Expression Of Recombinant Myosin Ⅱ Of Physarum Polycephalum And Measurement Mg～(2+)-ATPase Activity Of Recombinant Myosin Ⅱ

Fungoid is an important media used to study the motility of cells. For instance,plasmodia of Physarum polycephalum shows vigorous cytoplasmic streaming, and themotive force of which is supported by the actin-myosin interaction. Ca2+ inhibits theactin-myosin interaction in Physarum polycephalum which is diametrically opposite toCa2+ activation of the interaction found in muscle and nonmuscle cells of mammals. Aphenomena attracts scientists' interest: myosin II from Physarum polycephalum andscallop are Ca2+-binding and their subunits are similar; however, Ca2+ works as aninhibitor for Physarum myosin and as an activator for scallop myosin. This similarity inthe subunits composition has raised the question of what subunit determines theinhibitory and stimulatory effects of Ca2+. For revealing the possible mechanism,recombinant myosin II was used to study myosin II structure, function and the model ofregulation. The virus vectors of myosin heavy chain, light chains, heavy chain of heavymeromyosin (HMM) of Physarum polycephalum and scallop myosin light chains havebeen successfully constructed. Also, HMM physarum polycephalum has been expressedin sf-9 cells. Using this recombinant HMM, it was found that the motility was slowdown when the concentration of calcium was increased, however, Mg2+-ATPaseactivity of HMM was not changed, suggesting that light meromyosin (LMM) ofmyosin maybe affects the sensitivity of Ca2+. To reveal the relationship between LMMof recombinant Physarum polycephalum myosin and the sensitivity of calcium and Mg2+-ATPase activity of recombinant myosin II of Physarum polycephalum, we expressedthe myosin II in sf-9 cells and studied Mg2+-ATPase activity and the sensitivity ofrecombinant myosin II of Physarum polycephalum for Ca2+ .The results were asfollows: 1. Expression of recombinant myosin II of Physarum polycephalum Sf-9 cells were coinfected with three separate recombinant baculovirus vectors: HC,PLC, CaLC. The infected Sf-9 cells were grown for 3 days at 28℃. The recombinantmyosin II was purified from the cell culture. 2. Characters of Mg 2~+ -ATPase activity of recombinant myosin II ofPhysarum polycephalum (1) Ca2+ inhibited Mg 2~+-ATPase activity of recombinant myosin II of Physarumpolycephalum , suggesting recombinant myosin II there was biological activity andLMM maybe affected the sensitivity of myosin for Ca2+. (2) Mg2+-ATPase activity of phosphrylated recombinant myosin II was higherthan that of unphosphrylated recombinant myosin II, which was agree with thecharacters of Mg2+-ATPase activity of myosin II from nature Physarum polycephalum. (3) Mg2+-ATPase activity of mutant recombinant myosin II was inhibited bycalcium slightly, namely, recombinant myosin II decreased the sensitivity for Ca2+,suggesting that Ca2+ regulated Mg2+-ATPase activity by binding with Ca2+-bindinglight chain(CaLC). Our results suggested that recombinant myosin II of Physarum polycephalum therewas biological active, and the same extent of Mg2+-ATPase activities of bothrecombinant and nature Physarum polycephalum were observed. It was suggested thatboth of construction and expression of recombinant myosin II of Physarumpolycephalum were successful .These results provided valuable information for furtherstudy on the structure and function of recombinant myosin II subunits of Physarumpolycephalum.