⁺. However, the effect of Ca²⁺ on this myosin is in an opposite to the regulation of Physarum myosin; Ca²⁺ activates the activity. Because structure and function in relation to the regulation by Ca²⁺ are known better for scallop myosin than that of Physarum myosin. We adopted a strategy to compare the two myosins for a better understanding of Ca²⁺ regulation. As the first step to analyze how Ca²⁺ exerts a regulatory role on Physarum myosin through binding to CaLC, we tried to obtain recombinant myosin and heavy mero-myosin of Physarum myosin (HMM).

　

 We used baculovirus expression system. Sf9 cells were infected with the virus constructs.

　

(1) Ca²⁺ inhibited sliding velocity of actin filaments prorelled by recombinant full length myosin II and HMM of Physarum polycephalum, and as the concentration of calcium increased, the inhibition was stronger. But inhibition is stronger for full length myosin II, suggesting LMM maybe affected the sensitivity of myosin for Ca²⁺.

(2) The velocity of actin filaments caused by phosphorylated HMM was higher than unphosphorylated HMM, which agreed with the characters of Mg²⁺ -ATPase activity of myosin II before.

(3) Calcium could not impact the sliding velocity of actin filaments by mutant recombinant myosin II (it lacks calcium binding cite), suggesting that Ca²⁺ regulated the function of physarum myosin by binding with Ca²⁺-binding light chain (CaLC).

　

1. The effect of calcium on the sliding velocity of actin-filaments on a glass surface coated with unphosphorylated physarum myosin.

　

　

2. The effect of calcium on the sliding velocity of actin-filaments on a glass surface coated with unphosphorylated and phosphoryalted HMM.