Abstract
We have purified an actin-binding protein from the plasmodia of a lower eukaryote, Physarum polycephalum, with an apparent molecular mass of 210,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein bound to actin filaments with a stoichiometry of 1:7-8 in a Ca(2+)-calmodulin-dependent manner. Antibody raised against caldesmon from smooth muscle cross-reacted with the 210-kDa protein. In vitro motility assay revealed that the 210-kDa protein increased the sliding velocity of actin filaments on Physarum myosin. The 210-kDa protein more than doubled the actin-activated ATPase activity of Physarum myosin under comparative conditions of in vitro motility assay. Further increases in the concentration of the 210-kDa protein decreased its stimulatory effects. Ca(2+)-calmodulin prevented the stimulatory effects of the 210-kDa protein. Unexpectedly, smooth muscle caldesmon also increased the sliding velocity of actin filaments on smooth muscle myosin at lower concentrations. The well-known inhibitory effect of smooth muscle caldesmon on the actin-myosin interaction was observed with this motility assay when the concentration of the caldesmon was increased further. The stimulatory and inhibitory effects were confirmed by measurements of actin-activated ATPase activity of smooth muscle myosin. From estimations of the intracellular concentrations of the 210-kDa protein and smooth muscle caldesmon in vivo, it appears that effects of the former and the latter on actin-myosin interactions in vivo are stimulatory and inhibitory, respectively.
Highlights
We have purified an actin-binding protein from the linked regulation has received less attention and is poorly plasmodia of a lower eukaryote, Physarum polyce- understood
In vitro motility assay revealedthat the 210- an in uitro motility assay, inwhich ATP-dependent movement kDa protein increased the slidingvelocity of actin of actinfilaments is measured on a coverslip coated with filaments on Physarum myosin
The heavy chain of Physarum myosin could not be separated from the 210-kDa protein by SDS-PAGEunderthe conditions described under "Materials and Methods.",amonoclonal antibody raised against heavy chain of Physarum myosin [27] did not cross-react with the 210-kDa protein
Summary
Monoclonal antibody raised against smooth musclecaldesmon [15] cross-reacted with 210- and 130-kDa bands in the heat-stableHSE (Fig. 1, lanes I and 3 ). When calmodulin a t 14 nM was mixed the 210-kDa protein, the average velocity was reduced with from lane 6, purified caldesmon from chicken gizzard. The 210-kDa protein a t 0.07 P M stimulated mM) reduced the activity to 87nmol min-'mg", a result that the ATPase activity 2.3-fold (Fig. 6 A ) .The 210-kDa protein confirms previous observations [4, 22]. Bound to actin ata level equivalent to 10% saturation under the 210-kDa protein effectively stimulated the activityof the similar conditions (Fig. GB). The average sliding velocity of actin filaments was0.69 pm/s (Fig. a),confirming our recently reported data [13]. The sliding velocity was decreased to 0.23 pm/s (Fig. 8 E ) ,which was only 33% of the control velocity of actin filaments in the absence of smooth muscle caldesmon. Myosin, and the 210-kDa proteinin H S E of Physarum plasmodia
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