Piperno G, Fuller M T
J Cell Biol. 1985 Dec;101(6):2085-94. doi: 10.1083/jcb.101.6.2085.
Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.
七种针对海胆精子鞭毛轴丝微管蛋白产生的单克隆抗体，识别多种生物体轴丝中存在的一种乙酰化形式的α-微管蛋白。在多种细胞的可溶性细胞质α-微管蛋白同工型中未检测到该抗原。抗体的特异性通过对海胆和衣藻粗提物中的细胞质微管蛋白进行体外乙酰化来确定。在提取物中的所有乙酰化多肽中，只有α-微管蛋白具有抗原性。在衣藻微管蛋白同工型中，抗体仅识别在赖氨酸的ε-氨基上进行了体内乙酰化的轴丝α-微管蛋白同工型（L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473 - 478）。这些抗体不识别未修饰的轴丝α-微管蛋白、鞭毛基质加膜部分中存在的未组装α-微管蛋白，或衣藻细胞体中的可溶性细胞质α-微管蛋白。该抗原存在于含有多种来源轴丝微管的蛋白质组分中，这些来源包括海胆囊胚和四膜虫的纤毛、果蝇的精子和睾丸以及人类精子。相比之下，在可溶性细胞质微管蛋白制剂中未检测到该抗原，这些制剂不包含来自稳定微管阵列（如中心粒）的微管蛋白，这些制剂来自未受精的海胆卵、果蝇胚胎和HeLa细胞。尽管抗体识别的乙酰化α-微管蛋白存在于多种来源的轴丝中，并且可能是轴丝形成所必需的，但它并非仅存在于形态上不同的轴丝微管的任何一个子集中。在富含中央微管对或外二联体B或外二联体A微管的海胆精子轴丝组分中，以相似的比例发现了该抗原。因此，α-微管蛋白的乙酰化并未提供指定任何一类轴丝微管结构的机制。初步证据表明，乙酰化α-微管蛋白并不局限于轴丝。本报告中描述的抗体可能使我们能够推断微管蛋白乙酰化在体内微管结构和功能中的作用。
