The CheY protein, a signaling protein involved in bacterial chemotaxis, was purified using Affi-gel blue column and Sephacryl S-200 gel filtration column. Purified CheY proteins were acetylated in vitro by acetyl-CoA synthetase(ACS; isolated from yeast), and coenzyme A inhibited the acetylation of CheY. Urea gel system can separate modified form unmodified one by charge differences between protein species. When the acetylated CheY was subjected to urea polyacylamide gel lectrophoresis(urea-PAGE), two types of acetylated form detected. This result indicates that the CheY protein have two sites for acetylation, and that the sites are basic amino acid(likely to be Lys.). Cibacron blue dye-binding proteins were frationated from E. coli mutant strain lacking all the chemotaxis components that were induced by acetate. This fraction could acetylate CheY in the absence of ACS(isolated from yeast). This result implies that the acetylation of CheY can take place in E. coli. The CheY protein is phosphorylated by CheA as a part of the chemotactic signal transtuction pathway of E.coli. Phosphorylation of CheY has been proposed to occur in Asp57. When phosphorylated CheY were subjected to urea-PAGE, three types of modified forms were detected. This result suggests that CheY has more than one site for phosphorylation.
