研究業績・活動報告2013

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(1)東北大学多元物質科学研究所. 研究業績・活動報告 2013 年 (平成 25 年)12 月.

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(3) 多元物質科学研究所. 研究業績・活動報告目次研究活動報告 ·············································································· 有機・生命科学研究部門 ········································································································. 1 1. 無機材料研究部門 ················································································································· 16 プロセスシステム工学研究部門 ······························································································· 35 計測研究部門 ······················································································································· 54 サステナブル理工学研究センター ···························································································· 74 先端計測開発センター ··········································································································· 94 高分子・ハイブリッド材料研究センター ··················································································· 102 新機能無機物質探索研究センター ···························································································· 115 研究ステーション ················································································································· 126. 研究会報告 ·················································································131 学会発表講演目録 ········································································176 有機・生命科学研究部門 ········································································································ 176 無機材料研究部門 ················································································································· 178 プロセスシステム工学研究部門 ······························································································· 181 計測研究部門 ······················································································································· 184 サステナブル理工学研究センター ···························································································· 187 先端計測開発センター ··········································································································· 189 高分子・ハイブリッド材料研究センター ··················································································· 191 新機能無機物質探索研究センター ···························································································· 193. 研究業績目録 ··············································································196 有機・生命科学研究部門 ········································································································ 196 無機材料研究部門 ················································································································· 198 プロセスシステム工学研究部門 ······························································································· 202 計測研究部門 ······················································································································· 207 サステナブル理工学研究センター ···························································································· 212 先端計測開発センター ··········································································································· 218 高分子・ハイブリッド材料研究センター ··················································································· 221 新機能無機物質探索研究センター ···························································································· 225 その他 ································································································································ 230. 業績目録著者索引 ········································································232.

(4) . ◊ ✲ ά ື ሗ ࿌. . 研究活動報告. ࠙◊✲άືሗ࿌ࠚ. ⏕࿨ᶵ⬟ศᏊྜᡂ໬Ꮫศ㔝㸦2013.. 1㹼2013. 12㸧. ᩍ ᤵ㸸Ọḟ ྐ ຓ ᩍ㸸㨣ሯ࿴ග㸦ᖺ᭶㹼㸧ⴗཎఙஓ㸦㹼ᖺ᭶㸧 బࠎᮌせ㸦㹼ᖺ᭶㸧 ≉ ௵ ຓ ᩍ㸸㎷ཝ୍㑻㸦ᖺ᭶㹼㸧 ஦ົ⿵బဨ㸸ᮌᮧ⌮㤶 ኱ Ꮫ 㝔 ⏕㸸ⲡ㔝ಟᖹబ⸨᠇኱▼⃝ᝆᶞᒾᮏ┤⏕ ⱱᮌ⌶┿ 㹼ᖺ᭶

(5) ஭⏣⿱ኴ ⛅⃝ᣅஓ ᖺ᭶㹼

(6) ▼ᒣ⩧༗ ᖺ᭶㹼

(7) బࠎᮌḠᏹ ᖺ᭶㹼

(8) ᫓ᒣᣅဢ ᖺ᭶㹼

(9) ᮏ◊✲ศ㔝䛷䛿䠈᭷ᶵ໬Ꮫ䜢䝧䞊䝇䛻ᶵ⬟ᛶศᏊ䜢タィ䞉ྜᡂ䛧䚸䛭䜜䜙䜢⏝䛔䛶㑇ఏᏊⓎ⌧䜢໬Ꮫⓗ䛻 ไᚚ䛩䜛᪉ἲㄽ䛾㛤Ⓨ䜢┠ᣦ䛧䛶◊✲άື䜢⾜䛳䛶䛔䜛䚹2013ᖺ䛾ᮏ◊✲ᡤ䛻䛚䛡䜛୺䛺◊✲άື䛸䛧䛶䛿䠈 ௨ୗ䛾䜘䛖䛻ᴫᣓ䛥䜜䜛䠊㻝㻚㻌㓟໬䛻䜘䜚άᛶ໬䛥䜜䜛᪂つᯫᶫ཯ᛂᛶ᰾㓟䛾㛤Ⓨ㻌 ᡃ䚻䛿᫖ᖺᗘ䚸㧗䛔཯ᛂᛶ䜢ᣢ䛴᪂つᯫᶫ཯ᛂᛶ᰾㓟䛸䛧䛶 ASVP(1)䜢㛤Ⓨ䛧䛯䚹1䜢⤌䜏㎸䜣䛰 2’-OMe RNA䛿୰ᛶ᮲௳ୗ䚸䝅䝖䝅䞁䛻ᑐ䛧䛶1᫬㛫௨ෆ䛻60%䛸㠀ᖖ䛻㧗䛔཯ᛂᛶ䜢ᣢ䛴䛣䛸ཬ䜃䛭䛾๓㥑 య䛷䛒䜛䝇䝹䝣䜱䝗య2䛿཯ᛂᛶ䜢ᣢ䛯䛪㠀ᖖ䛻Ᏻᐃ䛷䛒䜛䛣䛸䛜䜟䛛䛳䛯䚹 ᅗ㸯 ཯ᛂᛶྥୖࢆ┠ᣦࡋࡓ᪂つᯫᶫ཯ᛂᛶ᰾㓟. ᅗ㸰άᛶ໬ࡋࡓᯫᶫ཯ᛂᛶ᰾㓟ࡢ཯ᛂᛶ. 㟁Ꮚ྾ᘬᇶ. 50 mM MES 100 mM NaCl. pH 7, 37 ºC, 12 h. RNA target. RNA target. 㟁Ꮚồᘬᇶ࡟ࡼࡾ཯ᛂᛶࡢຍ ㏿ࡀᮇᚅࡉࢀࡿ࡜ᮇᚅࡉࢀࡿ. ཯ᛂᛶࡣ࡞ࡃ㠀ᖖ࡟Ᏻᐃ ࡛࠶ࡿ . Yield (%). 80. 1: 2-Amino-6-(1-ethylsulfinylvinyl)purine (ASVP)㻌. 2: 2-Amino-6-(1-ethylthio vinyl)purine (ATVP) 㻌. rC. 60 40. 20 rA, rG, U. 0. Y. A. G. C. U. ௒ᖺᗘ䛿䛣䛾ᯫᶫ཯ᛂᛶ᰾㓟䜢⏝䛔䛯䚸ᶆⓗRNA䛾ᶵ⬟㜼ᐖ䛻䛴䛔䛶᳨ウ䛧䛯䚹䜎䛪ᶆⓗRNA䛸䛧䛶䚸. 䝹䝅䝣䜵䝷䞊䝊䜢Ⓨ⌧䛩䜛mRNA䜢ヨ㦂⟶ෆ䛷ㄪᩚ䛧䚸ேᕤ᰾㓟䠄1: ASVP䠅䜢ྵ䜐䜸䝸䝂2’-OMeRNA䛸1᫬ 㛫཯ᛂ䛥䛫䛯ᚋ䚸⣽⬊ᢳฟᾮ䜢⏝䛔䛶ヨ㦂⟶ෆ⺮ⓑ㉁ྜᡂ⣔䜢⏝䛔䛶⺮ⓑ㉁Ⓨ⌧䛻୚䛘䜛ᙳ㡪䛻䛴䛔䛶᳨ ᅗ㸱 LQYLWUR ࡟࠾ࡅࡿ࢔ࣥࢳࢭࣥࢫຠᯝ㻌. ᅗ ⣽⬊ᢳฟᾮ୰ࡢࢸ࣓࣮ࣟࣛࢮ㜼ᐖ㻌 Cell lysate: 1µL (CHAPS Lysis Buffer) CFO (0.1-1000nM): 1µL 37 ˚C, 30 min. Target: mRNA coding Fireflyluciferase m7G. 5’-2‘OMe(CUCAG UUAXG GUUAG ACAA)-3’. . AAA...... 120. ASVP. ASO:. X=. CFO2sulfide:. X=. AVP. 100. TRAP Assay. CFO2sulfoxide: X=. 80 60. 40. 䖣ASO ᯫᶫ཯ᛂ (30ศ䠅. 20. IC50 (nM). 0 ON: -. 0.2 1.0 5.0. 0.2 1.0 5.0. 0.2 1.0 5.0. ASO. ATVP. ASVP. 0.2 1.0 5.0 [mM]. AVP. 26.0. 䕔CFO2sulfide. 䕔CFO2sulfoxide. 0%. 55%. 29.1. 1.41. Telomerase activity. Relative Luciferase activity (%). 1. 䖣 ASO. 1. 䕔 CFO2sulfide 䕔 CFO2sulfoxide. 0.75 0.5 0.25 0 -2. 0. 2. log [ON]. 4.

