tailieunhanh - Báo cáo khoa học: Enzymatic actions of Pasteurella multocida toxin detected by monoclonal antibodies recognizing the deamidated a subunit of the heterotrimeric GTPase Gq

The DNA binding stringency of restriction endonucleases is crucial for their proper function. The X-ray structures of the specific and non-cognate complexes of the restriction nucleaseEcoRV are considerably different sug-gesting significant differences in the hydration and binding free energies. | IFEBS Journal Enzymatic actions of Pasteurella multocida toxin detected by monoclonal antibodies recognizing the deamidated a subunit of the heterotrimeric GTPase Gq Shigeki Kamitani1 Shinpei Ao2 Hirono Toshima1 Taro Tachibana2 Makiko Hashimoto1 Kengo Kitadokoro3 Aya Fukui-Miyazaki1 Hiroyuki Abe1 and Yasuhiko Horiguchi1 1 Department of Molecular Bacteriology Research Institute for MicrobialDiseases Osaka University Japan 2 Department of Bioengineering Graduate Schoolof Engineering Osaka City University Japan 3 Department of Biomolecular Engineering Graduate Schoolof Science and Technology Kyoto Institute of Technology Japan Keywords bacterialtoxin deamidation GTPase heterotrimeric in vitro assay monoclonal antibody Pasteurella multocida toxin Correspondence S. Kamitani Department of Molecular Bacteriology Research Institute for Microbial Diseases Osaka University 3-1 Yamada-oka Suita-shi Osaka 565-0871 Japan Fax 81 6 6879 8283 Tel 81 6 6879 8285 E-mail skami@ Received 12 October 2010 revised 9 May 2011 accepted 25 May 2011 doi Pasteurella multocida toxin PMT is a virulence factor responsible for the pathogenesis of some Pasteurellosis. PMT exerts its toxic effects through the activation of heterotrimeric GTPase Gq G12 13 and Gi -dependent pathways by deamidating a glutamine residue in the a subunit of these GTPases. However the enzymatic characteristics of PMT are yet to be analyzed in detail because the deamidation has only been observed in cellbased assays. In the present study we developed rat monoclonal antibodies specifically recognizing the deamidated Gaq to detect the actions of PMT by immunological techniques such as western blotting. Using the monoclonal antibodies we found that the toxin deamidated Gaq only under reducing conditions. The C-terminal region of PMT C-PMT was more active than the full-length PMT. The C3 domain possessing the enzyme core catalyzed the deamidation in vitro without any other .