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Purification and biochemical, cell biological analysis o a new human GTP-binding protein

一种新型人GTP结合蛋白的纯化及生化、细胞生物学分析

基本信息

批准号：
01580199
负责人：
TAKANO Toshiya
金额：
$ 1.47万
依托单位：
Keio university School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01580199/
关键词：
Rab-2 gene GTP-binding protein Transcription Complementary DNA Messenger RNA Polyadenylation site Antiserum Modification of protein GTP結合蛋白質 cDNA 蛋白質のりん酸化 rabー2蛋白質ウエスタンブロック法免疫沈降法蛍光抗体法組織特異的発現 poly A 付加部位 houseーkeeping

项目摘要

The human rab-2 gene codes a 24-kDa YPT1-related GTP-binding protein. In all the human cell lines examined, including hematopoietic, fibroblastic and tumor cells, three rab-2 transcripts of 3.5, 2.4 and 1.4 kb were detected. The 2.4- and 3.5-kb rab-2 RNA hybridized with the probe specific for the 3'-non-coding region of 2.4-kb transcript, but not the 1.4-kb RNA. These transcripts seemed to differ in the length of their 3'-non-translated regions due to terminations of the transcription and/or processing of the primary transcript at the alternative polyadenylation sites. The smaller 1.4-kb transcript was the most predominant in Molt-4 and U-937, while the 2.4-kb RNA was dominant in the other cell lines. The degradation rates of all the rab-2 transcripts wbre similarly as slow as beta-actin RNA, even in the two cell lines whose predominant species of the transcripts were different. Rab-2 protein was detected in the extracts of human cultured cells as one major band of 24 kDa and a more faint band of 25 kDa by both immunoblotting and immunoprecipitation. Only the 25-kDa protein was detected in HeLa and KOBK101 cells. In the COS-1 cells transfected with an expression vector carrying rab-2 cDNA, the rab-2 protein was mainly located in the cytoplasm.

人rab-2基因编码24 kDa YPT 1相关GTP结合蛋白。在所有的人类细胞系，包括造血细胞，成纤维细胞和肿瘤细胞，三个RAB-2转录检测3.5，2.4和1.4 kb。2.4-kb和3.5-kb的rab-2 RNA与2.4-kb转录物的3 '-非编码区特异性探针杂交，但不与1.4-kb RNA杂交。这些转录物的3 '-非翻译区的长度似乎不同，这是由于初级转录物的转录和/或加工在替代的多聚腺苷酸化位点处终止。在Molt-4和U-937中，较小的1.4-kb转录物占主导地位，而在其他细胞系中，2.4-kb RNA占主导地位。所有rab-2转录物的降解速率与β-肌动蛋白RNA的降解速率一样慢，即使在转录物的主要物种不同的两个细胞系中也是如此。通过免疫印迹和免疫沉淀，在人培养细胞提取物中检测到Rab-2蛋白，其为一条24 kDa的主要条带和一条25 kDa的较弱条带。在HeLa和KOBK 101细胞中仅检测到25-kDa蛋白。转染rab-2 cDNA的COS-1细胞中，rab-2蛋白主要定位于细胞质中。