(10) . ◊ ✲ ά ື ሗ ࿌. 2. 研究活動報告. ウ䛧䛯䚹䛭䛾⤖ᯝ䚸㠀ᖖ䛻ຠ⋡ⓗ䛻⺮ⓑ㉁Ⓨ⌧䜢㜼ᐖ䛩䜛䛣䛸䛜䜟䛛䛳䛯(ᅗ3䠅䚹䛥䜙䛻䝔䝻䝯䝷䞊䝊䛻ྵ䜎䜜䜛㗪ᆺRNA䛻ᑐ䛧䛶┦⿵ⓗ䛺㓄ิ䛻1䜢ྵ䜐䜸䝸䝂2’-OMeRNA䜢ྜᡂ䛧䚸䝔䝻䝯䝷䞊䝊䜢ྵ䜐⣽⬊ᢳฟᾮ୰30ศ䜲䞁䜻䝳䝧䞊䝖䛧䚸䝔䝻䝯䝷䞊䝊άᛶ䜢ㄪ䜉䛯䛸䛣䜝䚸ᯫᶫ཯ᛂᛶ䜢ᣢ䛯䛺䛔䜸䝸䝂2’-OMeRNA䛻ẚ㍑䛧䛶䚸⣙20ಸ䛾㜼ᐖάᛶ䜢♧䛩䛣䛸䛜䜟䛛䛳䛯䚹 ሷᇶḞᦆ㒊఩ࢆ᭷ࡍࡿᮏ㙐'1$࡟ᑐࡍࡿ໬Ꮫⓗಟ㣭ἲࡢ㛤Ⓨ ⏕యศᏊ䛻ᑐ䛧䛶㑅ᢥⓗ䛻໬Ꮫಟ㣭䜢᪋䛩᪉ἲㄽ䛾㛤Ⓨ䛿䚸⏕యศᏊ䛾ᶵ⬟ゎ᫂䜔ᶵ⬟ไᚚ䜢⾜䛖ୖ 䛷㠀ᖖ䛻㔜せ䛷䛒䜛䚹䛩䛷䛻ᡃ䚻䛿ᶆⓗ䛸䛺䜛᰾㓟ሷᇶ䛸Ỉ⣲⤖ྜ䜢ᙧᡂ䛩䜛䛣䛸䛷άᛶ໬䛥䜜䚸ᯫᶫ䜢ᙧ ᡂ䛩䜛ேᕤ᰾㓟ሷᇶ 2-Amino-6-vinylpurine(AVP, ᅗ5)䜢㛤Ⓨ䛧䛶䛚䜚䚸AVP䜢⤌䜏㎸䜣䛰䜸䝸䝂䛜䚸DNA 䜔RNA୰䛾䝏䝭䞁䛒䜛䛔䛿䜴䝷䝅䝹䛸㧗䛔㑅ᢥᛶ䛷ᯫᶫ཯ᛂ䛩䜛䛣䛸䜢ሗ࿌䛧䛶䛔䜛1, 2) ᅗ㸬ሷᇶḞᦆ㒊఩࡟࠾ࡅࡿ཯ᛂࡢᴫᛕ࡜ࣉ࣮ࣟࣈࡢタィ. ᅗ㸬ሷᇶ㑅ᢥⓗ࡞཯ᛂᛶ᰾㓟ሷᇶ . ཯ᛂᛶሷᇶ㒊ศ O. 㻭㻼㼟㼕㼠㼑. N. H N. N. H N. N. N. :. O. N. O. T N. N H H. O. '1$ ࡟ᑐࡋ࡚ぶ࿴ᛶࢆᣢࡘ ࢢ࣮ࣝࣉ ࢝ࢳ࢜ࣥᛶ࣌ࣉ ࢳࢻࠊ࢔ࢡࣜࢪࣥ࡞࡝㸧㻌. AP ࢧ࢖ࢺࢆ᭷ࡍࡿ 2 ᮏ㙐 DNA. AVP. ᮏ◊✲䛷䛿䛣䛾⤖ᯝ䛻ᇶ䛵䛝䚸AVPሷᇶ䜢⏝䛔䛯DNA 䛻ᑐ䛩䜛᪂つ㒊఩≉␗ⓗಟ㣭᪉ἲ䛾㛤Ⓨ䜢┠ⓗ 䛸䛧䛯䚹AVPሷᇶ䛿ᶆⓗሷᇶ䛸Ỉ⣲⤖ྜ䜢ᙧᡂ䛧άᛶ໬䛥䜜཯ᛂ䛩䜛䛸⪃䛘䜙䜜䜛䛣䛸䛛䜙䚸DNA ෆ䛷␯Ỉ ⎔ቃ䜢ᙧᡂ䛷䛝䜛ሷᇶḞᦆ㒊఩(AP䝃䜲䝖)䛻╔┠䛧䚸ሷᇶḞᦆ㒊఩䛾ྥ䛛䛔䛾ሷᇶ䛻ᑐ䛧䛶Ỉ⣲⤖ྜ䜢ᙧᡂ 䛧཯ᛂ䛩䜛䝥䝻䞊䝤䜢タィ䛧䛯䠄ᅗ6䠅䚹DNA䛻ᑐ䛧䛶ぶ࿴ᛶ䜢ᣢ䛴䜾䝹䞊䝤䛸䛧䛶䚸䜹䝏䜸䞁ᛶ䝨䝥䝏䝗ཬ䜃䜰䜽䝸䝆䞁䜢ᑟධ䛧䛯䝥䝻䞊䝤䜢ྜᡂ䛧䠄ᅗ7䠅䚸AP䝃䜲䝖䜢᭷䛩䜛䠎ᮏ㙐DNA䛻ᑐ䛩䜛཯ᛂᛶ䜢ẚ㍑䛧䛯䚹䛭䛾 ⤖ᯝ䚸䛔䛪䜜䛾䝥䝻䞊䝤䜒AP䝃䜲䝖䛾ྥ䛔䛻䛒䜛䝏䝭䞁䛻ᑐ䛧䛶㠀ᖖ䛻㑅ᢥⓗ䛻཯ᛂ䛩䜛䛣䛸䚸䛥䜙䛻䝣䝹䝬䝑䝏䛾2ᮏ㙐䛻ᑐ䛧䛶䛿཯ᛂ䛧䛺䛔䛣䛸䛜䜟䛛䛳䛯䚹䛥䜙䛻཯ᛂᛶ䜢ẚ㍑䛧䛯䛸䛣䜝䚸䜰䝹䜼䝙䞁䜢5䛴ᣢ䛴䝥䝻䞊䝤䛾཯ᛂᛶ䛜୍␒㧗䛔䛣䛸䛜䜟䛛䛳䛯䚹䛧䛛䛧䚸䛣䛾䝥䝻䞊䝤䛿1ᮏ㙐DNA䛻䜒཯ᛂ䛩䜛䛣䛸䛜䜟䛛䛳䛯䚹୍᪉䚸䜰䜽䝸䝆䞁䜢ᣢ䛴䝥䝻䞊䝤䛿䚸䛭䜜䜋䛹཯ᛂᛶ䛿㧗䛟䛺䛔䜒䛾䛾䚸1ᮏ㙐DNA䛻䛿཯ᛂ䛫䛪䚸AP䝃䜲䝖䜢ᣢ䛴2 ᮏ㙐DNA䛻ᑐ䛧䛶㑅ᢥⓗ䛻཯ᛂ䛩䜛䛣䛸䛜䜟䛛䛳䛯䚹⌧ᅾ䚸௜ຍయ䛾ᵓ㐀Ỵᐃ䛺䛹ヲ⣽䛻䛴䛔䛶᳨ウ䜢⾜䛳䛶䛔䜛䚹 ᅗ㸬ྜᡂࡋࡓࣉ࣮ࣟࣈ . dsDNA 5 mM. 3: R=. ᅗ㸬ྜᡂࡋࡓࣉ࣮ࣟࣈࡢ཯ᛂᛶࡢẚ㍑ 3’-d(GTC GCG XTT AAG CGC TCA)-5’ 50 mM MES, 100 mM + 5’-6FAM-d(CAG CGC NAA TTC GCG AGT)-3’ O. PRGRP. O. dSpacer (dS). 3: R=PRGRP. . 5: R=. 40 30 20. T. 10 50. 100. 150. Reaction time (h). . T. 60 50. ssDNA (N = T). 40 30 20. G C A. 10. AC G 0. Acr. 2). 50. 0. . 1). 60. 5: R=Acr 20. . 70. Alkylation yield (%). Penta-R. Alkylation yield (%). . 4: R=Penta-R. . 70. 4: R=. 100 mM. 200. 0. 0. 20. 40. 60. 80. Reaction time (h). 100. Alkylation yield (%). . Adduct. NaCl, pH 7.0, 37 Υ. O. X=. . Abasic T. 15. 10. ss-DNA T, C, A, G. 5. 0. 0. 20. 40. 60. Full matched 80 (X=A. N=T). Reaction time (h). Imoto, S., Hori, T., Hagihara, S., Taniguchi,Y., Sasaki, S., Nagatsugi, F. Bioorg. Med. Chem. Lett. 20 6121-6124 (2010) Hagihara, S., Kusano, S., Lin, W.C., Chao, X.G., Hori, T., Imoto, S., Nagatsugi, F., Bioorg. Med. Chem. Lett., 22, 3870-3872 (2012). .

(11) 3. ◊㻌 ✲㻌 ά㻌 ື㻌 ሗ㻌 ࿌ 研究活動報告. 䛆◊✲άືሗ࿌䛇㻌㻌 ⏕࿨ᶵ⬟ไᚚ≀㉁໬Ꮫ◊✲ศ㔝䠄2013䠊1䡚2013䠊12䠅㻌 ᩍ ᤵ㻌㻦㻌 ࿴⏣೺ᙪ㻌 ෸ ᩍ ᤵ㻌㻦㻌 Ⲩᮌಖᖾ㻌㻔㻞㻜㻝㻟㻚㻝㻜㻚㻝ࠥ㻕㻘㻌 ຓ㻌㻌 ᩍ䠖㻌 ᆏᮏΎᚿ㻌 ኱ Ꮫ 㝔 ⏕㻌㻦㻌 ᮧୖ㻌 ៅ㻘㻌 ℧㻌㞟స㻘㻌 ୖᯇுᖹ㻘㻌 బ⸨㎮ஓ㻘㻌 ℈⏣ⰾ⏕㻘㻌㻌 ‖㻌 ဏ⤮㻘㻌 ዟᮌ㻌 ᬸ㻘㻌 Ⳣ஭⚈ຍ㻘㻌 ᮧୖ┿ዉ⨾㻌㻌㻌㻌㻌㻌㻌㻌㻌㻌㻌㻌㻌㻌㻌 ᮏ◊✲ศ㔝䛷䛿 DNA 䜔 RNA䚸䛭䛧䛶䝍䞁䝟䜽㉁䛺䛹⏕య㧗ศᏊ䛾䛜䜣⣽⬊≉␗ⓗ᰾㓟་⸆䜈䛾ᒎ㛤䜔 ⣽⬊ෆྍど໬ヨ⸆䜈䛾ᒎ㛤䛺䛹ḟୡ௦䜲䞁䝔䝸䝆䜵䞁䝖ᆺ䝘䝜䝞䜲䜸ᶵ⬟ᮦᩱ䜈䛾ᛂ⏝䚸䛥䜙䛻⏕యศᏊ䛾 ᵓ㐀ኚ໬䛸ᶵ⬟ไᚚ䛾ゎ᫂䜢┠ᣦ䛧䛯㧗᫬㛫ศゎ⬟෇஧Ⰽ䝇䝨䜽䝖䝹 ᐃ⿦⨨䛾㛤Ⓨ䛺䛹⏕࿨໬Ꮫ䛛䜙≀ ⌮໬Ꮫ䜎䛷ศ㔝ᶓ᩿ⓗ䛻ᇶ♏䛛䜙ᛂ⏝䜎䛷ᖜᗈ䛔◊✲άື䜢ᒎ㛤䛧䛶䛔䜛䚹2013 ᖺ䛾◊✲άື䛸䛧䛶䛿䚸௨ ୗ䛾䜘䛖䛻ᴫᣓ䛥䜜䜛䚹㻌 1. ࡀࢇ⣽⬊≉␗ⓗ᰾㓟་⸆๰ᡂࢆᣦྥࡋࡓ࣌ࣉࢳࢻࣜ࣎᰾㓟-࣌ࣉࢳࢻ᰾㓟-DNA ࣓࢟ࣛேᕤ᰾㓟 (PRPD) ࡢྜᡂ࡜ RNase H άᛶ࡟㛵ࡍࡿ◊✲ 㻌 ᡃ䚻䛿䚸๪స⏝䛜ᑡ䛺䛟㐺⏝⠊ᅖᣑ኱䛜ᮇᚅ䛷䛝䜛ḟୡ௦䛾᰾㓟་⸆๰ 〇䜢┠ᣦ䛧䚸ᶆⓗ RNA 䛸䛾㘒యᙧᡂ䋽ゎ㞳䛾⮬ᅾ䛺ไᚚᶵ⬟䜢᭷䛩䜛䝨䝥䝏䝗䝸䝪᰾㓟 (PRNA)䛾㛤Ⓨ䛻ྲྀ⤌䜣䛷䛔䜛䚹≉䛻⣽⬊ෆ⎔ቃኚ໬ᛂ⟅ ⬟䜢௜୚䛧䛯 PRNA 䛾ᵓ㐀ኚ໬䜢䝖䝸䜺䞊䛸䛧䛯ᶆⓗ RNA 䛻ᑐ䛩䜛ㄆ㆑䞉」ྜయᙧᡂᶵ⬟䛾 on-off ไᚚ䜢㐩ᡂ䛧䚸⾑⟶᪂⏕䛜㏣䛔䛴䛛䛺䛔ቑᖜᮇ䛾䛜䜣⣽⬊ෆ≉᭷䛾ప㓟⣲⃰ᗘ䛻䜘䜚ㄏ㉳䛥䜜䜛ప pH 䛻ᛂ⟅䛩䜛䛜䜣⣽⬊ ≉␗ⓗ᰾㓟་⸆䜈䛾ᒎ㛤䜢┠ᣦ䛧◊✲䜢᥎㐍䛧䛶䛔䜛䚹PRNA 䛿䛜䜣⣽⬊ ෆ≉␗ⓗ⎔ቃ䠄⣽⬊㉁ pH=6.2䠅䜢䝖䝸䜺䞊䛸䛧䚸䛜䜣⣽⬊ෆ䛷䛾䜏 miRNA 䜔 mRNA 䛺䛹ᶆⓗ RNA 䛻ᑐ䛩䜛㧗䛔ሷᇶ㓄ิ㑅ᢥⓗᏳᐃ㘒యᙧᡂ⬟䜢 ᭷䛧䚸㑇ఏᏊ἞⒪⸆䛸䛧䛶䛾㧗䛔ຠᯝ䜢Ⓨ⌧䛩䜛䛾䛻ᑐ䛧䚸ṇᖖ⣽⬊ෆ (pH 7.2)䛷䛿 RNA 䛾䜏䛺䜙䛪 DNA 䛸䜒┦஫స⏝䛫䛪๪స⏝䜢Ⓨ⌧䛧䛺䛔 ⌮᝿ⓗ䛺䛜䜣⣽⬊≉␗ⓗ᰾㓟་⸆䛸䛧䛶ᮇᚅ䛥䜜䛶䛔䜛᪂つேᕤ᰾㓟䛷䛒 Fig.1. Structure of PRNA-PNADNA Chimera. 䜛䚹㻌䛣䛾 PRNA 䛸䚸ᶆⓗ RNA 䛾㘒య䛜䚸RNase H 䛾ᇶ㉁䛸䛺䜜䜀 PRNA 䛿ゐ፹ⓗ䛻ᶵ⬟䛧ຠᯝⓗ䛺⸆ຠⓎ⌧ 䛜ᮇᚅ䛥䜜䚸䜘䜚ᐇ⌧ᛶ䛾㧗䛔㑇ఏᏊ἞⒪⸆䛸䛧䛶䛾ᒎ㛤䛜ᮇᚅ䛥䜜䜛䚹ᮏᖺᗘ䛿ከᙬ䛺ᶵ⬟䜢⌮ㄽⓗ䛛䛴 ⡆౽䛻௜୚ྍ⬟䛺”䝰䝆䝳䞊䝹ἲ”䜢 PRNA ྜᡂ䛻㐺⏝䛧䚸PRNA 䜢䛜䜣⣽⬊≉␗ⓗᶵ⬟Ⓨ⌧䝰䝆䝳䞊䝹䚸 DNA 䜢ゐ፹ⓗ RNA ศゎᶵ⬟Ⓨ⌧䝰䝆䝳䞊䝹䚸PNA 䜢Ᏻᐃ㘒యᙧᡂ䝰䝆䝳䞊䝹䛸䛧䛶䛸䜙䛘䚸ᶆⓗ RNA 䛸Ᏻ ᐃ㘒య䜢ᙧᡂ䛧䚸䛛䛴㘒య䛜 RNaseH 䛾ᇶ㉁䛸䛺䜚䚸ゐ፹㔞䛷㧗䛔㑇ఏᏊ἞⒪ຠᯝⓎ⌧䛜ᮇᚅ䛷䛝䜛 PRNA-PNA-DNA 䜻䝯䝷ேᕤ᰾㓟䠄PRPD䠅䜢タィ䞉ྜᡂ䛧䛯(ᅗ 1)䚹 ᚓ䜙䜜䛯 PRPD 䛿ᶆⓗ RNA 䛻ᑐ䛧䚸㧗䛔ሷᇶ㓄ิ≉␗ⓗᏳᐃ㘒యᙧᡂᶵ⬟䜢♧䛧䛯䚹ITC 䜢⏝䛔䛶䚸䛭䛾」ྜయᙧᡂ㐣⛬䛻䛴䛔䛶ヲ⣽䛻᳨ウ䛧䛯⤖ᯝ䚸PRPD-RNA 」ྜయ䛻䛚䛔䛶䛿䚸Ᏻᐃᛶ䛻ཬ䜌䛩䜶䞁䝖䝻䝢䞊㡯䛾ᐤ୚䛜┦ᑐⓗ䛻኱䛝䛔஦䛜᫂䜙䛛䛸䛺䛳䛯䚹CD 䝇䝨䜽䝖䝹䛺䛹䜢ヲ⣽䛻᳨ウ䛧䛯⤖ᯝ䚸PRNA ᑟධ 䛻క䛔 PRPD 䛾ᵓ㐀䛜 RNA ྠᵝ A ᆺ㢮ఝ䛾ᵓ㐀䜢ඃඛ䛧䚸䛭䛾⤖ᯝ」ྜయᙧᡂ㐣⛬䛻䛚䛡䜛䜶䞁䝖䝻䝢䞊 ⓗ䛺ῶᑡ䛜ᑠ䛥䛟䛺䛳䛯஦䛻ᇶ䛵䛟䛣䛸䛜ண᝿䛥䜜䛯䚹⥆䛔䛶ᶆⓗ RNA ᚓ䜙䜜䛯 PRPD 䛾ሷᇶㄆ㆑⬟䜢䚸䝭䝇䝬䝑䝏ሷᇶᑐ䜢ྵ䜐 DNA 䛸䛾 Tm ᐃ䛻䜘䜚᳨ウ䛧䛯⤖ᯝ䚸ᮎ➃௨እ䛾఩⨨䛷䛿ሷᇶ㓄ิ≉␗ⓗ䛻Ᏻᐃ䛺」 ྜయ䜢ᙧᡂ䛧䛯䚹䜎䛯䚸PRPD 䛿䝥䝻䝔䜰䞊䝊⪏ᛶ䜢᭷䛧䛶䛔䜛䛣䛸䛜᫂䜙䛛䛻䛺䛳䛯䚹㻌 ḟ䛻 PRPD/RNA 㘒య 䛻ᑐ䛩䜛 RNase H άᛶ䜢᳨ウ䛧䛯䚹⺯ගᶆ㆑ RNA 䛻ᑐ䛧䚸1/100䚸1/1000䠈1/10000 䛾⃰ᗘ᮲௳䛷᳨ウ䛧䛯⤖ ᯝ䚸ཧ↷ DNA 㘒య䛾ሙྜ䛿 1/100 䛻䛚䛔䛶 6䠂䛾ษ᩿䛥䜜䛯䛾䛻ᑐ䛧䚸PRPD-RNA ΰྜ⣔䛷䛿 1/10000 䛾 ᮲௳ୗ䛷 5䠂䛾ษ᩿䛜ほ䛥䜜䚸⣙ 100 ಸ䛾ษ᩿άᛶྥୖ䛜ྍ⬟䛻䛺䜛䛣䛸䛜♧䛥䜜䛯䚹1/100 䛾᮲௳ୗ䛻䛚䛡䜛䝍䞊䞁䜸䞊䝞䞊ᩘ䛻䛴䛔䛶᳨ウ䛧䛯⤖ᯝ䚸PRPD 䛾ሙྜ䚸⣙ 98 ᅇ䛾ゐ፹䝃䜲䜽䝹䛜ほ䛥䜜䚸㠀ᖖ䛻ຠ ⋡䛾Ⰻ䛔 RNA ≉␗ⓗษ᩿䛾㐍⾜䛜☜ㄆ䛥䜜䛯䚹䛥䜙䛻⯆࿡῝䛔䛣䛸䛻䚸RNA ษ᩿཯ᛂ䛿 PRPD 䛾 PNA-DNA 䝆䝱䞁䜽䝅䝵䞁㒊఩௜㏆䛷≉␗ⓗ䛻㐍⾜䛧䛯䚹㻌䛣䜜䜙䛾⤖ᯝ䛿䚸PRPD 䛜ゐ፹ⓗ㑇ఏᏊ἞⒪⸆䛸䛧䛶䛾㧗䛔䝫䝔䞁䝅䝱䝹䜢᭷䛩䜛䛣䛸䜢♧䛧䛶䛔䛶䛚䜚䚸ᮏἲㄽ䛾᭷ຠᛶ䛾ᐇド䛻ᡂຌ䛧䛯䚹.

