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GTP hydrolysis during microtubule assembly
E T O'Brien1, W A Voter, H P Erickson
1Duke University Medical Center, Durham, North Carolina 27710.
Biochemistry
|June 30, 1987
Summary
The GTP cap model explains microtubule dynamics. This study found GTP hydrolysis is coupled to microtubule assembly, not delayed, challenging previous findings on GTP cap stability.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Dynamics
Background:
- The GTP cap model suggests GTP-tubulin subunits stabilize microtubules, preventing rapid disassembly.
- This model assumes GTP hydrolysis occurs after subunit assembly, not coupled to it.
- Previous studies reported a lag in GTP hydrolysis and a slow hydrolysis rate constant (kh).
Purpose of the Study:
- To investigate the timing of GTP hydrolysis relative to microtubule assembly.
- To determine the rate constant for GTP hydrolysis (kh) under various conditions.
- To evaluate the GTP cap model's validity based on experimental findings.
Main Methods:
- Utilized tubulin free of microtubule-associated protein at concentrations of 18.5-74 microM.
- Performed microtubule assembly experiments with and without glycerol.
- Employed two independent assays to measure GTP hydrolysis rates.
Main Results:
- No lag between GTP hydrolysis and microtubule assembly was observed under any tested conditions.
- Initial rates of GTP hydrolysis were directly proportional to microtubule assembly rates.
- Estimated kh to be at least 2.5/min, significantly faster than previously reported.
Conclusions:
- GTP hydrolysis is coupled to microtubule assembly, contradicting the uncoupled hydrolysis assumption of the GTP cap model.
- A faster kh value supports the dynamic instability of microtubules.
- A preliminary model of assembly coupled to hydrolysis was proposed, explaining GTP cap formation and loss.