Xiang H, MacRae T H
Department of Biology, Dalhousie University, Halifax, NS, Canada.
Biochem Cell Biol. 1995 Sep-Oct;73(9-10):673-85. doi: 10.1139/o95-075.
The reversible, enzymatically driven removal and readdition of its carboxy-terminal tyrosine are major posttranslational modifications of alpha-tubulin. To study these processes isoform-specific antibodies were produced and subsequently used to characterize tyrosinated and detyrosinated tubulin in the brine shrimp, Artemia. Tyrosinated tubulin existed in relatively constant amounts on western blots of cell-free protein extracts from Artemia at all developmental stages examined, whereas detyrosinated tubulin was present after 20-24 h of postgastrula growth. In agreement with the blots, the detyrosinated isoform was observed in immunofluorescently stained larvae after 24 h of incubation, appearing first in structures of a transient nature, namely spindles and midbodies. The elongated muscle cells encircling the gut and the epithelium bordering the gut lumen were stained extensively with antibody to detyrosinated tubulin. Detyrosination was accompanied by the appearance of a tubulin-reactive carboxypeptidase, which used both nonpolymerized and polymerized tubulin as substrate. The enzyme bound to microtubules very poorly, if at all, under conditions used in this work. Several inhibitors of carboxypeptidase A had no effect on the carboxypeptidase from Artemia and revealed similarities between this enzyme and others thought to be tubulin specific. The use of inhibitors also indicated that the carboxypeptidase from Artemia recognized aspects of tubulin structure in addition to the carboxy-terminal tyrosine. Our results support the idea that detyrosinated tubulin appears in microtubules of varying stability, and they demonstrate that Artemia possess a carboxypeptidase with the potential to detyrosinate tubulin during growth of larvae.
α-微管蛋白的可逆性、酶促驱动的羧基末端酪氨酸去除和重新添加是主要的翻译后修饰。为了研究这些过程，制备了亚型特异性抗体，随后用于表征卤虫（Artemia）中的酪氨酸化和去酪氨酸化微管蛋白。在所有检测的发育阶段，卤虫无细胞蛋白质提取物的蛋白质印迹上，酪氨酸化微管蛋白的含量相对恒定，而去酪氨酸化微管蛋白在原肠胚后期生长20 - 24小时后出现。与印迹结果一致，在孵育24小时后的免疫荧光染色幼虫中观察到去酪氨酸化亚型，首先出现在具有短暂性质的结构中，即纺锤体和中间体。环绕肠道的细长肌肉细胞和与肠腔相邻的上皮细胞被抗去酪氨酸化微管蛋白抗体广泛染色。去酪氨酸化伴随着一种微管蛋白反应性羧肽酶的出现，该酶以未聚合和聚合的微管蛋白为底物。在本研究使用的条件下，该酶与微管的结合非常差，甚至根本不结合。几种羧肽酶A抑制剂对卤虫的羧肽酶没有影响，并揭示了该酶与其他被认为是微管蛋白特异性酶之间的相似性。抑制剂的使用还表明，卤虫的羧肽酶除了识别羧基末端酪氨酸外，还识别微管蛋白结构的其他方面。我们的结果支持去酪氨酸化微管蛋白出现在稳定性不同的微管中的观点，并且表明卤虫在幼虫生长过程中拥有一种有潜力使微管蛋白去酪氨酸化的羧肽酶。
