Assembly of pure tubulin in the absence of free GTP: effect of magnesium, glycerol, ATP, and the nonhydrolyzable GTP analogues.
We describe in vitro microtubule assembly that exhibits, in bulk solution, behavior consistent with the GTP cap model of dynamic instability. Microtubules assembled from pure tubulin in the absence of free nucleotides could undergo one cycle of assembly, but could not sustain an assembly plateau. After the initial peak of assembly was reached and bound E-site GTP hydrolyzed to GDP, the microtubules gradually disassembled. We studied buffer conditions that maximized this disassembly while still allowing robust assembly to take place. While both glycerol and glutamate increased the rate of initial assembly and then slowed disassembly, magnesium promoted initial assembly and, surprisingly, enhanced disassembly. After cooling, a second cycle of assembly was unsuccessful unless GTP or the hydrolyzable GTP analogue GMPCPOP was readded. The nonhydrolyzable GTP analogues GMPPNP and GMPPCP could not support the second assembly cycle in the absence of E-site GTP. Analysis using HPLC found no evidence that GMPPNP, GMPPCP, or ATP could bind to free tubulin, and these nucleotides did not compete with GTP for the E-site. We have, however, demonstrated that the nonhydrolyzable GTP analogues and ATP do have an important effect on microtubule assembly. GMPPNP, GMPPCP, and ATP could each enhance the rate of assembly and stabilize the plateau of assembled microtubules against disassembly, while not binding appreciably to free tubulin. We conclude that these nucleotides, as well as GTP itself, enhance assembly by binding to a site on microtubules that is not present on free, unpolymerized tubulin. We estimate the affinity (KD) of the polymeric site for nucleotide triphosphates to be approximately 10(-4)M.
O'Brien
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Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710.
Erickson
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D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology*; D013552:Swine; D014404:Tubulin / Q000378:metabolism*
D046911:Macromolecular Substances; D014404:Tubulin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium; D005990:Glycerol