Fong K C, Babitch J A, Anthony F A
Chemistry of Behavior Program, Texas Christian University, Forth Worth 76129.
Biochim Biophys Acta. 1988 Jan 4;952(1):13-9. doi: 10.1016/0167-4838(88)90096-9.
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 MgSO4, 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 alpha- and beta-subunits, respectively, forming major fragments of 35 kDa and 20 kDa from the alpha-subunit, and major fragments of 31 kDa and 22 kDa from the beta-subunit. The high-affinity calcium-binding sites were detected 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.
使用流动透析法，我们在微管蛋白上发现了两类钙结合位点：高亲和力结合位点（每微管蛋白二聚体有1.56±0.38个），解离常数为(4.86±0.12)×10⁻⁶ M；低亲和力结合位点（每微管蛋白二聚体有5.82±0.50个），解离常数为(6.4±0.4)×10⁻⁵ M。在6×10⁻⁵ M MgSO₄存在的情况下，我们发现每微管蛋白二聚体有0.64±0.18个钙结合位点，解离常数为(4.7±0.5)×10⁻⁶ M，每二聚体还有1.2±0.2个位点，解离常数为(3.5±0.4)×10⁻⁵ M。在可控条件下，胰蛋白酶和胰凝乳蛋白酶分别选择性地切割α和β亚基，从α亚基形成35 kDa和20 kDa的主要片段，从β亚基形成31 kDa和22 kDa的主要片段。在每个微管蛋白亚基的羧基末端区域检测到了高亲和力钙结合位点。对亚基氨基酸序列的计算机分析表明了假定钙结合位点的可能位置。
