Eddé B, Rossier J, Le Caer J P, Berwald-Netter Y, Koulakoff A, Gros F, Denoulet P
Laboratoire de Biochimie Cellulaire, Collège de France, Paris.
J Cell Biochem. 1991 Jun;46(2):134-42. doi: 10.1002/jcb.240460207.
We describe the presence of alpha-tubulin and MAP2 acetyltransferase activities in mouse brain. The enzyme(s) copurified with microtubules through two cycles of assembly-disassembly. Incubation of microtubule proteins with [3H]acetyl CoA resulted in a strong labeling of both alpha-tubulin and MAP2. To determine the site of the modification, tubulin was purified and digested with Glu-C endoproteinase. A unique radioactive peptide was detected and purified by HPLC. Edman degradation sequencing showed that this peptide contained epsilon N-acetyllysine at position 40 of the alpha-tubulin molecule. This result demonstrates that mouse brain alpha-tubulin was acetylated at the same site as in Chlamydomonas. Isoelectric focusing analysis showed that acetylated alpha-tubulin was resolved into five isoelectric variants, denoted alpha 3 and alpha 5 to alpha 8. This heterogeneity is not due to acetylation of other sites but results from a single acetylation of Lys40 of an heterogeneous population of alpha-tubulin isoforms. These isoforms are produced by posttranslational addition of one to five glutamyl units. Thus, neuronal alpha-tubulin is extensively modified by a combination of modifications including acetylation, glutamylation, tyrosylation, and other yet unknown modifications.
我们描述了小鼠脑中α-微管蛋白和微管相关蛋白2（MAP2）乙酰转移酶活性的存在。该酶通过两轮组装-拆卸与微管共纯化。微管蛋白与[3H]乙酰辅酶A孵育导致α-微管蛋白和MAP2均被强烈标记。为了确定修饰位点，纯化微管蛋白并用Glu-C内肽酶消化。通过高效液相色谱法检测并纯化了一种独特的放射性肽。埃德曼降解测序表明，该肽在α-微管蛋白分子的第40位含有ε-N-乙酰赖氨酸。该结果表明，小鼠脑α-微管蛋白的乙酰化位点与衣藻中的相同。等电聚焦分析表明，乙酰化的α-微管蛋白可解析为五个等电变体，分别表示为α3和α5至α8。这种异质性不是由于其他位点的乙酰化，而是由α-微管蛋白同工型异质群体中赖氨酸40的单一乙酰化导致的。这些同工型是通过翻译后添加一至五个谷氨酰基单位产生的。因此，神经元α-微管蛋白通过包括乙酰化、谷氨酰化、酪氨酰化和其他未知修饰在内的多种修饰组合而被广泛修饰。
