Interactions of tubulin with guanylyl-(beta-gamma-methylene)diphosphonate. Formation and assembly of a stoichiometric complex

Complete replacement of the nucleotide on the exchangeable binding site of purified calf brain tubulin by the non-hydrolyzable GTP-analogue guanylyl-(beta,gamma-methylene)diphosphonate (GMPPCP) has been achieved by treatment of tubulin-GDP with phosphodiesterase-free alkaline phosphatase. GMPPCP bin...

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Bibliographic Details
Published in:The Journal of biological chemistry 1990-05, Vol.265 (13), p.7655-7661
Main Authors: Seckler, R, Wu, G M, Timasheff, S N
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Brain - metabolism
Cattle
Chromatography, High Pressure Liquid
Fundamental and applied biological sciences. Psychology
Glycerol - pharmacology
Guanine Nucleotides - isolation & purification
Guanosine Diphosphate - metabolism
Guanosine Triphosphate - analogs & derivatives
Guanosine Triphosphate - metabolism
guanylyl-( beta - gamma -methylene)diphosphonate
Interactions. Associations
Intermolecular phenomena
Kinetics
Microscopy, Electron
Molecular biophysics
Protein Binding
Tubulin - isolation & purification
Tubulin - metabolism
Tubulin - ultrastructure
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Summary:Complete replacement of the nucleotide on the exchangeable binding site of purified calf brain tubulin by the non-hydrolyzable GTP-analogue guanylyl-(beta,gamma-methylene)diphosphonate (GMPPCP) has been achieved by treatment of tubulin-GDP with phosphodiesterase-free alkaline phosphatase. GMPPCP binds to tubulin with a low affinity relative to GTP or GDP. Binding of the analogue is linked to magnesium ion concentration and, like the binding of other guanine nucleotides, is promoted by high concentrations of glycerol. The complex of pure tubulin and GMPPCP readily assembles at 37 degrees C into microtubules or curled ribbons of protofilaments, depending on buffer composition. Assemblies are cold-reversible at 0-2 degrees C, and multiple reversible assemblies can be observed during repeated heating/cooling cycles.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)39164-1