Related Experiment Videos
Properties of Physarum myosin purified by a potassium iodide procedure
Abstract:
Myosin has been purified free of actin from Physarum actomyosin by a two step adaptation of the classical potassium iodide method for depolymerizing actin. On 12% sodium dodecyl sulfate (SDS) gels, the single major slowly moving protein band present in the calcium activated adenosine triphosphatase peak (90% pure) is associated with two fast moving bands of molecular weights of approximately 17,000 and 21,000 daltons, respectively. Densitometry shows the molar ratio of heavy chains to the 21,000 and 17,000 dalton chains on the gels to be 1:2:1. The highly purified myosin forms filaments up to 2.5 microm long in the presence of 5 mM magnesium and 0.05 M KCl. Calcium ions were not required for the formation of long filaments from this highly purified myosin. At low ionic strength (0.05 M KCl) the magnesium ATPase of the highly purified myosin is activated four- to tenfold by muscle actin. The extent of activation is a function of the actin concentration and levels off at high levels of actin. In 0.1 mM calcium salts the ATPase activity is approximately 60% of that in 1 mM EGTA. In summary, Physarum myosin is similar to a number of muscle myosins as well as to platelet and fibroblast myosin, which all possess light chains of two different molecular weights associated with the heavy chains. Under ionic conditions close to those in vivo, highly purified Physarum myosin aggregates into long filaments.
Insights
Physarum myosin, purified using a modified potassium iodide method, forms long filaments and shows magnesium ATPase activation by actin. This myosin shares similarities with muscle and other non-muscle myosins.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Motors
Background:
- Myosin is a crucial protein for muscle contraction and cellular movement.
- Understanding myosin structure and function is key to deciphering cellular mechanics.
- Physarum polycephalum offers a unique model for studying non-muscle myosin.
Purpose of the Study:
- To purify and characterize myosin from Physarum actomyosin.
- To investigate the structural and functional properties of purified Physarum myosin.
- To compare Physarum myosin with myosins from other sources.
Main Methods:
- Purification of myosin using a modified potassium iodide method.
- Analysis of purified myosin by SDS-PAGE and densitometry.
- Filament formation studies under varying ionic conditions.
- Actin-activated magnesium ATPase assays.
Main Results:
- Highly purified Physarum myosin consists of heavy chains and two light chains (17,000 and 21,000 daltons) in a 1:2:1 molar ratio.
- Purified myosin forms long filaments (up to 2.5 microm) independent of calcium ions.
- Physarum myosin's magnesium ATPase activity is significantly activated by muscle actin in a concentration-dependent manner.
- Calcium ions slightly inhibit ATPase activity.
Conclusions:
- Physarum myosin shares structural and functional similarities with muscle, platelet, and fibroblast myosins, particularly in its light chain composition.
- Highly purified Physarum myosin can self-assemble into filaments under physiological ionic conditions.
- This study provides insights into the fundamental properties of Physarum myosin, relevant to understanding cellular motility.