Purification and characterization of a basal body-associated Ca2+-binding protein.
Purification and characterization of a basal body-associated Ca2+-binding protein.
复制标题
基础与身体相关的Ca2+结合蛋白的纯化和表征。
DOI：
10.1083/jcb.107.1.121
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发表时间：
1988-07
影响因子：
7.8
通讯作者：
Schibler, M J
中科院分区：
文献类型：
--
作者：
Huang, B;Watterson, D M;Lee, V D;Schibler, M J
从单细胞藻类中分离出的莱茵衣藻基体复合物含有一种低分子质量的酸性多肽，不同于钙调蛋白，但具有与钙结合蛋白钙调蛋白家族成员共同的生化特征。这些共同特征包括相对较低的分子质量为20 kD，实验确定的酸性pI为5.3，在添加钙的情况下sds -聚丙烯酰胺凝胶中的电泳迁移率改变，以及钙依赖与疏水配体苯基- sepharose的结合，使其能够通过亲和层析纯化。基础体相关的20-kD钙结合蛋白（CaBP）与钙调蛋白的相关性通过氨基酸组成分析和部分肽测序得到证实。培养了一种针对20-kD CaBP的兔抗体，并用间接免疫荧光法确定了该蛋白在衣藻细胞中的细胞定位。在间期细胞中，抗体强烈地染色成对的基底体之间的区域、延伸在基底体和底层细胞核之间的两条纤维以及围绕细胞核的一列纵向细丝。在薄层电子显微镜下，两条基底体核连接纤维为狭窄的条纹纤维根。在有丝分裂细胞中，20-kD的CaBP特异性地与有丝分裂纺锤体的两极在复制的基底体复合体的位置相关。
Isolated basal body complexes from the unicellular alga, Chlamydomonas reinhardtii were found to contain a low molecular mass acidic polypeptide, distinct from calmodulin, but with biochemical features in common with members of the calmodulin family of calcium-binding proteins. These common characteristics included a relative low molecular mass of 20 kD, an experimentally determined acidic pI of 5.3, an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of added calcium, and a calcium-dependent binding to the hydrophobic ligand phenyl-Sepharose which allowed its purification by affinity chromatography. The relatedness of the basal body-associated 20-kD calcium-binding protein (CaBP) to calmodulin was confirmed by amino acid compositional analysis and partial peptide sequencing of the isolated protein. A rabbit antibody specific for the 20-kD CaBP was raised and used to determine by indirect immunofluorescence the cellular localization of the protein in Chlamydomonas cells. In interphase cells the antibody stained intensely the region between the paired basal bodies, two fibers extending between the basal bodies and the underlying nucleus, and an array of longitudinal filaments surrounding the nucleus. The two basal body-nuclear connecting fibers were identified in thin-section electron micrographs to be narrow striated fiber roots. In mitotic cells the 20-kD CaBP was specifically associated with the poles of the mitotic spindle at the sites of the duplicated basal body complexes.