Botulinum ADP-ribosyltransferase and its substrate, Gb.

1. 从C型肉毒杆菌培养液中纯化出一种adp -核糖基转移酶，鉴定为24 kDa蛋白，命名为肉毒杆菌C3外切酶。根据纯化蛋白的部分氨基酸序列克隆出该酶基因，并在大肠杆菌中表达。本研究表明，该酶是一种244个氨基酸的蛋白，在n端40个氨基酸肽的信号肽被剪切后分泌。从牛肾上腺细胞质中纯化了一种底物gtp结合蛋白Gb，根据其部分氨基酸序列克隆了其cDNA。通过这些程序，它被鉴定为rho基因的产物，rho基因是ras癌基因相关基因之一。使用纯化的底物，确定adp核糖基化的氨基酸位点为位于蛋白质氨基末端第41位的天冬酰胺残基。利用纯化的或重组的C3外酶，分析了rho基因产物（Gb）在不同类型的培养细胞和血小板中的细胞功能。添加到这些细胞中的C3外泌酶被时间依赖性地掺入细胞中并使细胞中的rho蛋白发生adp -核糖基化，并且伴随着adp -核糖基化，细胞发生了明显的形态学变化。从这些分析中，我们得出结论，rho蛋白参与了细胞内肌动蛋白丝的组装和细胞-底物的粘附。以rho蛋白的催化活性为参数，筛选与该蛋白相互作用的分子，鉴定并纯化了rho特异性GTPase激活蛋白。

1. An ADP-ribosyltransferase was purified from culture fultrate of type C Clostridium botulinum and identified as a 24 kDa protein named botulinum C3 exoenzyme. This enzyme gene was cloned based on the partial amino acid sequence of the purified protein and expressed in Esherichia coli . This study revealed that this enzyme is a protein of 244 amino and secreted after the signal peptide of N-terminal 40 amino acid peptide is clipped.2. Gb, a subsrate GTP-binding protein, was purified from the cytosol of bovine adrenal glands, and its cDNA was cloned based on the partial amino acid sequences. By these procedures, it was identified as a product of rho genes, one of the ras oncogene-related genes. Using the purified subsrate, the amino acid site for ADP-ribosylation was determined as an asparagine residue located at the 41st position from the amino terminal of the protein.3. Using purified or recombinant C3 exoenzyme, cellular function of the rho gene product (Gb) was analyzed in various types of cultured cells as well as blood platelets. C3 exoenzyme added to these cells was time-dependently incorporated into the cells and ADP-ribosylated the rho protein in the cells, and concomitant with this ADP-ribosylation the cells underwent marked morphological changes. From these analyses, we concluded that the rho protein is involoved in assembly of actin filament in the cells and in cell-substrate adhesion.4. Using catalytic activities of the rho protein as parameters, molecules interacting with this protein was screened and rho-specific GTPase activating protein was identified and purified to apparent homogeneity.