(12) ◊㻌 ✲㻌 ά㻌 ື㻌 ሗ㻌 ࿌ 研究活動報告. 4. እ㒊่⃭ᛂ⟅ᆺࢱࣥࣃࢡ㉁ࡢᵓ⠏࡜⏕యෆࣉࣟࢸ࢔࣮ࢮάᛶ᳨ฟ࡬ࡢᛂ⏝࡟㛵ࡍࡿ◊✲ 㻌㻌 ⏕యෆ䛻䛿ከᵝ䛺䝥䝻䝔䜰䞊䝊䛜Ꮡᅾ䛧䚸⏕≀䛾Ⓨ⏕䚸⣽⬊ศ໬䚸⣽⬊࿘ᮇ䚸⒴໬䚸⪁໬䛺䛹ᵝ䚻䛺⏕࿨ ⌧㇟䜔⑌ᝈ䛻㛵୚䛧䛶䛔䜛䛣䛸䛜ุ᫂䛧䛶䛔䜛䚹ᚑ䛳䛶䚸⣽⬊ෆ䛻Ꮡᅾ䛩䜛䝥䝻䝔䜰䞊䝊䛾άᛶ䜢㑅ᢥⓗ䛻ྍ ど໬ 䈄᳨ฟ䛩䜛䛣䛸䛿䚸⣽⬊ᶵ⬟䜢ศᏊ䝺䝧䝹䛷ゎᯒ 䈄⌮ゎ䛩䜛ୖ䛷㠀ᖖ䛻㔜せ䛺◊✲ㄢ㢟䛷䛒䜛䛸⪃䛘䜙䜜䜛䚹䛣䜜䜎䛷䛻䜒䝥䝻䝔䜰䞊䝊ᇶ㉁䝨䝥䝏䝗䛸⺯ගศᏊ䜢」ྜ໬䛧䛯ከ䛟䛾໬Ꮫྜᡂ䝥䝻䞊䝤ศᏊ䛜㛤Ⓨ䛥䜜䚸㓝⣲άᛶ◊✲䛻䛚䛔䛶ከ኱䛺㈉⊩䜢ᯝ䛯䛧䛶䛝䛯䛜䚸እ㒊䛛䜙䛾⣽⬊ෆᑟධ䛻䛿ᮍ䛰ᅔ㞴䜢క䛖䛣䛸䛜 ከ䛔䚹ᚑ䛳䛶䚸㑇ఏᏊ䜶䞁䝁䞊䝕䜱䞁䜾䛧䛯≧ែ䛷⏕⣽⬊ෆ䛻ᑟධྍ⬟䛺䝍䞁䝟䜽㉁ᆺ䝥䝻䞊䝤䛿䚸໬Ꮫྜᡂ 䝥䝻䞊䝤䜢⿵᏶䛩䜛ୖ䛷㔜せ䛺ᙺ๭䜢ᯝ䛯䛩䛣䛸䛜ᮇᚅ䛥䜜䜛䚹௨ୖ䛾⫼ᬒ䜢㋃䜎䛘䚸䛣䜜䜎䛷䛻䚸䝍䞊䝀䝑䝖䛸䛺䜛䝥䝻䝔䜰䞊䝊Ꮡᅾୗ䛻䛚䛔䛶⺯ග䜔Ⓨගᙉᗘ䛾ቑຍ䜢♧䛩 GFP 䛚䜘䜃䝹䝅䝣䜵䝷䞊䝊ኚ␗య䛾タィ䜢ヨ䜏䛶䛝䛯䚹1 ௒ᖺᗘ䛿䚸᫖ᖺᗘ䛻ᘬ䛝⥆䛝䚸ศ๭ᆺⓎග䝍䞁䝟䜽㉁䜢⏝䛔䛯䝥䝻䝔䜰䞊䝊άᛶ᳨ฟ䝅䝇䝔䝮䛾ᵓ⠏䛸䛥䜙䛺䜛䝞䝑䜽䜾䝷䜴䞁䝗Ⓨග䛾ᢚไ䛻ᇶ䛵䛟᳨ฟឤᗘ䛾ྥୖ䜢ヨ䜏䛯䚹䛣䛾⣔䛷䛿䚸ศ๭ᆺ䜴䝭䝅䜲䝍䜿䝹䝅䝣䜵䝷䞊䝊䛾 N 䛚䜘䜃 C ᮎ➃ഃ䝣䝷䜾䝯䞁䝖䛻ᑐ䛧䚸ᑐ㇟䛸䛺䜛䝥䝻䝔䜰䞊䝊≉␗ⓗᇶ㉁㓄ิ䜢௜ ຍ䛧䚸N- 䛚䜘䜃 C- ᮎ➃䜢ศ๭ᆺ䜲䞁䝔䜲䞁䛾ศᏊෆ䝇䝥䝷䜲䝅䞁䜾άᛶ䛻䜘䛳䛶ᯫᶫ䛩䜛䛣䛸䛷⎔≧໬䜢⾜䛔䚸䝥䝻䝔䜰䞊䝊㠀Ꮡᅾୗ䛷䛾෌ᵓᡂ཯ᛂ䛾㜼ᐖ䛸ゐ፹ᶵ⬟䛾ᢚไ䜢ᅗ䛳䛶䛔䜛䚹ᐇ㝿䛻䚸୧䝣䝷䜾䝯䞁䝖䛾 ⎔≧໬䛻䜘䛳䛶䚸䝥䝻䝔䜰䞊䝊㠀Ꮡᅾୗ䛻䛚䛡䜛Ⓨග䝅䜾䝘䝹䛿䚸㢧ⴭ䛻ᢚไ䛥䜜䛯䚹୍᪉䚸┠ⓗ䛸䛩䜛䝥䝻䝔䜰䞊䝊Ꮡᅾୗ䛷䛿䚸⎔≧໬䝣䝷䜾䝯䞁䝖୰䛾ᇶ㉁㓄ิ㒊఩䛜≉␗ ⓗ䛻ษ᩿䛥䜜䚸䝍䞁䝟䜽㉁᩿∦㛫䛾෌⤖ྜ䛜ྍ⬟䛸䛺䜚䚸㓝⣲άᛶ 䛻≉␗ⓗ䛻౫Ꮡ䛧䛯Ⓨග䝅䜾䝘䝹䛜ほ䛥䜜䛯䚹᳨ฟឤᗘ䛿䚸㐣ཤ 䛻ሗ࿌䛥䜜䛯䝍䞁䝟䜽㉁ᆺ䝥䝻䞊䝤䛸ྠ⛬ᗘ䛻䜎䛷ྥୖ䛧䛯䚹௒ᚋ 䛿䚸ᮏ䝅䝇䝔䝮䜢⏕⣽⬊䛻㐺⏝䛩䜛䛣䛸䛷䚸᫬✵㛫≉␗ⓗ䛺⏕యෆ 䝥䝻䝔䜰䞊䝊άᛶ᳨ฟ⣔䛻Ⓨᒎ䛥䛫䜛ணᐃ䛷䛒䜛䚹 Fig. 2 Protease activity sensing system with split luciferase system.. 3. ⏕య㧗ศᏊ䛾ືⓗᣲື䛾ゎ ᯒ䜢┠ᣦ䛧䛯㧗ឤᗘ䞉㧗᫬㛫᫬㛫ศゎ෇஧Ⰽ ᐃἲ䛾㛤Ⓨ䛸ᛂ⏝㻌䛣䜜䜎䛷ᡃ䚻䛜ᥦ᱌䛧䛶䛝䛯ᴃ෇೫ග䜢⏝䛔䜛 CD ᳨ฟ⣔䛿䚸ᴃ෇೫ග䛾ᴃ෇⋡䜢䝁䞁䝖䝻䞊䝹䛩䜛ᐇ㦂䝟䝷䝯䞊䝍䛷䛒䜛೫ගᏊ᪉఩ゅ䜢⢭ᐦ䛻䝁䞁䝖䝻䞊䝹䛩䜛䛣䛸䛷䚸ಙ ྕᙉᗘ䛾ぢ䛛䛡ୖ䛾ቑ኱䚸䛩䛺䜟䛱 CD 䛾㧗ឤᗘ᳨ฟ䛜ྍ⬟ 䛻䛺䜛䛣䛸䜢ᐇド䛧䛶䛝䛯䚹2 CD 䛾᳨ฟឤᗘ䛾ྥୖ䛿䚸 ᐃ䛻౑ ⏝䛩䜛䝃䞁䝥䝹㔞䛾ῶᑡ䛻䛴䛺䛜䜚䚸CD ᐃ䛾ᐇ⏝ୖ䛾䜰䝗䝞䞁䝔䞊䝆䛸䛺䜛䛸ᮇᚅ䛥䜜䜛䛸ඹ䛻䚸ᚤᑠ㔞⏕ᡂ䛩䜛䜘䛖䛺཯ᛂ ୰㛫య䛾䜻䝷䝸䝔䜱䛾᳨ฟ䛻ጾຊ䜢Ⓨ᥹䛩䜛䛸ᮇᚅ䛥䜜䛶䛔䜛䚹 ᮏᖺᗘ䛿䚸᫖ᖺ䛻ᘬ䛝⥆䛝㧗ឤᗘ䞉㧗᫬㛫ศゎ⬟䜢᭷䛩䜛 CD ᐃᡭἲ䜢㛤Ⓨ䛩䜛䛸䛸䜒䛻䚸D,L-[Ru(bpy)3]Cl2 ᕼⷧỈ⁐ᾮ 䛾 CD ᐃ䛛䜙䚸ᡃ䚻䛾 CD ᳨ฟἲ䛾᭷⏝ᛶ䛾ᐇド䜢⾜䛳䛯䚹1 ᅗ 3 䛻 ᐃ⤖ᯝ䛾୍౛䜢♧䛩䚹ᡃ䚻䛾 ᐃἲ䛻䜘䜜䜀䚸2 mM 䛾D-[Ru(bpy)3]Cl2 Ỉ⁐ᾮ䛾 CD 䝇䝨䜽䝖䝹䛿䚸ᅗ 3 䛾㯮䛷♧䛥䜜䜛䚹䛣䛾䝇䝨䜽䝖䝹䛾ྲྀᚓ᫬㛫䛿⣙ 200 ⛊䛷䛒䛳䛯䚹୍᪉䚸ᕷ㈍䛾⿦⨨䠄J-820䠅䛷 ᐃ䛩䜛䛸䚸3000 ⛊௨ୖ䛾 ᐃ᫬㛫䛷䛩䜙ᅗ 3 䛾㉥䛷♧䛥䜜䜛䝇䝨䜽䝖䝹䛧䛛ᚓ䜙䜜䛺䛔䚹䛣䜜䜙䛾⤖ᯝ䚸ྠ S/N 䜢୚䛘䜛 ᐃ᫬㛫䛾ほⅬ䛛䜙䛿䚸ᡃ䚻䛾 ᐃἲ䛜ᚑ᮶ἲ䛻 Fig.3 Comparison of CD spectrum of ẚ䜉ከ኱䛺䜰䝗䝞䞁䝔䞊䝆䛜䛒䜛䛸ᐇド䛩䜛䛣䛸䛜䛷䛝䛯䚹 D-[Ru(bpy)3]Cl2 (2 mM) in deionized water 㻌 ୖグᡂᯝ䜢㋃䜎䛘䚸ᮏᖺᗘ䛛䜙ᡃ䚻䛾 CD ᐃᢏ⾡䜢㢧ᚤ㙾 obtained by our equipment (black, azimuth of ගᏛ⣔ CD ᐃ䜈䛾ᒎ㛤䜢┠ᣦ䛧䚸ி㒔኱Ꮫ㟷ᮌ෸ᩍᤵ䛸䛾ඹ retarder was 4° and retardance of retarder was ྠ◊✲䛻ྲྀ䜚⤌䜣䛷䛔䜛䚹 1. 2.. around 10°) with one by conventional CD spectrometer (J-820).. Sakamoto, S.; Terauchi, M.; Hugo, A.; Tanner, K.; Araki, Y.; Wada, T. Chem. Commun., 2013, 49, 10323-10325. Murakami, M.; Araki, Y.; Sakamoto, S.; Hamada, Y.; Wada, T. Chem. Lett., 2013, 42, 261-262..

