| Co-Investigator(Kenkyū-buntansha) |
BUHLMANN Philippe The University of Tokyo, Graduate School of Science, Assistant, 大学院・理学系研究科, 助手 (20262149)
TOHDA Koji The University of Tokyo, Graduate School of Science, Assistant, 大学院・理学系研究科, 助手 (60212065)
SUGAWARA Masao Nihon University.College of Humanities and Sciences, Professor, 文理学部, 教授 (50002176)
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| Budget Amount *help |
¥38,000,000 (Direct Cost: ¥38,000,000)
Fiscal Year 2000: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1999: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1998: ¥30,700,000 (Direct Cost: ¥30,700,000)
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| Research Abstract |
In most of chemical methods of analysis, selectivity of analytes against interfering substances is essentially governed by the respective binding constant, K_f, between the analyte, A, and its molecular recognition reagent, B, K_f=[AB]/[A][B] This is generally the basis for binding assay. A typical example is immunoassay. Recently, we have been studying analytical methods for bioactive substances, being based not only on binding to receptors but also the following biological process of signal transduction is, in part, taken into account. The idea has lead us to explore some new analytical methods that can evaluate physiologically more relevant selectivity of analytes (agonists or antagonists). Typical examples are ion channel, transporter proteins and kinase-type proteins embedded in lipid bilayer membranes. In contrast to the binding assay and related techniques, these sensors utilize the corresponding downstream signals, such as ion-channel currents or the extent of phosphorylation o
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f the receptor itself and its target proteins for yielding physiologically more relevant signals compared to those with the binding assay approach. This is achieved by both in vitro and in vivo analytical approaches using surface plasmon resonance (SPR) and fluorescent probe reagents involving part of these cellular signaling mechanisms. As to intracellular receptor proteins, Ca^<2+> signaling pathways for example based on a Ca^<2+>-dependent on/off switching of calmodulin, the agonist selectivity in terms of ion selectivity has been evaluated by SPR for the formation of a Ca^<2+> calmodulin target peptide ternary complex. By this method, not only Ca^<2+> ion, but also Sr^<2+>, Pb^<2+>, Cd^<2+> and many of lanthunum ions have been found to ride on "Ca^<2+> signaling" and behave as a strong agonist toward the Ca^<2+> signaling. Screening of agonists concerning their physiologically relevant selectivity is very important for biological studies as well as for pharmaceutical needs. The conventional binding assays can neither give sufficient information on the agonist activity nor discriminate between agonists and antagonists. Assay procedures based on cellular signal transduction mechanisms thus appear to become a general analytical method for bioactive analytes. Our further studies of this apporach include screening of antigen-specific IgEs based on intracellular Ca^<2+> singnaling, and also screening of anticancer drugs based on active transport involving multi-drug resistance proteins. Less
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