Lee S Y, Kim H J, Yoo S Y, Ahn T I
Department of Biology Education, Seoul National University, Korea.
J Eukaryot Microbiol. 1998 Jul-Aug;45(4):431-8. doi: 10.1111/j.1550-7408.1998.tb05095.x.
A monoclonal antibody was obtained that reacts with many different proteins (14-200 kDa) of Amoeba proteus. By indirect immunofluorescence microscopy we found the antigens to be dispersed throughout the cytoplasm but were more concentrated in the nucleus. The antibody cross-reacted with proteins of Tetrahymena, Xenopus embryo, and mouse macrophages. Using the antibody as a probe we cloned a cDNA of 1.2 kb coding for ubiquitin in five repeats. Amino acid sequences of ameba's polyubiquitin showed the most variations among the nineteen polyubiquitins of other organisms compared. The well-conserved 20Ser and 55Thr residues were replaced with Gly and Ser, respectively. The 28Ala residue found in most organisms was replaced with Gln or Glu in the amoeba. Amoebae contained two ubiquitin-mRNAs that could be detected by Northern blot analysis using the cDNA as a probe. In an analysis for specificity, the antibody reacted with polyubiquitin and ubiquitin-fusion proteins larger than 14 kDa but not with monomeric ubiquitin. The antibody is a useful probe in the detection and characterization of proteins ubiquitinated in response to cellular stresses.
获得了一种单克隆抗体，它能与变形虫的许多不同蛋白质（14 - 200 kDa）发生反应。通过间接免疫荧光显微镜观察，我们发现抗原分散在整个细胞质中，但在细胞核中更为集中。该抗体与四膜虫、非洲爪蟾胚胎和小鼠巨噬细胞的蛋白质发生交叉反应。以该抗体为探针，我们克隆了一个1.2 kb的cDNA，其编码五个重复的泛素。与其他生物的19种多聚泛素相比，变形虫的多聚泛素氨基酸序列变化最大。保守的20位丝氨酸和55位苏氨酸残基分别被甘氨酸和丝氨酸取代。大多数生物中存在的28位丙氨酸残基在变形虫中被谷氨酰胺或谷氨酸取代。变形虫含有两种泛素mRNA，使用该cDNA作为探针通过Northern印迹分析可以检测到。在特异性分析中，该抗体与大于14 kDa的多聚泛素和泛素融合蛋白发生反应，但不与单体泛素反应。该抗体是检测和鉴定细胞应激反应中泛素化蛋白质的有用探针。
