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  • Purification and biochemical characterization of tubulin from the budding yeast Saccharomyces cerevisiae.

Purification and biochemical characterization of tubulin from the budding yeast Saccharomyces cerevisiae.

Biochemistry (1993-08-31)
A Davis, C R Sage, L Wilson, K W Farrell
ABSTRACT

We describe a method for isolating milligram quantities of assembly-competent tubulin from the budding yeast Saccharomyces cerevisiae. The tubulin is > 95% purified and free of contaminating enzyme activities. As a result, it has been possible to determine the yeast tubulin equilibrium-binding constant for Mg-GTP and the tubulin GTPase activity under nonassembling and assembling conditions. We also determined the critical concentration for yeast tubulin polymerization and found it to be significantly lower than that for bovine brain tubulin under identical conditions. Similarly, the dynamic properties both of individual yeast microtubules and of bulk microtubule suspensions were significantly different from those of bovine brain microtubules free of microtubule-associated proteins. The data suggest that the properties of the yeast tubulin may reflect the particular functional requirements of the yeast cell. With this method, it is now possible to introduce any desired tubulin gene mutation into the budding yeast and correlate the phenotypic effects of the mutation in cells with the effects of the mutation on the biochemical and polymerization properties of the tubulin.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Guanosine 5′-triphosphate sodium salt hydrate, ≥95% (HPLC), powder
Sigma-Aldrich
Guanosine 5′-triphosphate sodium salt solution, HPLC purified, aqueous solution for RNA polymerase transcription
Sigma-Aldrich
Guanosine 5′-triphosphate sodium salt hydrate, ≥90% (HPLC)