Sioussat T M, Boekelheide K
Department of Pathology, Brown University, Providence, Rhode Island 02912.
Biochemistry. 1989 May 16;28(10):4435-43. doi: 10.1021/bi00436a047.
Following a 16-h incubation with a large excess of 2,5-hexanedione (2,5-HD) while in the assembled state, bovine brain tubulin contained a powerful nucleating component, the presence of which lowered the dissociation rate from 83 s-1 for untreated tubulin to 13 s-1 for 2,5-HD-treated tubulin. This nucleating component could be selectively concentrated by sequential stringent (conditions of low temperature and low tubulin concentration) cycles of assembly and disassembly. In 2-(N-morpholino)ethanesulfonic acid buffer without glycerol, the critical concentration of assembly of untreated tubulin (2.4 mg/mL) was 19 times higher than that of 2,5-HD-treated tubulin subjected to three sequential stringent cycles of assembly and disassembly (0.13 mg/mL). This highly nucleating 2,5-HD-treated tubulin preparation could both copolymerize with untreated tubulin and seed subcritical concentration assembly of untreated tubulin. Experiments to define the assembly-altering component have identified structural alterations to the alpha-tubulin monomer. While the alpha-tubulin subunit of native untreated tubulin dimer contained no chymotryptic cleavage sites, the native 2,5-HD-treated alpha-tubulin subunit was cleaved by chymotrypsin to yield a 37-kDa C-terminal fragment.
在组装状态下与大量过量的2,5 - 己二酮（2,5 - HD）孵育16小时后，牛脑微管蛋白含有一种强大的成核成分，其存在将解离速率从未处理微管蛋白的83 s⁻¹降低至2,5 - HD处理微管蛋白的13 s⁻¹。这种成核成分可以通过连续严格的（低温和低微管蛋白浓度条件下）组装和解聚循环进行选择性浓缩。在不含甘油的2 - （N - 吗啉代）乙磺酸缓冲液中，未处理微管蛋白（2.4 mg/mL）的组装临界浓度比经过三个连续严格组装和解聚循环的2,5 - HD处理微管蛋白（0.13 mg/mL）高19倍。这种高度成核的2,5 - HD处理微管蛋白制剂既能与未处理微管蛋白共聚，又能引发未处理微管蛋白亚临界浓度的组装。确定改变组装成分的实验已确定α - 微管蛋白单体的结构改变。虽然天然未处理微管蛋白二聚体的α - 微管蛋白亚基不含胰凝乳蛋白酶切割位点，但天然2,5 - HD处理的α - 微管蛋白亚基被胰凝乳蛋白酶切割产生一个37 kDa的C末端片段。