(13) ◊㻌 ✲㻌 ά㻌 ື㻌 ሗ㻌 ࿌ 研究活動報告. 5. ࠙◊✲άືሗ࿌ࠚ㻌 ᩍ ෸. ⏕࿨㢮ఝᶵ⬟໬Ꮫ◊✲ศ㔝. 䠄2013䠊1䡚2013䠊12䠅㻌. ᤵ㸸㔠ཎ ᩘ ᩍ ᤵ㸸⛅ᒣ බ⏨. ຓ ᩍ㸸ᮧᒸ ㈗༤, Ᏹ஭ ⨾✑Ꮚ, Nabanita Sadhukhan ᢏ ⾡ ⫋ ဨ㸸㧗ᶫ Ὀே ◊✲⿵ຓဨ㸸㐲⸨ ᑑ⨾♩, Rainy Chowdhury, ᕷᮧ ࿴᫂ ኱ Ꮫ 㝔 ⏕㸸Ἑᓮ ಇ୍, ᔱ ೺ஓ, Adam Wawro, Rui Li, ᱵὠ ࠿࠾ࡾ, ᮏከ ⚽㝯, ୕ᾆ ⣪⌮, ᐑෆ భ㍜, 㔝ཱྀ ኱ᶞ㻌㻌. ᮏ◊✲ศ㔝䛷䛿䚸⏕యศᏊ䛾ᶵ⬟䛸䛭䛾Ⓨ⌧䝯䜹䝙䝈䝮䛻╔┠䛧䚸᭷ᶵྜᡂ໬Ꮫⓗ䛺ᡭἲ䛻䜘䜚඲䛟᪂䛧䛔 ᶵ⬟䜢᭷䛩䜛᪂≀㉁䚸䛒䜛䛔䛿䛭䛾ᶵ⬟䜢⮬⏤⮬ᅾ䛻ไᚚ䛷䛝䜛ேᕤ≀㉁䛾タィ䛸ྜᡂ䜢⾜䛺䛳䛶䛔䜛䚹䜎䛯䚸ู䛾䜰䝥䝻䞊䝏䛸䛧䛶⏕యศᏊ䜢໬Ꮫಟ㣭䛩䜛䛣䛸䛻䜘䜚䚸⏕యศᏊ䛸ேᕤศᏊ䛾฼Ⅼ䜢ྲྀ䜚ධ䜜䛯䝴䝙䞊䜽䛺ᶵ⬟䜢᭷䛩䜛ศᏊ䛾ྜᡂ䜒⾜䛺䛳䛶䛔䜛䚹䜎䛯䚸ᶵ⬟ᛶศᏊ䛾≀ᛶ䛻╔┠䛧䛯◊✲䛸䛧䛶䚸ගᶵ⬟ᮦᩱ 䛾䝇䝢䞁ග໬Ꮫ䛻㛵䛩䜛◊✲⾜䛳䛶䛔䜛䚹2012 ᖺ䛾◊✲άື䛸䛧䛶䛿䚸௨ୗ䛾䜘䛖䛻ᴫᣓ䛥䜜䜛䚹㻌㻝㻚. ගཷᐜ⺮ⓑ㉁㻼㼅㻼⼥ྜ䛻䜘䜛⺮ⓑ㉁ᶵ⬟䛾ගไᚚ⣔ᵓ⠏㻌㏆ᖺ䚸⏕≀Ꮫ䚸⬻⚄⤒⛉Ꮫ䛷䛾᪂䛧䛔䜰䝥䝻䞊䝏䛸䛧䛶䚸⏕య䛾ග᧯సᢏ⾡䛜┒䜣䛻◊✲䛥䜜䛶䛚䜚䚸䛭. 䛾୰䛾䜂䛸䛴䛸䛧䛶ගᛂ⟅ᛶ䜢᭷䛧䛯ᶵ⬟ᛶศᏊ䛾㛤Ⓨ䛜ồ䜑䜙䜜䛶䛔䜛䚹ගཷᐜ⺮ⓑ㉁ photoactive yellow protein (PYP)䛿䚸⣚Ⰽගྜᡂ⣽⳦䜘䜚༢㞳䛥䜜䛯ගཷᐜ⺮ⓑ㉁䛷䛒䜚䚸ẚ㍑ⓗᑠᆺ䛾Ỉ⁐ᛶ⺮ⓑ㉁䛷䛒䜛䛣䛸䛛䜙ග᝟ሗఏ㐩䛾ศᏊᶵᵓ䜢㆟ㄽ䛩䜛䛾䛻㐺䛧䛯䝰䝕䝹⺮ⓑ㉁䛸䛧䛶┒䜣䛻◊✲䛥䜜䛶䛔䜛(ᅗ 1)䚹PYP 䛿䚸㟷Ⰽ ග䜢྾཰䛧䛶ⓎⰍᅋ䛾␗ᛶ໬䜢క䛳䛯㧗ḟᵓ㐀ኚ໬䜢ᘬ 䛝㉳䛣䛩䛯䜑䚸ග᝟ሗ䜢ືⓗ䛺ᵓ㐀ኚ໬䜈䛸ኚ᥮䛥䛫䜛䝖䝷䞁䝇䝕䝳䞊䝃䞊䛸䛧䛶⺮ⓑ㉁ᕤᏛⓗ䛺ほⅬ䛛䜙䜒᭷⏝ᛶ 䛜㧗䛔䚹᫖ᖺᗘ䜎䛷䛻䚸ᡃ䚻䛿Ꮝᙧᡂ⺮ⓑ㉁ẘ⣲ -hemolysin (Hla)䛾 N ᮎ➃䛚䜘䜃䛻 C ᮎ➃䛻 PYP 䜢ᑟධ 䛧䛯 N-, C-PYP-Hla 䜢ᵓ⠏䛧䚸ᶵ⬟䛾ගไᚚ䛻ᡂຌ䛧䛶䛔. ᅗ 1:㻌 PYP 䛾❧యᵓ㐀. 䜛䚹ᮏᖺᗘ䛿䚸✀䚻䛾ຍỈศゎ㓝⣲䛻ᑐ䛧䛶㑇ఏᏊᕤᏛ ⓗ䛻 PYP 䜢ᑟධ䛧䛯⼥ྜ⺮ⓑ㉁䜢๰〇䛧䚸䛭䛾㓝⣲άᛶ䜢ග䛻䜘䛳䛶䝁䞁䝖䝻䞊䝹䛩䜛䛣䛸䜢┠ᣦ䛧䛯䚹㻌 ┠ⓗ⺮ⓑ㉁䛿䚸䛔䛪䜜䜒኱⭠⳦Ⓨ⌧⣔䜢⏝䛔䛶኱㔞ㄪ〇䛧䚸p-䜽䝬䝹㓟↓Ỉ≀䜢ῧຍ䛩䜛䛣䛸䛷㻌 PYP 㒊఩ 䛾෌ᵓᡂ䜢⾜䛳䛯䚹⣸እྍど྾཰䝇䝨䜽䝖䝹 ᐃ䜢⾜䛳䛯䛸䛣䜝䚸䛒䜛✀䛾┠ⓗ⺮ⓑ㉁䛿㔝⏕ᆺ PYP 䛸ྠᵝ䛻 446 nm 䛻ᴟ኱䜢䜒䛴྾཰ᖏ䜢♧䛩䛸ྠ᫬䛻ග཯ᛂ䝃䜲䜽䝹䜢ᣢ䛴䛣䛸䛜᫂䜙䛛䛸䛺䛳䛯䚹䜎䛯䚸㓝⣲άᛶ䛻䛚䛔䛶䚸450 nm 䛾ග↷ᑕୗ䛷䛿ᬯ㜌᮲௳ୗ䛸䛿᫂䜙䛛䛻␗䛺䜛ᣲື䜢♧䛧䛯䚹䛧䛯䛜䛳䛶䚸PYP 䜢㑇ఏᏊᕤᏛ ⓗ䛻⼥ྜ䛥䛫䜛䛣䛸䛷㓝⣲άᛶ䛻ගᛂ⟅ᛶ䜢௜୚䛩䜛䛣䛸䛜ฟ᮶䛯䚹⌧ᅾ䚸䛣䛾᪂䛯䛻ᵓ⠏䛧䛯ගᛂ⟅ᛶศ Ꮚ䜢⏝䛔䛶ᚤ⏕≀䛾䝅䜾䝘䝹ఏ㐩᧯స䛻ᣮᡓ䛧䛶䛚䜚䚸ศᏊᶵᵓ䜒ྵ䜑䛶ゎᯒ䜢⾜䛳䛶䛔䜛ẁ㝵䛷䛒䜛䚹.

