Rüdiger M, Weber K
Max Planck Institute for Biophysical Chemistry, Department of Biochemistry, Göttingen, Germany.
Eur J Biochem. 1993 Nov 15;218(1):107-16. doi: 10.1111/j.1432-1033.1993.tb18357.x.
Tubulin purified from turkey erythrocytes was characterized by partial protein sequence data, high-resolution IEF and by its reaction with antibodies specific for certain post-translational modifications. The tubulin from the marginal band contains a single alpha and beta isotype, i.e. alpha 1 and beta 6. Partial protein sequences and immunoblotting with antibody 6-11B-1 show that erythrocyte alpha 1 tubulin is not acetylated at Lys40. The acidic carboxy-terminal peptides purified by Mono Q chromatography and reverse-phase HPLC were characterized by sequence analysis and mass spectrometry. Although erythrocyte alpha tubulin is almost completely detyrosinated it retains the penultimate glutamic acid residue, which is partially lost in brain tubulin. Thus erythrocyte tubulin is an excellent substrate for extensive in vitro tyrosination by tubulin-tyrosine ligase. Erythrocyte alpha and beta tubulin lack the side-chain polyglutamylation found in all major tubulins from adult brain. Finally we show that about 10% of the beta tubulin is phosphorylated at Ser441. Thus erythrocyte tubulin is an unusual homogeneous preparation. It contains the minimum possible number of tubulin isotypes and the only post-translational modifications detected (detyrosination and phosphorylation) are reversible.
从火鸡红细胞中纯化得到的微管蛋白通过部分蛋白质序列数据、高分辨率等电聚焦以及其与针对某些翻译后修饰的特异性抗体的反应进行了表征。边缘带中的微管蛋白包含单一的α和β同种型，即α1和β6。部分蛋白质序列以及用抗体6-11B-1进行的免疫印迹表明，红细胞α1微管蛋白在赖氨酸40处未被乙酰化。通过Mono Q色谱法和反相高效液相色谱法纯化得到的酸性羧基末端肽通过序列分析和质谱进行了表征。尽管红细胞α微管蛋白几乎完全去酪氨酸化，但它保留了倒数第二个谷氨酸残基，而该残基在脑微管蛋白中会部分丢失。因此，红细胞微管蛋白是微管蛋白-酪氨酸连接酶进行广泛体外酪氨酸化的优良底物。红细胞α和β微管蛋白缺乏在成人大脑所有主要微管蛋白中发现的侧链多聚谷氨酸化。最后，我们表明约10%的β微管蛋白在丝氨酸441处被磷酸化。因此，红细胞微管蛋白是一种不寻常的均匀制剂。它包含尽可能少的微管蛋白同种型，并且检测到的唯一翻译后修饰（去酪氨酸化和磷酸化）是可逆的。
