Related Experiment Videos
Outer arm dynein from trout spermatozoa. Purification, polypeptide composition, and enzymatic properties
J L Gatti1, S M King, A G Moss
1Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
The Journal of Biological Chemistry
|July 5, 1989
Summary
Trout sperm axonemes were analyzed to isolate and characterize the outer arm dynein. This two-headed 19S particle is crucial for sperm motility and likely represents a common structure in vertebrates.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Motors
Background:
- Sperm motility is essential for fertilization.
- Axonemal outer arms are key motor proteins driving flagellar movement.
- Understanding the structure and function of dynein is critical for reproductive biology.
Purpose of the Study:
- To isolate and characterize the 19S outer arm dynein from trout sperm axonemes.
- To determine the polypeptide composition and enzymatic properties of this dynein.
- To establish if this dynein is representative of other vertebrate outer arm dyneins.
Main Methods:
- Extraction of axonemes using 0.6 M NaCl.
- Sucrose density gradient centrifugation and DEAE-Sephacel chromatography for purification.
- Electrophoresis, quantitative densitometry, and electron microscopy for structural and compositional analysis.
- Enzymatic assays to determine MgATPase activity and substrate specificity.
Main Results:
- A 19S particle, representing the outer arm dynein, was successfully isolated.
- The 19S dynein comprises two heavy chains (alpha and beta), five intermediate chains, and six light chains.
- The purified dynein exhibits MgATPase activity, with optimal function at alkaline pH and preference for MgATP2-.
Conclusions:
- The isolated two-headed 19S particle is the complete trout sperm axonemal outer arm dynein.
- This dynein possesses specific enzymatic and structural characteristics.
- The findings suggest this trout sperm dynein is exemplary of outer arm dyneins in other vertebrates.