In vivo microtubules are copolymers of available beta-tubulin isotypes: localization of each of six vertebrate beta-tubulin isotypes using polyclonal antibodies elicited by synthetic peptide antigens.
In vivo microtubules are copolymers of available beta-tubulin isotypes: localization of each of six vertebrate beta-tubulin isotypes using polyclonal antibodies elicited by synthetic peptide antigens.
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体内微管是可用的β-微管蛋白同种型的共聚物：使用合成肽抗原引起的多克隆抗体的六个脊椎动物β-微管蛋白同种型的定位。
DOI：
10.1083/jcb.105.4.1707
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发表时间：
1987-10
影响因子：
7.8
通讯作者：
Cleveland, D W
中科院分区：
文献类型：
--
作者：
Lopata, M A;Cleveland, D W
β-微管蛋白在高等动物的基因组中由一个小的多基因家族编码，该家族包括大约7个功能基因。这些基因产生一个密切相关但不同的多肽同种型家族，其主要通过约15个羧基末端氨基酸残基内的序列来区分。通过用化学合成的与这些可变结构域序列相对应的肽免疫兔子，我们现在已经制备了针对六种不同同种型中的每一种的多克隆抗体。通过抗体与代表各同种型的细菌产生的克隆蛋白的结合，以及通过将各抗血清仅与免疫肽而不与异源肽预孵育来抑制这种结合，明确证明了各抗血清的特异性。已知量的克隆的、同种型纯多肽的蛋白质印迹已经允许精确定量测量在各种细胞中以可溶性和聚合物形式存在的每种β-微管蛋白同种型的量，但是没有揭示任何同种型的优先组装的偏差。在三种不同的细胞类型，使用间接免疫荧光的每种同种型的本地化已经证明，在体内的每一类微管可区分的光学显微镜组装作为共聚物的所有同种型在一个单一的细胞中表达。
beta-Tubulin is encoded in the genomes of higher animals by a small multigene family comprising approximately seven functional genes. These genes produce a family of closely related, but distinct polypeptide isotypes that are distinguished principally by sequences within the approximately 15 carboxy-terminal amino acid residues. By immunizing rabbits with chemically synthesized peptides corresponding to these variable domain sequences, we have now prepared polyclonal antibodies specific for each of six distinct isotypes. Specificity of each antiserum has been demonstrated unambiguously by antibody binding to bacterially produced, cloned proteins representing each isotype and by the inhibition of such binding by preincubation of each antiserum only with the immunizing peptide and not with heterologous peptides. Protein blotting of known amounts of cloned, isotypically pure polypeptides has permitted accurate quantitative measurement of the amount of each beta- tubulin isotype present in the soluble and polymer forms in various cells, but has not revealed a bias for preferential assembly of any isotype. Localization of each isotype in three different cell types using indirect immunofluorescence has demonstrated that in vivo each class of microtubules distinguishable by light microscopy is assembled as copolymers of all isotypes expressed in a single cell.