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Adenosine 5'-O-(3-thiotriphosphate) hydrolysis by dynein
T Shimizu1, T Katsura, P L Domanico
1Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Biochemistry
|August 22, 1989
Summary
Dynein
Area of Science:
- Biochemistry
- Molecular Biology
- Motor Proteins
Background:
- Dynein is a crucial motor protein involved in intracellular transport.
- Understanding dynein's interaction with ATP is key to elucidating its mechanism.
- ATP gamma S serves as a non-hydrolyzable ATP analog to study binding and dissociation.
Purpose of the Study:
- To investigate the interaction of dynein with ATP gamma S.
- To determine the mechanism and kinetics of microtubule-dynein complex dissociation induced by ATP gamma S.
- To characterize the hydrolysis of ATP gamma S by dynein and the role of microtubules.
Main Methods:
- Stopped-flow light-scattering assays to monitor complex dissociation.
- Rapid quench-flow experiments to study ATP gamma S hydrolysis.
- Kinetic analysis of ATP gamma S concentration dependence.
- Use of stereospecifically labeled ATP gamma S to determine reaction mechanism.
Main Results:
- ATP gamma S competitively inhibits ATP hydrolysis and induces complete dissociation of the microtubule-dynein complex.
- Dissociation kinetics follow a hyperbolic curve, indicating a multi-step process with an apparent Kd of 0.5 mM.
- ATP gamma S hydrolysis by dynein shows an initial burst of product formation, and microtubules activate thiophosphate release.
- The rate of ATP gamma S turnover is 4-8 times slower than ATP turnover, with thiophosphate release being rate-limiting.
Conclusions:
- ATP gamma S is a valuable tool for studying dynein's interaction with ATP and its dissociation mechanism.
- Microtubules play a role in activating the release of thiophosphate from dynein.
- The findings provide insights into the kinetics and mechanism of dynein motor function.