Geahlen R L, Haley B E
Proc Natl Acad Sci U S A. 1977 Oct;74(10):4375-7. doi: 10.1073/pnas.74.10.4375.
Tubulin dimers isolated from brain contain two GTP binding sites, a nonexchangeable site and an exchangeable site. To localize the exchangeable site, we used a photoaffinity analog of GTP, 8-azidoguanosine triphosphate (8-N3GTP), which supports tubulin polymerization in the absence of activating light. Photolysis of tubulin polymerized in the presence of 0.01 to 0.1 mM [beta, gamma-32P]8-N3GTP resulted in covalent incorporation of radioactivity only onto the beta monomer. Photolysis with 8-N3GTP also prevented any further repolymerization of the tubulin whereas like treatment in the presence of GTP had no effect. Preincubation of tubulin with GTP prevented photo-incorporation of [beta, gamma-32P]8-N3GTP whereas preincubation with ATP did not.
从大脑中分离出的微管蛋白二聚体含有两个GTP结合位点，一个不可交换位点和一个可交换位点。为了定位可交换位点，我们使用了一种GTP的光亲和类似物，8-叠氮鸟苷三磷酸（8-N3GTP），它在没有激活光的情况下支持微管蛋白聚合。在0.01至0.1 mM [β,γ-32P]8-N3GTP存在下聚合的微管蛋白经光解后，放射性仅共价结合到β单体上。用8-N3GTP进行光解也阻止了微管蛋白的任何进一步再聚合，而在GTP存在下进行类似处理则没有效果。微管蛋白与GTP预孵育可防止[β,γ-32P]8-N3GTP的光掺入，而与ATP预孵育则没有此作用。
