This chapter presents methods for probing tubulin-nucleotide interactions as well as a group of assays for evaluating the associated enzyme activities in a more quantitative fashion. The methods include the preparation of tubulin-nucleotide complexes, such as GTP regenerating systemsand labeled nucleotide complexes. It is necessary to devise adequate strategies for making tubulin-nucleotide complexes of known stoichiometry and stability. The tight binding of guanine nucleotides to tubulin was alluded to earlier, and this represents a major experimental hurdle for characterizing more weakly bound nucleotides with tubulin. From the experience, gel filtration and dialysis are practically ineffective for the purpose of freeing tubulin of the exchangeable nucleotide. Two less desirable methods have been perfected for this purpose: charcoal method and alkaline phosphatase method. The adsorption of nucleotides to charcoal at slightly acidic pH is a useful means to prepare nucleotide-depleted tubulin samples. The phosphatase treatment is a method for replacing guanine nucleotide by nonhydrolyzable analogs rather than a method for removal of the nucleotide from the tubulin sites.
ASJC Scopus subject areas
- Molecular Biology