(14) ◊㻌 ✲㻌 ά㻌 ື㻌 ሗ㻌 ࿌ 研究活動報告. 6. 㻞㻚ᵓ㐀໬䛧䛯㻼㻱㻳䛾㛤Ⓨ㻌䝫䝸䜶䝏䝺䞁䜾䝸䝁䞊䝹䠄PEG䠅䛿䚸௦⾲ⓗ䛺Ỉ⁐ᛶ䝫䝸䜶䞊䝔䝹䛷䛒䜚䚸䛭䛾㧗䛔⏕యぶ࿴ᛶ䛛䜙䚸᭷ᶵ䞉㧗ศᏊ໬Ꮫ䛾◊✲ᑐ㇟䛾䜏䛺䜙䛪䚸䝍䞁䝟䜽㉁ᕤᏛ䜔⸆Ꮫ䛻䛚䛔䛶䜒ᛂ⏝䛥䜜䛶䛔䜛≀ ㉁䛷䛒䜛䚹䛧䛛䛧䛣䜜䜎䛷⏝䛔䜙䜜䛶䛝䛯 PEG 䛾䜋䛸䜣䛹䛿┤㙐≧ 䛾䜒䛾䛷䛒䜚䚸䛛䛴ศᏊ㔞ศᕸ䜢᭷䛩䜛ከศᩓᛶ䛾ΰྜ≀䛷䛒䜛䚹㔜ྜᗘ䛾␗䛺䜛 PEG 䛿␗䛺䜛≀ᛶ䜢♧䛩䛣䛸䛜▱䜙䜜䜛䛜䚸ศᏊ 䝃䜲䝈䜔ᙧ≧䛾ຠᯝ䜢⢭ᐦ䛻ㄪ䜉䜛䛻䛿䚸ศᏊ㔞ศᕸ䜢ᣢ䛯䛺䛔䚸༢ศᏊ PEG 䜢⏝䛔䛯≀ᛶホ౯䛜ᚲせ䛷䛒䜛䚹㏆ᖺ䛻䛚䛔䛶䚸㛗㙐䛾ศᏊ PEG 䛾ྜᡂ䚸≀ᛶホ౯䛻䛴䛔䛶ሗ࿌䛿䛒䜛䜒䛾䛾䚸 ᮍ䛰䛭䛾౛䛿㝈䜙䜜䛶䛔䜛䚹䛣䛾䜘䛖䛺⫼ᬒ䛾䜒䛸ᡃ䚻䛿䚸PEG 䛾. ᅗ 2䠖 ୕ゅᙧᆺ PEG. ᙧ≧䛜≀ᛶ䛻୚䛘䜛ຠᯝ䜢᥈䜛䜉䛟䚸㠀┤㙐≧䛾༢ศᏊ PEG 㢮⦕య䜢㛤Ⓨ䛧䚸䛭䛾≀ᛶホ౯䚸ᶵ⬟㛤ᣅ䜢⾜䛺䛳䛶䛔䜛䚹䛭䛾୰䛷䚸ᅗ 2 䛻♧䛩୕ゅᙧᆺ䛻ᵓ㐀໬䛧䛯༢ศᏊ PEG 䜢㛤Ⓨ䛧䛯䚹VT 13C NMR ᐃ䛛䜙䚸䛣䛾୕ゅᙧᆺ PEG 䛿䚸 ᗘୖ᪼䛻క䛔䝁䞁䝣䜷䝯䞊䝅䝵䞁ኚ໬䚸䛴䜎䜚䜶䝏䝺䞁䜸䜻䝃䜲䝗㒊ศ䛾䝂䞊䝅䝳䛛䜙䜰䞁䝏ᙧ䜈䛾ኚ໬䜢㉳䛣䛩䛣䛸䛜♧၀䛥䜜䛯䚹䛣䛾䝁䞁䝣䜷䝯䞊䝅䝵䞁ኚ໬䛿䚸PEG 㒊ศ䛾ᴟᛶ䜢పୗ䛥䛫䜛䛣䛸䛜▱䜙䜜䜛䚹ᐇ㝿䚸1H NMR T1 ᐃ䛛䜙䚸୕ゅᙧᆺ PEG 䛿 60 ᗘ௜㏆䛷⬺Ỉ࿴䛩䜛䛣䛸䛜♧၀䛥䜜䛯䚹䜋䜌ྠ 䛨ศᏊ㔞䛾┤㙐≧ PEG 䛷䛿䚸20 °C 䛛䜙 80 °C 䛾 ᗘ⠊ᅖ䛻䛚䛔䛶⬺Ỉ࿴䛿ほ䛥䜜䛺䛔䚹ᚑ䛳䛶䚸PEG 䜢 ஧ḟඖⓗ䛻ᵓ㐀໬䛩䜛䛣䛸䛷䚸⬺Ỉ࿴ ᗘ䛾పୗ䛜ᘬ䛝㉳䛣䛥䜜䜛䛣䛸䛜♧၀䛥䜜䛯䚹䛣䛾ᛶ㉁䜢฼⏝䛧䚸୕ ゅᙧᆺ PEG 䛜䝸䝌䝏䞊䝮䛾จ㞟䜢ᢚไ䛩䜛ຠᯝ䜢᭷䛩䜛䛣䛸䜢ぢฟ䛧䛯䚹. ගᶵ⬟Ⓨ⌧䛻㛵୚䛩䜛άᛶ✀䛾㟁Ꮚ䝇䝢䞁ග໬Ꮫ◊✲ 䝺䞊䝄䞊䛸ྠᮇ䛧䛯✀䚻䛾᫬㛫ศゎ䝇䝨䜽䝖䝻䝇䝁䝢䞊䠄᫬㛫ศゎ EPR䚸䝟䝹䝇 EPR䚸㐣Ώ྾཰䚸Ⓨගศග ➼䠅䜢⏝䛔䚸㟁Ꮚ䝇䝢䞁䛜㛵୚䛩䜛ගᶵ⬟ᛶ䛚䜘䜃㛵㐃䛩䜛ග≀⌮໬Ꮫⓗ㐣⛬䛻䛴䛔䛶ゎ᫂䛩䜛◊✲άື䜢⾜䛳䛶䛔䜛䚹≉䛻䚸ගㄏ㉳㟁Ꮚ⛣ື཯ᛂึᮇ㐣⛬䛷㔜せ䛺ᙺ๭䜢ᢸ䛖㟁Ⲵศ㞳≧ែ䛾㟁Ꮚᵓ㐀䛸䛭䛾ᛶ㉁䞉 ືⓗᣲື䛻䛴䛔䛶◊✲䜢㐍䜑䛯䚹୍㐃䛾⣔⤫ⓗ䛻ศᏊタィ䛥䜜䛯㔜ཎᏊ䠄Pt䠅䜢ྵ䜐ບ㉳≧ែ䞉㟁Ⲵศ㞳≧ ែ䛾䝇䝢䞁ไᚚ䛻㛵䛩䜛◊✲䜢㐍䜑䚸㓄ྥ䞉㊥㞳ไᚚ䛾ᅉᏊ䜢௜୚䛩䜛䛣䛸䛻䜘䜚㧗ຠ⋡໬䛜㐩ᡂ䛷䛝䜛䛣䛸䜢᫂䜙䛛䛻䛧䛯䚹䛥䜙䛻䚸᪂つ䛾ศᏊෆ㟁Ⲵศ㞳⣔䛾᥈⣴䜢㐍䜑䛯⤖ᯝ䚸ᯫᶫ㒊䛾ᵓᡂཬ䜃 hole 䛾Ꮡᅾᵝᘧ 䜢ไᚚ䛩䜛䛣䛸䛻䜘䜚䚸䛣䛾≧ែ⏕ᡂ䛾㧗ຠ⋡໬䛸䛭䛾㛗ᑑ࿨໬䛜㐩ᡂ䛷䛝䜛䛣䛸䜢♧䛧䛯䚹ศᏊタィᣦ㔪䛸䛾㛵䜟䜚䛛䜙䚸㟁Ⲵศ㞳⣔䛻䛴䛔䛶≀⌮໬Ꮫⓗᛶ㉁䜢ヲ⣽䛻᫂䜙䛛䛻䛩䜛◊✲䜢㐍䜑䛶䛔䜛䚹䛣䜜䜙䛾◊✲䛸୪ ⾜䛧䛶䚸㧗䛔ගᶵ⬟ᛶ䜢᭷䛩䜛≀㉁⣔䜢᥈⣴䛩䜛◊✲䛾᪂䛯䛺ᒎ㛤䜢ᅗ䜛䛯䜑䛻䚸᫬㛫ศゎ EPR ἲ䛚䜘䜃☢ ሙຠᯝ䛻ຍ䛘䛶᪂つศගᡭἲ䛾㛤Ⓨ䜢㐍䜑䛶䛔䜛䚹䜎䛯䚸ගᶵ⬟䛸ᐦ᥋䛻㛵㐃䛩䜛ᖖ☢ᛶάᛶ✀䛾㟁Ꮚᵓ 㐀䛸ᛶ㉁䛻㛵䛩䜛◊✲䜢㐍䜑䛯䚹ඹྠ◊✲䛸䛧䛶䚸᪂つ䛾ཎᏊෆໟ䝣䝷䞊䝺䞁䛾᥈⣴䛸ගᶵ⬟ᛶᮦᩱ䜈䛾ᛂ ⏝◊✲䚸ᾮᬗศᏊ䛾ගຎ໬ᶵᵓ䛻㛵୚䛩䜛ບ㉳≧ែ䛚䜘䜃ᖖ☢ᛶ✀䛾ᛶ㉁䛻䛴䛔䛶᫂䜙䛛䛻䛩䜛◊✲䚸᭷ ᶵ༙ᑟయⷧ⭷୰䛷䛾㟁Ⲵ㍺㏦㐣⛬䛻㛵䛩䜛㟁Ꮚ䝇䝢䞁ග⛉Ꮫⓗ◊✲䜢㐍䜑䛶䛔䜛䚹.

