本文3/YA8183G
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1 pp. Na H Na H ph Na H NHE B CHP NHE NHE CHP NMR NHE NHE pcold-gst ph Na Na H Na H Na H Na H antiporter Nha Na H Na H Na H exchanger NHE C C C The regulation mechanism of Na H exchanger Kokoro Hayashi and Chojiro Kojima Laboratory of Biophysics, Graduate School of Biological Sciences, Nara Institute of Science and Technology NAIST, Takayama, Ikoma, Nara, Japan B calcineurin B homologous protein, CHPNHE NHE C CHP NHE NHE C CHP NMR NHE C CHP CHP NHE NHE Na H NHE NHE Na H Na NHE NHE NHE NHE NHE NHE NHE NHE NHE NHE NHE NHE NHE
2 NHE NHE NHE NHE NHE NHE NHE NHE NHE NHE ph phi Na NHE a phi phi NHE NHE ph NHE ph NHE NHE C C Na H NhaA NHE NHE a NHE b NHE PIP CHP B ERM CaM - - ROCK I Rho I NIK Nck CaII II.
3 NHE C NHE C C NHE C b NHE C Rho I ROCK I C PKC Nck NIK CaM CHP ERM PIP NHE CHP CHP NHE NHE phi NHE CHP CHP ph B CHP CHP CHP CHP CHP N GlyCHP CHP CHP CHPCHP NHE CHP CHP NHE ph ph ph ph CHP CHP CHP α B NHE C CHP NMR CHP NHE NHE CHP EF CHP EF N EF EF C EF EF CHP EF EF CHP NHE CHP NHEnM NHE CHP CHP N N C C CHP N C CHP B CHP CHP CHP unique region CHP NHE NHE phi CHP NHE NMR CHP NHE NMR CHP NHE NHE pcold-gst NHE NMR CHP NMR NMR CHP NHEkDa NMR PDB NMR kda NMR NMR CHP NHE C N NMR H H C N NOESY CYANA nuclear Overhauser effect NOE CANDID
4 9 2 8 生化学 第8 0巻 第1 0号 図2 CHP NHE1ペプチド複合体の 立体構造 CHP の N-ローブを青色 C-ローブ をピンク色 CHP-ループを灰色 NHE1ペプチドを緑色で示す NHE ペプチドは 5 1 6 5 4 0 の領域を表 示 し た 左 CHP1 NHE1ペ プ チ ドの溶液構造 PDB ID 2E3 0 右 CHP2 NHE1ペ プ チ ド の 結 晶 構造 PDB ID 2BEC CHP-ルー プの点線は構造が決定されていない 領域 図3 CHP1と CHP2の表面電荷マップ CHP NHE1複合体における CHP の表面電荷を示す 正電荷は青 負電荷は赤 緑色のリボン構造は NHE1ペプチドを示す 黄色い点線は CHP1と CHP2で電荷が反転している領域 a CHP1 NHE1ペ プチド複合体 b CHP2 NHE1ペプチド複合体
5 NMR NOE Güntert kda NMR NMR CHP NHE residual dipolar coupling, RDC NMR RDC NH NH RDC CHP NHE NMR CHP NHE NMR CHP NHE CHP NHE NMR CHP NHE X CHP NHE CHP NHE CHP NHECHP CHP NHE CHP NHE NHECHP NHE NHE α CHP CHP C NHE N CHP N NHE C NHE CHP CHP CHP NHE C N CHP CHP CHP NHENHE C CHP CHP NHE CHP NHE NHE α C C O CHP α NH CHP NHE NHE CHP CHP CHP CHP CHP NHE CHP NHE ph NHE NhaA ph NHE IL b RCHP CHP phi CHP CHP NHE IL CHP NHE ph CHP NHE b NHE CHP NHE CHP NHE pcold-gst NHE pcold-gst NHE C S- GST pcold I pcold I N His cspa lac operator a IPTG
6 pcold a pcold I b pcold-gst pcold I His GST pcold-gst b pcold-gst pcold I His Factor Xa human rhinovirus C protease HRV C GST HRV C GST PreScission protease pcold-gst pcold-gst kojima@bs.naist.jp NHE pcold-gst N C His N His a b NHE NMR c NMR NHE CHP α CHP NHE CHP NHE α
