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An axoplasmic myosin with a calmodulin-like light chain
E L Bearer1, J A DeGiorgis, H Jaffe
1Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912, USA.
Summary
A novel myosin (p235) was purified from squid giant axons and identified as the motor protein responsible for organelle movement along actin filaments. This myosin is associated with a unique calcium-binding light chain, distinct from conventional myosin II light chains.
Area of Science:
- Cell Biology
- Molecular Motor Proteins
- Neuroscience
Background:
- Organelle transport in the squid giant axon relies on motor proteins interacting with actin filaments.
- A ~235-kDa protein (p235) in axoplasmic organelles was previously hypothesized to be the motor driving this movement.
Purpose of the Study:
- To purify and characterize the ~235-kDa protein (p235) from squid axoplasm.
- To determine if p235 is a myosin responsible for organelle transport.
- To identify and characterize the light chain associated with p235.
Main Methods:
- Protein purification of p235 from squid axoplasm.
- Biochemical assays for actin-activated Mg-ATPase activity.
- Low-angle rotary shadowing for structural analysis.
- Western blotting with pan-myosin antibodies.
- Amino acid sequencing and protein homology analysis.
- Antibody generation and characterization.
- Reverse transcriptase-PCR for gene cloning.
Main Results:
- Purified p235 is recognized by a pan-myosin antibody and exhibits high actin-activated Mg-ATPase activity, confirming it as a myosin.
- Structural analysis indicates p235 differs from myosin II and does not form bipolar filaments.
- A copurifying 17-kDa protein is a squid calcium-binding protein, highly homologous to calmodulin but distinct.
- An antibody against the 17-kDa protein recognizes the purified protein and confirms its association with organelles.
Conclusions:
- The ~235-kDa protein (p235) is identified as a novel myosin motor responsible for organelle movement in the squid giant axon.
- p235 is associated with a unique calmodulin-like light chain, suggesting a specialized function in organelle transport.
- This myosin and its light chain are integral components of the organelle transport machinery in neurons.