(15) ◊㻌 ✲㻌 ά㻌 ື㻌 ሗ㻌 ࿌ 研究活動報告. 7. ⏕యศᏊᵓ㐀◊✲ศ㔝. ࠙◊✲άືሗ࿌ࠚ ᩍ ෸. 䠄2013䠊1䡚2013䠊12䠅㻌. ᤵ✄ⴥㅬḟ ᩍ. ᤵ㛛಴ᗈ. ຓ ᩍΏ㒊⪽ ༤ ኈ ◊ ✲ ဨ๓ᕝ᠇୍㸪஭ୖ㐨㞝㸪ዟᮧṇᶞ㸪బ⸨ྜྷ⨾㸪 ⴗཎㄔᬛ ◊ ✲ ⿵ ຓ ဨᰗ἟ᬛᏊ ኱ Ꮫ 㝔 ⏕ቑ஭⩧ྐ㻌㻌. ᮏ◊✲ศ㔝䛷䛿䚸⣽⬊䛻䛚䛡䜛䝍䞁䝟䜽㉁ရ㉁⟶⌮䝅䝇䝔䝮䛻䛴䛔䛶䚸ᵓ㐀⏕≀Ꮫ䚸⏕໬Ꮫ䚸䝥䝻䝔䜸䝭䜽䝇䚸⣽⬊⏕≀Ꮫ䛺䛹ከゅⓗ䜰䝥䝻䞊䝏䛻䜘䜚◊✲άື䜢⾜䛳䛶䛔䜛䠊2013 ᖺ䛾୺䛯䜛◊✲άື䛸䛧䛶䛿䠈௨ୗ 䛾䜘䛖䛻ᴫᣓ䛥䜜䜛䠊㻌㻌㻝㻚㻌 ᪂つ䝆䝇䝹䝣䜱䝗⤖ྜᙧᡂᅉᏊ 㻼㼞㼤㻠䜢㉳Ⅼ䛸䛧䛯㓟໬ⓗ䝣䜷䞊䝹䝕䜱䞁䜾⤒㊰䛾ᵓ㐀䛸స⏝ᶵᗎ䛻㛵䛩䜛 ◊✲㻌 ᑠ⬊యෆ䛷ྜᡂ䛥䜜䜛䝍䞁䝟䜽㉁䛾ከ䛟䛿䝆䝇䝹䝣䜱䝗⤖ྜ䛾ᑟධ䜢క䛖㓟໬ⓗ䝣䜷䞊䝹䝕䜱䞁䜾䜢ཷ䛡䜛䛸 ⪃䛘䜙䜜䛶䛚䜚䚸䛭䛾ᇶ㉁䛻䛿✀䚻䛾⣽⬊⾲ᒙཷᐜయ䚸ච␿䜾䝻䝤䝸䞁䚸⾑ᾮจᅛᅉᏊ䚸䜲䞁䝇䝸䞁䛺䛹䛜▱䜙䜜䜛䚹䛣䜜䜎䛷┿᰾⏕≀䛻䛚䛡䜛䝍䞁䝟䜽㉁䛾䝆䝇䝹䝣䜱䝗⤖ྜ䛾ᙧᡂ䛿୺䛸䛧䛶ᇶ㉁䛻䝆䝇䝹䝣䜱䝗⤖ྜ䜢ᑟ ධ䛩䜛 protein disulfide isomerase (PDI) 䛸 PDI 䜢෌㓟໬䛩䜛㓝⣲ ER oxidoreductin 1 (Ero1) 䛻䜘䜚⾜䜟䜜䜛䛸 ⪃䛘䜙䜜䛶䛝䛯䚹䛧䛛䛧㏆ᖺ Ero1 ௨እ䛾 PDI 㓟໬㓝⣲䛜┦ḟ䛔䛷ྠᐃ䛥䜜䚸㧗➼ື≀⣽⬊䛾ᑠ⬊య䛻䛿䝆䝇䝹䝣䜱䝗⤖ྜᙧᡂ䛾䛯䜑䛾」ᩘ䛾㓟໬⤒㊰䛜」㞧䛺䝛䝑䝖䝽䞊䜽䜢ᙧᡂ䛧䛶䛔䜛䛸䛔䛖᪂䛯䛺ᴫᛕ䛜☜❧䛥䜜䛴䛴䛒䜛䚹 ᡃ䚻䛿᪂つ䛻ぢ䛴䛛䛳䛯 PDI 㓟໬㓝⣲ peroxiredoxin-4 (Prx4) 䜢௓䛧䛯䝆䝇䝹䝣䜱䝗⤖ྜᑟධ⤒㊰䛾ྠᐃ䛸ᶵ⬟ⓗᙺ๭䛾ゎ᫂䜢 ┠ⓗ䛸䛧䛶◊✲䜢㐍䜑䛯䚹䛭䛾⤖ᯝ䚸Ero1 䛿 PDI 䜢≉␗ⓗ䛻㓟໬ 䛩䜛䛾䛻ᑐ䛧䚸Prx4 䛿 PDI 䝣䜯䝭䝸䞊䝍䞁䝟䜽㉁䛾䛖䛱 ERp46 䛸 P5 䛾㓟໬䛻≉໬䛧䛯㓟໬㓝⣲䛷䛒䜛䛣䛸䜢᫂䜙䛛䛻䛧䛯䚹䜎䛯 PDI 䛿 ᇶ㉁䛻ṇ䛧䛟䝆䝇䝹䝣䜱䝗⤖ྜ䜢ᑟධ䛩䜛䛾䛻ᑐ䛧䚸P5 䛒䜛䛔䛿 ERp46 䛿 PDI 䜘䜚䜒㧗ຠ⋡䛻䝆䝇䝹䝣䜱䝗⤖ྜ䜢ᑟධ䛩䜛䜒䛾䛾䚸 ṇ䛧䛔䝆䝇䝹䝣䜱䝗⤖ྜ䜢㑅ᢥⓗ䛻ᯫ䛡䜛⬟ຊ䛻䛿Ḟ䛡䜛䛣䛸䛜䜟䛛䛳䛯䚹䛣䜜䜙␗䛺䜛」ᩘ䛾㓟໬⤒㊰䛿⊂❧䛧䛶ാ䛟䜟䛡䛷䛿䛺䛟䚸 ༠ྠⓗ䛛䛴┦஌ⓗ䛻㓟໬ⓗ䝣䜷䞊䝹䝕䜱䞁䜾䜢ಁ㐍䛩䜛䛣䛸䜒᫂䜙䛛䛻䛧䛯䚹䛭䛾⤖ᯝ䚸ᅗ䠍䛻♧䛩့ஙື≀⣽⬊䛾ᑠ⬊య䛻䛚䛡䜛㓟໬ⓗ䝣䜷䞊䝹䝕䜱䞁䜾䝛䝑䝖䝽䞊䜽䛾䝰䝕䝹䜢ᥦၐ䛩䜛䛻⮳䛳䛶䛔䜛䚹. ᅗ䠍 ့ஙື≀⣽⬊䛾ᑠ⬊య䛻䛚䛡䜛㓟໬ⓗ 䝣䜷䞊䝹䝕䜱䞁䜾䝛䝑䝖䝽䞊䜽.

(16) ◊㻌 ✲㻌 ά㻌 ື㻌 ሗ㻌 ࿌ 研究活動報告. 䛥䜙䛻 Prx4 䛻䜘䜛 PDI 䝣䜯䝭䝸䞊䝍䞁䝟䜽㉁䛾ㄆ㆑ᶵᵓ䛻䛴䛔䛶䜒⏕໬Ꮫⓗゎᯒ䜢㐍䜑䚸ᮏ㓝⣲䛜 PDI 䝣䜯䝭䝸䞊䝍䞁䝟䜽㉁䜢䝏䜸䝺䝗䜻䝅䞁䝗䝯䜲䞁༢఩䛷ㄆ㆑䛩䜛䛣䛸䜢᫂䜙䛛䛻䛧䛯䚹䛥䜙䛻 P5 䛾䝏䜸䝺䝗䜻䝅䞁䝗䝯䜲䞁䛸 Prx4 䛾άᛶ㒊఩䝅䝇䝔䜲䞁䜢ྵ䜐 C ᮎ➃ tail 㡿ᇦ䛾」ྜయ䛾⤖ᬗᵓ㐀䜢 2.1䊅ศゎ⬟䛷ゎ䛟䛣䛸䛻䜒ᡂ ຌ䛧䚸䛣䜜䛻䜘䜚 Prx4 䛻䜘䜛䝏䜸䝺䝗䜻䝅䞁䝗䝯䜲䞁ㄆ㆑䛾ヲ⣽䛺ศᏊᶵᵓ䛜ゎ᫂䛥䜜䛯䚹㻌㻞㻚㻌㻱㻾㼜㻠㻢䛸㻼㻰㻵䛾␗䛺䜛ᵓ㐀䛸ᶵ⬟ⓗᙺ๭䛻㛵䛩䜛◊✲㻌 ့ஙື≀⣽⬊䛾ᑠ⬊య䛻䛿䠎䠌✀㢮௨ୖ䛾 PDI 䝣䜯䝭䝸䞊䝍䞁䝟䜽㉁䛜Ꮡᅾ䛩䜛䛜䚸ಶ䚻䛾ᅉᏊ䛾⏕⌮ⓗ ᶵ⬟䛿䜋䛸䜣䛹ゎ᫂䛥䜜䛶䛔䛺䛔䚹๓⠇䛷㏙䜉䛯䜘䛖䛻䚸᪂䛯䛻ぢ䛴䛛䛳䛯 PDI 㓟໬㓝⣲ Prx4 䛿 ERp46 䛻ᑐ 䛧䛶ᴟ䜑䛶㧗䛔㓟໬άᛶ䜢᭷䛩䜛䛣䛸䜢ᡃ䚻䛿᫂䜙䛛䛻䛧䛯䚹䛭䛣䛷ᮏ◊✲䛷䛿䚸ERp46 䛚䜘䜃 Prx4–ERp46 」ྜయ䛾ᵓ㐀ᶵ⬟ゎᯒ䜢㐍䜑䚸Prx4 䛸 ERp46 䜢௓䛧䛯ᇶ㉁䜈䛾䝆䝇䝹䝣䜱䝗ᑟධ⤒㊰䛾ᵓ㐀ᇶ┙䜢☜❧䛩䜛䛻⮳䛳䛯䚹ERp46 䛿 3 䛴䛾䝏䜸䝺䝗䜻䝅䞁䝗䝯䜲䞁(Trx)䛛䜙ᡂ䜛䛜䚸䛣䛾䛖䛱 Trx1, Trx2 䛾 X ⥺⤖ᬗᵓ㐀䜢䛭䜜䛮䜜 2.5 䠈0.95 䛾ศゎ⬟䛷Ỵᐃ䛧䛯䚹䜎䛯䚸Trx2 䛸 Prx4 䛾 C ᮎ➃㡿ᇦ䛾」ྜయ䛾⤖ᬗᵓ㐀䜢 0.92 䛾ศゎ ⬟䛷Ỵᐃ䛧䚸䛭䛾⤖ྜᵝᘧ䜢᫂䜙䛛䛻䛩䜛䛸䛸䜒䛻䚸Prx4 䛜₯ᅾⓗ䛻ᖜᗈ䛟 PDI 䝣䜯䝭䝸䞊䝍䞁䝟䜽㉁䛾 Trx 䜢ㄆ㆑䛧㓟໬䛩䜛ᶵᵓ䜢ゎ᫂䛧䛯䚹䛥䜙䛻䚸X ⥺ᑠゅᩓ஘ἲ䛻䜘䜚 ERp46 䛾඲㛗ᵓ㐀䛾䝰䝕䝸䞁䜾䜢⾜䛳䛯⤖ᯝ䚸 ERp46 䛿௚䛾 PDI 䝣䜯䝭䝸䞊䝍䞁䝟䜽㉁䛻䛿䜏䜙䜜䛺䛔᪂つ䛺䛂㛤䛔䛯 V Ꮠᵓ㐀䛃䜢䛸䛳䛶䛔䜛䛣䛸䛜᫂䜙䛛䛻䛺䛳䛯䚹䛥䜙䛻⣔⤫ⓗ䛺ᶵ⬟ゎᯒ䛻䜘䜚䚸Prx4-ERp46 ⤒㊰䛿㓟໬ⓗ䝣䜷䞊䝹䝕䜱䞁䜾ึᮇ䛻䛚䛡䜛䝆䝇䝹䝣䜱䝗 ⤖ྜᑟධ䛾䝇䝔䝑䝥䜢ಁ㐍䛩䜛䛾䛻ᑐ䛧䚸Ero1-PDI ⤒㊰䛿㓟໬ⓗ䝣䜷䞊䝹䝕䜱䞁䜾ᚋᮇ䛻䛚䛡䜛୰㛫≧ែ䛛䜙᭱ ⤊ኳ↛≧ែ䜈䛾⛣⾜䝇䝔䝑䝥䜢ಁ㐍䛩䜛䛣䛸䜢ぢ䛔ฟ䛧䛯䠄ᅗ䠎䠅䚹ERp46 䛿㛤䛔䛯 V Ꮠᵓ㐀ୖ䛷άᛶ㒊఩䜢⁐፹ 䛻㟢ฟ䛥䛫䚸䜰䞁䝣䜷䞊䝹䝗䛧䛯ᇶ㉁䛻䝷䞁䝎䝮䛛䛴㎿㏿䛻䝆䝇䝹䝣䜱䝗⤖ྜ䜢ᑟධ䛩䜛䛾䛻ᑐ䛧䚸PDI 䛿 U Ꮠᵓ㐀 ෆ㒊䛾␯Ỉᛶ䝫䜿䝑䝖䛻䝣䜷䞊䝹䝕䜱䞁䜾୰㛫≧ែ䜢ྲྀ䜚㎸䜏䚸஫䛔䛻ྥ䛝ྜ䛳䛯άᛶ㒊఩䛜༠ㄪⓗ䛻䛿䛯䜙䛟䛣䛸䛷ຠ⋡䜘䛟䝆䝇䝹䝣䜱䝗⤖ྜ䛾⤌᥮䛘䜢⾜䛖䛸⪃ᐹ䛧䛶䛔䜛䚹ᮏ◊✲䛻䜘䜚䚸့ஙື≀⣽⬊䛾௦⾲ⓗ䛺 PDI 䝣䜯䝭䝸䞊䝍䞁䝟䜽㉁ ERp46 䛸 PDI 䛾␗䛺䜛ᵓ㐀䛸ᶵ⬟ⓗᙺ๭䛜 ᫂䜙䛛䛸䛺䛳䛯䚹. ᅗ䠎 ERp46 䛸 PDI 䛾㓟໬ⓗ䝣䜷䞊䝹䝕䜱䞁䜾䛻䛚䛡䜛␗䛺䜛ᶵ⬟ⓗᙺ๭. 㻌㻟㻚 ᑠ⬊య䠉䝂䝹䝆య㛫䛻Ꮡᅾ䛩䜛㼜㻴໙㓄䛻౫Ꮡ䛧䛯᪂䛯䛺䝍䞁䝟䜽㉁ရ㉁⟶⌮ᶵᵓ䛻㛵䛩䜛◊✲㻌 ᑠ⬊య䛷ṇᖖ䛺㧗ḟᵓ㐀䜢⋓ᚓ䛧䛯䝍䞁䝟䜽㉁䛿ศἪ⤒㊰䛻ἢ䛳䛶䝂䝹䝆య䜈䛸㍺㏦䛥䜜䜛䚹୍᪉䛷䚸PDI 䜔Ero1 䛾䜘䛖䛺ᑠ⬊యᒁᅾ䝍䞁䝟䜽㉁䜒ศἪ⤒㊰䛻ἢ䛳䛶䝂䝹䝆య䜈䛸㍺㏦䛥䜜䜛䛣䛸䛜䛒䜛䚹䛭䛾䜘䛖䛺ሙ ྜ䛷䜒䚸ᑠ⬊యᒁᅾ䝍䞁䝟䜽㉁䛾ከ䛟䛿ᑠ⬊యᒁᅾ㓄ิ(KDEL㓄ิ)䜢䜒䛴䛯䜑䚸COP-I⿕そᑠ⬊䛻䛾䛳䛶㏫⾜㍺㏦䜢ཷ䛡ᑠ⬊య୰䛻䝸䝔䞁䝅䝵䞁䛥䜜䜛䚹⯆࿡῝䛔䛣䛸䛻䚸PDI㓟໬㓝⣲䛷䛒䜛Ero1 䛿ᑠ⬊యᒁᅾ䝍䞁䝟䜽㉁䛷䛒䜛䛻䜒㛵䜟䜙䛪䚸KDEL㓄ิ䜢䜒䛯䛺䛔䚹㐣ཤ䛾◊✲䛛䜙䚸Ero1 䛿KDEL㓄ิ䜢䜒䛴ERp44䛸䝂䝹䝆 య䛻䛚䛔䛶⤖ྜ䛩䜛䛣䛸䛻䜘䜚䚸ᑠ⬊య䜈㏫⾜㍺㏦䛥䜜䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛䚹ᮏ◊✲䛷䛿䚸ᑠ⬊య୰䛷䛿㓟໬㓝⣲䛸䛧䛶PDI䛸≉␗ⓗ䛻⤖ྜ䛩䜛Ero1 䛜ఱᨾ䝂䝹䝆య䛷䛿ERp44䛸⤖ྜ䛩䜛䛾䛛䚸䛭䛾ぶ࿴ᛶไᚚᶵ ᵓ䛾ゎ᫂䛻ྲྀ䜚⤌䜣䛰䚹. 8.