7 NHE MrkDaa GST-NHE SDS-PAGE S P W E GST-NHE b GST NHE SDS-PAGE NHE c MHz NMR NHE H- NHSQC NHE CHP PIP ERM CHP
8 NHE NHE NHE NHE NHE moloney sarcoma virus NHE NHE NHE CHP NMR NHE CHP NHE NHE CHP NHE NHE NHE CHP NHE CHP NHE CHP ph NHE CHP NHE C Na H C C Wakabayashi, S., Shigekawa, M., & Pouyssegur, J. Physiol. Rev.,,. Orlowski, J. & Grinstein, S.Pflugers Arch.,,. Pang, T., Su, X., Wakabayashi, S., & Shigekawa, M.J. Biol. Chem.,,. Mishima, M., Wakabayashi, S., & Kojima, C.J. Biol. Chem.,,. Ammar, Y.B., Takeda, S., Hisamitsu, T., Mori, H., & Wakabayashi, S.EMBO J.,,. Sardet, C., Franchi, A., & Pouyssegur, J.Cell,,. Nakamura, N., Tanaka, S., Teko, Y., Mitsui, K., & Kanazawa, H.J. Biol. Chem.,,. Lee, S.H., Kim, T., Park, E.S., Yang, S., Jeong, D., Choi, Y., & Rho, J.Biochem. Biophys. Res. Commun.,,. Orlowski, J., Kandasamy, R.A., & Shull, G.E.J. Biol. Chem.,,. Noel, J., Roux, D., & Pouyssegur, J.J. Cell Sci., Pt,. Baird, N.R., Orlowski, J., Szabo, E.Z., Zaun, H.C., Schultheis, P.J., Menon, A.G., & Shull, G.E.J. Biol. Chem.,,. Numata, M. & Orlowski, J. J. Biol. Chem.,,. Pang, T., Wakabayashi, S., & Shigekawa, M.J. Biol. Chem.,,. Cardone, R.A., Casavola, V., & Reshkin, S.J.Nat. Rev. Cancer,,. Naoe, Y., Arita, K., Hashimoto, H., Kanazawa, H., Sato, M., & Shimizu, T.J. Biol. Chem.,,. Shimizu, T. & Mishima, M.,,. Pang, T., Hisamitsu, T., Mori, H., Shigekawa, M., & Wakabayashi, S.Biochemistry,,. Wakabayashi, S., Hisamitsu, T., Ben Ammar, Y., Nakamura- Nishitani, T.Y., & Iwata, Y.,,. Hunte, C., Screpanti, E., Venturi, M., Rimon, A., Padan, E., & Michel, H.Nature,,. Wakabayashi, S., Hisamitsu, T., Pang, T., & Shigekawa, M. J. Biol. Chem.,,. Wakabayashi, S., Hisamitsu, T., Pang, T., & Shigekawa, M. J. Biol. Chem.,,. Hayashi, K. & Kojima, C.Protein Expr. Purif., in press. Qing, G., Ma, L.C., Khorchid, A., Swapna, G.V., Mal, T.K., Takayama, M.M., Xia, B., Phadtare, S., Ke, H., Acton, T., Montelione, G.T., Ikura, M., & Inouye, M. Nat. Biotechnol.,,. Bell, S.M., Schreiner, C.M., Schultheis, P.J., Miller, M.L., Evans, R.L., Vorhees, C.V., Shull, G.E., & Scott, W.J. Am. J. Physiol.,, C. Hashimoto, K.,,. Engelhardt, S., Hein, L., Keller, U., Klambt, K., & Lohse, M.J. Circ. Res.,,. Kilic, A., Velic, A., De Windt, L.J., Fabritz, L., Voss, M., Mitko, D., Zwiener, M., Baba, H.A., van Eickels, M., Schlatter, E., & Kuhn, M.Circulation,,. Iwata, Y., Katanosaka, Y., Hisamitsu, T., & Wakabayashi, S. Am. J. Pathol.,,.
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