Inhibition of tubulin self-assembly and tubulin-colchicine GTPase activity by guanosine 5'-(gamma-fluorotriphosphate).
The inhibitory effects of guanosine 5'-(gamma-fluorotriphosphate) [GTP(gamma F)] on both the polymerization and the colchicine-dependent GTPase activity of calf brain tubulin have been studied. The results demonstrate that this analogue of GTP, with a fluorine atom on the gamma-phosphate, is a reversible competitive dead-end inhibitor of the colchicine-induced GTPase activity with a K1 value of (1.8 +/- 0.6) X 10(-4) M. GTP(gamma F) did not promote assembly of tubulin from which the E-site guanine nucleotide had been removed. It binds to the exchangeable nucleotide site competitively with respect to GTP, diminishing both the rate and extent of tubulin polymerization. Treatment in terms of the Oosawa-Kasai model of the inhibitory effect of GTP(gamma F) on the assembly led to a value of Kdis = 1.1 X 10(-6) M for the complex GTP(gamma F)-tubulin. This analogue does not bind to the postulated third site. The growing of tubulin polymers at 37 degrees C was arrested by GTP(gamma F), and only limited depolymerization was induced by the addition of this analogue after assembly in the presence of GTP. This result confirms that the E-site is blocked in the polymer and that this analogue can bind only to the ends of the polymers. Sedimentation velocity and circular dichroism studies showed that the conformation of the tubulin-GTP(gamma F) complex is not identical with that of tubulin-GTP. This is caused by the replacement of the hydroxyl group in the gamma-phosphate by the fluorine group, which have 2.20- and 1.35-A van der Waals radii, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Monasterio
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Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Timasheff
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D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D003078:Colchicine; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D011487:Protein Conformation; D014404:Tubulin / Q000302:isolation & purification; D050257:Tubulin Modulators
D046911:Macromolecular Substances; D014404:Tubulin; D050257:Tubulin Modulators; C054439:guanosine 5'-(gamma-fluorotriphosphate); D006160:Guanosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D003078:Colchicine