BINDING CONSTANTS AND STOICHIOMETRIES OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE-TUBULIN COMPLEXES

The catalytic activity of glyceraldehyde 3-phosphate dehydrogenase (GA PDH) decreased (almost linearly) as a function of increasing concentra tions of tubulin; the total loss in activity was attained at a ratio o f 1.2 to 1.8 tubulin dimer to GAPDH tetramer. Based on the inhibition data, a dissociation constant for the tubulin-GAPDH complex was calcul ated to be about 0.73 nM. The stoichiometry and the dissociation const ants of the tubulin-GAPDH complex were found to be dependent upon the ionic strength of the assay media. Qualitatively similar results were obtained (i.e., inhibition and ionic strength effect) when the GAPDH-c atalyzed reaction was measured in the presence of Sepharose-immobilize d tubulin. The physical interaction between these two proteins, i.e., GAPDH and tubulin, was measured by the ability of one protein (immobil ized on a Sepharose matrix) to copellet the other protein. By employin g this copelleting technique, we measured the dissociation constant an d stoichiometry of the immobilized tubulin-GAPDH complex to be about 6 .4 nM and 0.91 tubulin dimer/GAPDH tetramer, respectively. The dissoci ation constant and stoichiometry thus obtained were found to be remark ably similar to those obtained by the tubulin-dependent GAPDH inhibiti on data. In contrast to these results, (soluble) tubulin had no effect on the catalytic activity of the immobilized GAPDH, albeit the solubl e tubulin copelleted with the immobilized GAPDH. The dissociation cons tant and stoichiometry of immobilized GAPDH-tubulin complex were calcu lated to be 0.76 +/- 0.13 mu M and 3.23 +/- 0.16 tubulin dimer/GAPDH t etramer, respectively. These data suggest that there are two classes o f binding sites for tubulin on a tetrameric GAPDH; high-affinity and l ow-affinity sites. The enzyme is inhibited when tubulin binds at the h igh-affinity site while the catalytic function of the enzyme is unaffe cted when the tubulin binds at the low-affinity site. The latter site is suggested herein to be responsible for the crosslinking (bundling) of microtubules. (C) 1994 Academic Press,Inc.