Monoclonal antibodies Ki-S3 and Ki-S5 yield new data on the 'Ki-67' proteins
Monoclonal antibodies Ki-S3 and Ki-S5 yield new data on the 'Ki-67' proteins
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DOI：
10.1111/j.1365-2184.1996.tb00984.x
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发表时间：
1996-07-01
影响因子：
8.5
通讯作者：
Parwaresch, R
中科院分区：
文献类型：
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作者：
Heidebrecht, HJ;Buck, F;Parwaresch, R
单克隆抗体 (mab) Ki-67 已使用约 10 年，主要用于组织切片，以监测增殖细胞，但迄今为止，人们对其识别的蛋白质知之甚少。新的单克隆抗体 Ki-S3 和 Ki-S5 可检测冷冻和石蜡包埋组织中的增殖细胞。他们在蛋白质印迹中以及首次在免疫沉淀实验中识别出与 Ki-67 具有相同分子量的蛋白质。利用这些单克隆抗体，我们能够富集和纯化 ki-67 蛋白。消化蛋白的四个肽的蛋白质测序与已经发表的 ki-67 蛋白的 cDNA 推导的氨基酸序列相对应。由于我们能够免疫沉淀 Ki-67 蛋白，因此我们进行了各种免疫沉淀实验以获得有关这些蛋白性质的更多信息。用 [S-35]-甲硫氨酸放射性标记 L428 细胞后，我们仅在标记时间 5 分钟后就能够免疫沉淀 Ki-67 蛋白。在周转实验中，标记结束后 3 小时无法检测到 Ki-67 蛋白。这些数据表明 Ki-67 蛋白的半衰期约为 90 分钟。[P-32]-正磷酸盐标记实验表明 Ki-67 蛋白被磷酸化。用冈田酸阻断去磷酸化或用秋水仙胺抑制细胞生长后，Ki-67蛋白的磷酸化大大增加，表明ki-67蛋白是通过丝氨酸和苏氨酸磷酸化的，并且Ki-67蛋白的磷酸化在循环细胞中增加。 [H-3]-甘露糖和[H-3]-葡萄糖标记实验表明该蛋白质具有弱N-糖基化。
The monoclonal antibody (mab) Ki-67 has been used for about 10 years, mainly in tissue sections, to monitor proliferating cells, but so far only very little is known about the proteins it recognizes. The new mabs Ki-S3 and Ki-S5 detect proliferating cells in frozen and paraffin-embedded tissues. They recognize proteins with the same molecular mass as Ki-67 in western blot and for the first time also in immunoprecipitation experiments. With these mabs we were able to enrich and purify the ki-67 proteins. Protein sequencing of four peptides of the digested proteins corresponded to the cDNA-deduced amino acid sequence already published for the ki-67 proteins.Since we were able to immunoprecipitate the Ki-67 proteins, we performed various immunoprecipitation experiments to obtain more information about the nature of these proteins. After radiolabelling L428 cells with [S-35]-methionine we were able to immunoprecipitate the Ki-67 proteins after only 5 minutes of labelling time. In turnover experiments the Ki-67 proteins could not be detected 3 hours after the end of labelling. These data indicate a half-life of the Ki-67 proteins of about 90 minutes.Labelling experiments with [P-32]-orthophosphate revealed that the Ki-67 proteins are phosphorylated. After dephosphorylation was blocked with okadaic acid or cell growth was arrested by means of Colcemid, the phosphorylation of the Ki-67 proteins was greatly increased, indicating that the ki-67 proteins are phosphorylated via serine and threonine, and that the phosphorylation of the Ki-67 proteins increases in cycling cells. Labelling experiments with [H-3]-mannose and [H-3]-glucose revealed that the protein is weakly N-glycosylated.