In this application, we outline a series of experiments designid to provide a more in-depth understanding of the factors controlling the binding and hydrolytic steps which together constitute the tubulin GTPase reaction and their relationship to microtubule self-assembly: (1) Examination of the medium and intermediate oxygen-18 exchange reactions associated with the assembly-induced GTPase (The goal here is to learn about the reversibility of certain steps in the hydrolytic mechanism and the rate constants which define their rates and fluxes.); (2) determination of the stereochemical course of the hydrolysis using GTP Lambda S labeled with oxygen-16, -17 and -18 stereospecifically in the Lambda-phosphate (This will help discriminate between reactions involving a single in-line attack or two inversions, the latter characteristic of a phosphoryl-protein intermediates); (3) Characterization of the binding mechanism for guanine nucleotides (The objective is to learn more about the tubulin conformational changes preceding or attending ligand binding.); (4) Application of chromium (III) GTP complexes to define the stereochemistry of tubulin binding of metal-neucleotide complexes (The approach taken advantage of the invertness of chromium-nucleotide complexes to ligand exchange to help derive otherwise nearly inaccessible information about the binding of short-lived MgGTP2-binding.); (5) Studies of the time-course of hydrolysis during a single elongation step (This will help to distinguish between prompt and delayed DTP hydrolysis with repsect to tubulin binding to an elongating microtubule.); (6) Use of 5'-guanylyl peroxy-diphosphate to potentially modify the chemical course of GTPase action or to inhibit GTP bining to tubulin (This would represent the first attempt to utilize another hydrolyzable analogue of GTP at the exchangeable nucleotide site); (7) Reexamination of the 32Pi less than greater than GTP and 32Pi less than greater than GDP exchange reactions of porcine microtubule protein (This study promises to resolve the conflicting exchange properties of bovine and porcine brain microtubule proteins); (8) Development of an isothermal polymerization method to eliminate potentially misleading cold-stable forma of microtubule protein (This new approach of rapid isothermal concentration may be helpful in studying the regulation of assembly.). The overall goals of the project will be realized when results of these and other studies integrate into a global mechanism.
| Yamauchi, P S; Purich, D L (1987) Modulation of microtubule assembly and stability by phosphatidylinositol action on microtubule-associated protein-2. J Biol Chem 262:3369-75 |
