ALPHA-TUBULIN INFLUENCES NUCLEOTIDE BINDING TO BETA-TUBULIN - AN ASSAY USING PICOMOLES OF UNPURIFIED PROTEIN

Tubulin binds guanine nucleotides tightly within its β subunit. Whether the a subunit influences binding to this site has been unknown. This question was addressed by comparing the nucleotide binding properties of the free β subunit with those of the heterodimer. The free β subunit was obtained from an in vitro expression system and its nucleotide binding properties were determined by an assay that requires ≈100-fold less protein than conventional assays. This assay exploits the observation that the recovery of β-tubulin from Mono Q anion-exchange columns is dependent on added nucleotide. Our results demonstrate that the newly synthesized β subunit and the heterodimer bind nucleotides with similar specificity. We found that in the presence of magnesium the α subunit enhances GTP binding to the β subunit ≈4-fold. However, in the absence of magnesium the α subunit appears to specifically weaken GTP binding to the β subunit. Thus, nucleotide binding to the E site in the heterodimer may not be solely defined by the β subunit.