(17) 9. ◊㻌 ✲㻌 ά㻌 ື㻌 ሗ㻌 ࿌ 研究活動報告. PDI䝣䜯䝭䝸䞊䝍䞁䝟䜽㉁䛾1䛴䛷䛒䜛ERp44䛿䚸a-b-b’䛾3䛴䛾䝏䜸䝺䝗䜻䝅䞁ᵝ䝗䝯䜲䞁䛸Cᮎ➃䛾tail (C-tail) 䛛䜙ᵓᡂ䛥䜜䛶䛚䜚䚸a䝗䝯䜲䞁୰䛾Cys29䛾࿘䜚䛻䛿␯Ỉᛶ䝟䝑䝏䛜Ꮡᅾ䛩䜛䚹䜎䛯ERp44䛾⤖ᬗᵓ㐀୰䛻䛚䛔䛶䚸Cys29䛸C-tailୖ䛻Ꮡᅾ䛩䜛Thr369䛸䛾㛫䛻Ỉ⣲⤖ྜ䛜ᙧᡂ䛥䜜䚸䛣䜜䛻䜘䜚C-tail㡿ᇦ䛿㛢䛨䛯≧ែ䛻䛒䜛䚹䛭䛾⤖ᯝ䚸ᇶ㉁䛸䛾⤖ྜ䛻㛵䜟䜛䛸⪃䛘䜙䜜䜛Cys29䛸␯Ỉᛶ㡿ᇦ䛿そ䜟䜜䛯≧ែ䛸䛺䛳䛶䛔䜛䚹 ௨ୖ䛾ERp44䛾ᵓ㐀ⓗ≉ᚩ䛸ᑠ⬊య–䝂䝹䝆య 㛫䛻Ꮡᅾ䛩䜛pH໙㓄䛻╔┠䛧䚸⣔⤫ⓗ䛺ᵓ㐀⏕໬ Ꮫᐇ㦂䜢⾜䛳䛯䚹䛭䛾⤖ᯝ䚸ᑠ⬊య䛾pH䛻┦ᙜ䛩䜛pH7.2䛷䛿ERp44䛾C-tail䛜㛢䛨Ero1 䛸ERp44䛜ゎ㞳䛩䜛䛾䛻ᑐ䛧䚸䝂䝹䝆య䛾pH䛻┦ᙜ䛩䜛pH6.5 䛷䛿ERp44䛾C-tail䛜㛤䛝Ero1 䛸ERp44䛜ᙉ䛟⤖ྜ 䛩䜛䛣䛸䛜ุ᫂䛧䛯䚹䛩䛺䜟䛱䚸pH౫Ꮡⓗ䛺ERp44 䛾C-tail䛾㛤㛢䛜ERp44-Ero1 㛫䛾ぶ࿴ᛶ䜢ไᚚ 䛩䜛䛣䛸䜢᫂䜙䛛䛻䛧䛯䚹⣽⬊䜢⏝䛔䛯ᐇ㦂⤖ᯝ䛸 ᅗ䠏 ERp44 䛾 pH ౫Ꮡⓗ䛺ᵓ㐀ኚ໬䛻ᇶ䛵䛟ᮍᡂ⇍ ే䛫䚸௨ୗ䛻グ䛩ERp44䜢௓䛧䛯᪂䛯䛺䝍䞁䝟䜽㉁䝍䞁䝟䜽㉁䛾䝂䝹䝆య—ᑠ⬊య㏫⾜㍺㏦ᶵᵓ ရ㉁⟶⌮ᶵᵓ䜢ᥦၐ䛩䜛䛻⮳䛳䛯䠄ᅗ䠏䠅䚹 (i) 䝂䝹䝆య䛾పpH䛻䛚䛔䛶ERp44䛾Cys29䛜䝥䝻䝖䞁໬䛥䜜䜛䛣䛸䛻䜘䜚䚸C-tail䛜㛢䛨䛯≧ែ䜢Ᏻᐃ໬䛩䜛䛾䛻ᚲせ䛺Ỉ⣲⤖ྜ䝛䝑䝖䝽䞊䜽䛜◚ቯ䛥䜜䜛䚹 (ii) 䛭䛾⤖ᯝ䚸C-tail䛜㛤䛝䚸ERp44䛜Ero1 䜔ᵓ㐀ᮍᡂ⇍䛺䝍䞁䝟䜽㉁䜢ᤕ⋓䛧䚸䛥䜙䛻KDELཷᐜయ䛸䜒⤖ ྜ䛩䜛䚹 (iii) COP-I⿕そᑠ⬊䛻䜘䜚䚸ᇶ㉁䜢ᤕ⋓䛧䛯ERp44䛸KDELཷᐜయ䛾」ྜయ䛜ᑠ⬊య䜈㏫⾜㍺㏦䛥䜜䜛䚹 (iv) ᑠ⬊య䛾୰ᛶpH䛷䛿䚸C-tail䛿㛢䛨䛯≧ែ䛻ᡠ䜚䚸ERp44, ᇶ㉁, KDELཷᐜయ䛿ゎ㞳䛩䜛䚹㏫⾜㍺㏦䛥䜜䛯ᇶ㉁䛿䚸ᑠ⬊య䛻䛚䛔䛶෌ᗘṇ䛧䛔❧యᵓ㐀ᙧᡂ䠄ᡂ⇍໬䠅䛜ಁ䛥䜜䜛䚹 ⌧ᅾ䛿ERp44䛻䜘䜛ᵓ㐀ᮍᡂ⇍䛺ᇶ㉁ㄆ㆑ᶵᵓ䜢ㄪ䜉䜛䛯䜑䚸X⥺⤖ᬗᵓ㐀ゎᯒ䜔X⥺ᑠゅᩓ஘ἲ䛻䜘䜛 ERp44–ᇶ㉁」ྜయ䛾ศᏊᵓ㐀䛾Ỵᐃ䛻ྲྀ䜚⤌䜣䛷䛔䜛䚹㻌㻠㻚 ့ஙື≀⣽⬊ᑠ⬊య䛻ᒁᅾ䛩䜛㻱㻾㼐㼖㻡䛾⏕⌮ⓗᇶ㉁ೃ⿵䝍䞁䝟䜽㉁䛾⥙⨶ⓗゎᯒ䛻㛵䛩䜛◊✲㻌䝆䝇䝹䝣䜱䝗⤖ྜ䛾ᙧᡂ䛿䚸ከ䛟䛾ศἪ䝍䞁䝟䜽㉁䛻䛸䛳䛶❧యᵓ㐀ᙧᡂୖ䚸㔜せ䛺཯ᛂ䝇䝔䝑䝥䛷䛒䜛䚹ୖ ㏙䛾䜘䛖䛻䚸့ஙື≀⣽⬊䛾 PDI 䝣䜯䝭䝸䞊䝍䞁䝟䜽㉁䛾ከ䛟䛿ศἪ䝍䞁䝟䜽㉁䛻䝆䝇䝹䝣䜱䝗⤖ྜ䜢ᑟධ䛩䜛㐣⛬䜔䝆䝇䝹䝣䜱䝗⤖ྜ䜢ษ᩿䛩䜛㐣⛬䛻㛵䜟䜛䛸ண᝿䛥䜜䜛䚹䛧䛛䛧䚸ಶ䚻䛾㓝⣲䛾⏕⌮ⓗ䛺ᙺ๭䛻䛴䛔䛶䛿䜋䛸䜣䛹ศ䛛䛳䛶䛔䛺䛔䚹䛭䛾᭱䜒኱䛝䛺せᅉ䛸䛧䛶䛿ྛ㓝⣲䛾⏕యෆ䛻䛚䛡䜛ᇶ㉁䛜୙᫂䛷䛒䜛䛣䛸䛜ᣲ 䛢䜙䜜䜛䚹ᮏ◊✲䛷䛿䛣䜜䜙䛾㓝⣲䛾⏕యෆ䛻䛚䛡䜛ാ䛝䜢⌮ゎ䛩䜛䛯䜑䛾➨୍Ṍ䛸䛧䛶䚸PDI 䝣䜯䝭䝸䞊䝍䞁䝟䜽㉁䛾୍䛴䛷䛒䜛 ERdj5 䛾䝬䜴䝇ಶయ⤌⧊୰䛻䛚䛡䜛ᇶ㉁䜢ྠᐃ䛩䜛䛣䛸䛻䛧䛯䚹 PDI 䝣䜯䝭䝸䞊䛻ᒓ䛩䜛㓝⣲䛜䚸䝆䝇䝹䝣䜱䝗⤖ྜ䛾ᙧᡂ䚸㑏ඖ䚸␗ᛶ໬䜢⾜䛖㝿䛻䛿䚸㓝⣲䛸ᇶ㉁䛜ศᏊ㛫䛾䝆䝇䝹䝣䜱䝗⤖ྜ䛷㐃⤖䛧䛯୰㛫య䜢ᙧᡂ䛩䜛䛣䛸䛜▱䜙䜜䜛䚹ERdj5 䛾ᇶ㉁䜢ྠᐃ䛩䜛䛯䜑䛻䚸䛣䛾ᛶ㉁䜢 ฼⏝䛩䜛䛣䛸䛻䛧䛯䚹䛯䛰䛧䚸䛣䛾䜘䛖䛺㓝⣲䞉ᇶ㉁」ྜయ䛿㏻ᖖ୍㐣ⓗ䛻䛾䜏ᙧᡂ䛩䜛䚹䜘䛳䛶䚸䛣䛾䜘䛖䛺」 ྜయ䜢⢭〇䛧ᇶ㉁䜢ྠᐃ䛩䜛䛯䜑䛻䛿䚸䛔䛛䛻䛣䛾」ྜయ䜢Ᏻᐃ໬䛥䛫䜛䛛䛜㘽䛻䛺䜛䚹ᮏ◊✲䛷䛿䚸䝖䝸䜽䝻䝻㓑㓟䛸 N-䜶䝏䝹䝬䝺䜲䝭䝗䜢฼⏝䛩䜛䛣䛸䛻䜘䛳䛶䝬䜴䝇ಶయ䛾⤌⧊୰䛷⏕ᡂ䛩䜛䛣䛾䜘䛖䛺」ྜయ䜢Ᏻᐃ໬ 䛩䜛䛣䛸䛻ᡂຌ䛧䛯䚹ERdj5 䛻ᑐ䛩䜛ᢠయ䜢฼⏝䛧䛶ㄪ䜉䛯䛸䛣䜝䚸ERdj5 䛿䝬䜴䝇䛾⢭ᕢୖయ䛷ᙉ䛟Ⓨ⌧䛧䛶䛚䜚䚸䛣䛾⤌⧊୰䛷䚸ୖ㏙䛾୰㛫య䛸ண᝿䛥䜜䜛」ྜయ䛜ከᩘ᳨ฟ䛥䜜䛯䚹䛭䛣䛷䚸䛣䜜䜙䛾」ྜయ䜢䚸ERdj5 䛻ᑐ䛩䜛ᢠయ䜢฼⏝䛧䛶⢭〇ᚋ䚸㉁㔞ศᯒἲ䛻䜘䛳䛶ゎᯒ䛧䛯䚹䛭䛾⤖ᯝ䚸ERdj5 䛾ᇶ㉁䛾ೃ⿵䛸䛺䜛䝍䞁䝟䜽㉁䜢ከᩘྠᐃ䛩䜛䛣䛸䛻ᡂຌ䛧䛯䚹᭦䛻䚸䛣䜜䜙䛾䝍䞁䝟䜽㉁䛾ᗄ䛴䛛䛿ศᏊ㛫䛾䝆䝇䝹䝣䜱䝗⤖ྜ䜢௓䛧䛶 ERdj5 䛸┦஫స⏝䛧䛶䛔䜛䛣䛸䜒✺䛝Ṇ䜑䛯䚹ྠᐃ䛥䜜䛯䝍䞁䝟䜽㉁䛛䜙ᮏ㓝⣲䛾⏕యෆ䛻䛚䛡䜛ᶵ⬟䜢ண 䛩䜛ୖ䛷ᴟ䜑䛶᭷⏝䛺▱ぢ䛜ᚓ䜙䜜䛯䚹.

