| Affiliation: | (1) Graduate School of Environmental Studies, Tohoku University, Aoba 6-6-20, Aramaki, Aoba, Sendai 980-8579, Japan;(2) Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba 305-8572, Japan |
| Abstract: | We carried out a comprehensive study of proteins that exhibit specific interactions with a naturally occurring toxin, microcystin (MC)-LR, in order to gain insight into the unknown underlying mechanism of MC virulence. This audacious study employed a simple affinity test that used MC-LR immobilized on an original ethylene oxide based monolithic solid phase (Moli-gel), and swine liver lysate. Some of the proteins that interacted with MC-LR on this original affinity resin were separated by SDS-PAGE, measured by nano-LC/MS/MS after trypsin digestion, and identified using a Mascot database search. Protein sequence analyses revealed that glutathione S-transferase (GST) was one of the candidate target proteins for MC-LR. This protein was confirmed as a target protein for MC-LR based on the results of for the inhibition of an enzymatic reaction by Dhb-MC-LR. Moreover, L-3-hydroxyacyl coenzyme A dehydrogenase (HDHA) was shown to be one of the proteins that specifically interacts with MC-LR. Our results demonstrated that our analytical systems based on an original affinity resin and nano-LC/MS/MS were effective for target protein research. |
