Calcium binding to tubulin

Abstract

Using flow dialysis, we found two classes of calcium-binding sites on tubulin: high-affinity binding sites ( 1.56 ± 0.38 per tubulin dimer) with a dissociation constant of ( 4.86 ± 0.12) · 10 −6 M and low-affinity binding sites ( 5.82 ± 0.50 per tubulin dimer) with a dissociation constant of ( 6.4 ± 0.4) · 10 −5 M. In the presence of 6 · 10 −5 M MgSO 4, we found 0.64 ± 0.18 calcium-binding sites per tubulin dimer with a dissociation constant of ( 4.7 ± 0.5) · 10 −6 M and 1.2 ± 0.2 sites per dimer with a dissociation constant of ( 3.5 ± 0.4) · 10 −5 M. Under controlled conditions, trypsin and chymotrypsin selectively cleaved α- and β-subunits, respectively, forming major fragments of 35 kDa and 20 kDa from the α-subunit, and major fragments of 31 kDa and 22 kDa from the β-subunit. The high-affinity calcium-binding sites were detecte in the carboxyl-terminal region of each tubulin subunit. Computer analysis of the subunit amino-acid sequences suggested possible locations of the putative calcium=binding sites.