(18) ◊㻌 ✲㻌 ά㻌 ື㻌 ሗ㻌 ࿌ 研究活動報告. ࠙◊✲άືሗ࿌ࠚ. 10. ࢱࣥࣃࢡᶵ⬟ゎᯒ◊✲ศ㔝㸦2013㸬1㹼2013㸬12㸧㻌 ᩍ ෸ ຓ. ᤵ㰻⸨ṇ⏨ ᩍ. ᤵᯇ஭ᩄ㧗 ᩍ༡㒊࿘௓. ᢏ ⾡ ⫋ ဨ༓ᅜ཭Ꮚ ኱ Ꮫ 㝔 ⏕すᒣᓫᕝෆ㇂ᗣ ᮏ◊✲ศ㔝࡛ࡣࠊ㔠ᒓࢱࣥࣃࢡ㉁ࡢᵓ㐀࡜ᶵ⬟࡟ࡘ࠸࡚◊✲άືࢆ⾜ࡗ࡚࠸ࡿࠋ2013 ᖺࡢ◊✲άື ࡜ࡋ࡚ࡣࠊ୺࡟࣒࣊ศゎ㓝⣲࡟㛵ࡍࡿ◊✲ࢆ㐍ࡵࠊᡂᯝࡣ௨ୗࡢࡼ࠺࡟ᴫᣓࡉࢀࡿࠋ 㻌 1. HO 䛾ᇶ㉁ྲྀ䜚㎸䜏ᶵᵓ ⏕య䛻䛚䛡䜛䝦䝮䠄㕲—䝫䝹䝣䜱䝸䞁㘒య䠅䛾ศゎ཯ᛂ 䛿䚸䝦䝮௦ㅰ䞉ᢠ㓟໬䝇䝖䝺䝇䞉䝅䜾䝘䝹ఏ㐩䛺䛹䛾㔜せ䛺⏕⌮ᶵ⬟䜢ᢸ䛳䛶䛔䜛䚹䜋ங㢮䛛䜙Ⓨぢ䛥䜜䛯䝦䝮䜸䜻䝅䝀䝘䞊䝊䠄HO䠅䛿ྂ䛟䛛䜙▱䜙䜜䛯䝦䝮ศゎ㓝⣲䛷䛒䜚䚸䝦䝮䜢 CO䞉㕲䞉䝡䝸䝧䝹䝆䞁䛻ศゎ䛩䜛䠄ᅗ䠍䠅䚹HO ཯ᛂ䛿ᇶ㉁⮬㌟䛻䜘䜛㓟⣲άᛶ໬䛷㐍⾜䛧䚸䛭䛾≉Ṧ䛺 ⮬ᕫ㓟໬ᶵᵓ䛿䜋䜌ゎ᫂䛥䜜䛶䛝䛯䚹䛧䛛䛧䚸HO 䛻䛚䛡䜛ᇶ㉁ྲྀ䜚㎸䜏䜔䚸άᛶ୰ᚰᙧᡂ䛾ᶵᵓ䛿䜋䛸䜣䛹୙᫂. ᅗ 㻝㻌㻴㻻䛻䜘䜛䝦䝮ศゎ཯ᛂ. 䛷䛒䛳䛯䚹䛭䛣䛷ᮏᖺ䛿䚸䝆䝣䝔䝸䜰⳦⏤᮶䛾 HO䠄HmuO䠅䛾⤖ᬗᵓ㐀䜢䚸ᇶ㉁㠀⤖ྜᆺ䞉Fe-䝡䝸䝧䝹䝆䞁㘒 య⤖ྜᆺ䞉䝡䝸䝧䝹䝆䞁⤖ྜᆺ䛻䛴䛔䛶ྛ䚻1.80, 1.90, 1.85 Å ศゎ⬟䛷Ỵᐃ䛧䛯䚹 ᪤ሗ䛾䝦䝮⤖ྜᆺ HmuO 䛷䛿䚸䝦䝮䛿䠎䛴䛾䝦䝸䝑䜽䝇䠄㐲఩䞉㏆఩䝦䝸䝑䜽䝇䠅䛻ᣳ䜎䜜䜛ᙧ䛷⤖ྜ䛧䛶䛔䜛䠄ᅗ 2A䠅䚹䛣䜜䜙䛾䝦䝸䝑䜽䝇䛿ᇶ㉁㠀⤖ྜᆺ HmuO䠄ᅗ 2B䠅䛷䛿䛚஫䛔䛻㐲䛦䛛䜚䚸䛥䜙䛻䝦䝮㍈㓄఩Ꮚ䛾䝠䝇䝏䝆䞁䠄His20䠅䜢ྵ䜐㏆఩䝦䝸䝑䜽䝇䛜㒊ศⓗ䛻䜋䛹䛡䛶䛔䛯䚹䛣䜜䜙䛾ᵓ㐀ኚ໬䛻䜘䛳䛶ᇶ㉁㠀⤖ྜᆺ HmuO 䛾㛤ཱྀ㒊䛿኱䛝䛟ᗈ䛜䛳䛶䛚䜚䚸䝦䝮䛾ྲྀ䜚㎸䜏䜢ᐜ᫆䛻䛩䜛䛸⪃䛘䜙䜜䛯䚹䛥䜙䛻཯ᛂ䛻㔜せ䛺㐲఩ 䝦䝸䝑䜽䝇࿘㎶䛷䛿䚸ỈศᏊ䜢ྵ䜐Ỉ⣲⤖ྜ䝛䝑䝖䝽䞊䜽䛾ᵓ㐀䛜኱䛝䛟ኚ໬䛧䛶䛔䛯䠄ᅗ 2C䠅䚹䛣䛾⤖ᯝ䛛䜙䚸 HmuO 䛾άᛶ୰ᚰᵓ㐀䛿䝦䝮䛜⤖ྜ䛧䛶ึ䜑䛶ᙧᡂ䛥䜜䜛䛣䛸䛜♧䛥䜜䛯䚹ྠᵝ䛾ᵓ㐀ኚ໬䛿ศᏊືຊᏛィ ⟬䛻䛚䛔䛶䜒ぢ䜙䜜䚸ୖグ䛾ᥦ᱌䛜ᙉ䛟ᨭᣢ䛥䜜䛯䚹 ୍᪉䚸䝡䝸䝧䝹䝆䞁」ྜయ䛷䛿 His20䠄㍈㓄఩Ꮚ䠅䛜≉ᚩⓗ䛺ື䛝䜢♧䛧䛯䚹Fe3+䝡䝸䝧䝹䝆䞁㘒య⤖ྜᆺ䛷䛿 His20 䛿㕲䛻㓄఩䛧䚸䜲䝭䝎䝌䞊䝹ᇶ䛿䝢䝻䞊䝹㠃䛻ᑐ䛧䛶䜋䜌ᆶ┤ 䛸䛺䜛䠄ᅗ 3-1䠅䚹䛧䛛䛧䚸㕲䛜⬺㞳䛧䛯䝡䝸䝧䝹䝆䞁⤖ྜᆺ䛷䛿 His20 䛿⣙ 90 ᗘᅇ㌿䛧䚸䝢䝻. ᅗ 㻞㻌 ᇶ㉁䛾᭷↓䛻䜘䜛㻴㼙㼡㻻䛾ᵓ㐀ኚ໬㻌.

(19) ◊㻌 ✲㻌 ά㻌 ື㻌 ሗ㻌 ࿌ 研究活動報告. 11. 䞊䝹㠃䛻ᖹ⾜䛺䝁䞁䝣䜷䝯䞊䝅䝵䞁䛻ኚ໬䛧䛶䛔䛯䠄ᅗ 3-4䠅䚹 ⯆࿡῝䛔䛣䛸䛻䚸Fe 䝡䝸䝧䝹䝆䞁㘒య⤖ྜᆺ䛾୍㒊䛷䛿୰ ᚰ㕲䛜Ꮡᅾ䛩䜛䛻䜒ᣊ䜟䜙䛪䚸His20 䛜ᅇ㌿䛧䛶䛔䛯䠄ᅗ 3-2䠅䚹䛣䜜䛿 Fe2+䝡䝸䝧䝹䝆䞁㘒య䛾ᵓ㐀䛸⪃䛘䜙䜜䚸䝡䝸䝧䝹䝆䞁䛛䜙䛾㕲⬺㞳୰㛫య䛸᝿ᐃ䛥䜜䜛䚹䛴䜎䜚䚸Fe3+䝡䝸䝧䝹䝆䞁䛜㑏ඖ䛥䜜䜛䛸ึ䜑䛻 His 䛜⬺㞳䛧䚸Fe2+䜲䜸䞁䛜ᨺ ฟ䛥䜜䛯ᚋ䛻䝡䝸䝧䝹䝆䞁䛜㐟㞳䛩䜛䛸⪃䛘䜙䜜䜛䚹 ௨ୖ䛾⤖ᯝ䛻䜘䜚䚸HO 䛻䛚䛡䜛ᇶ㉁⤖ྜ䜔⏕ᡂ≀⬺㞳. ᅗ 㻟㻌㻴㼙㼡㻻䛻䛚䛡䜛㕲㐟㞳ᶵᵓ㻌. 䛾ྛᶵᵓ䛻䛴䛔䛶䚸ヲ⣽䛺ᵓ㐀Ꮫⓗ▱ぢ䛜ᚓ䜙䜜䛯䚹. 㻞㻚㻌 ᪂つ MhuD ᆺ䝦䝮ศゎ㓝⣲䛻㛵䛩䜛◊✲㻌 HO 䛿䜋ங㢮䚸᳜≀䚸⣽⳦䛺䛹ከ䛟䛾⏕≀䛻 ᖜᗈ䛟ศᕸ䛧䛶䛚䜚䚸䛭䛾䜋䛸䜣䛹䛿㢮ఝ䛾䜰䝭䝜㓟㓄ิ䞉❧యᵓ㐀䜢᭷䛧䛶䛔䜛䚹䛧䛛䛧㏆ᖺ䚸. ᅗ 㻠㻌㻹㼔㼡㻰䛻䜘䜛≉Ṧ䛺䝦䝮௦ㅰ཯ᛂ. ୍㒊䛾⣽⳦䛛䜙᪂䛯䛺䝦䝮ศゎ㓝⣲䠄MhuD䠅䛜Ⓨぢ䛥䜜䚸䛭䛾≉␗䛺ᵓ㐀䠄䝦䝮䛜␗ᖖ䛻ṍ䜣䛷⤖ྜ䠅䛻ὀ ┠䛥䜜䛶䛔䜛䚹ᙜ◊✲ᐊ䛷䛿๓ᖺ䜎䛷䛻䚸䝦䝮䛾ṍ䜏䛜 MhuD ཯ᛂ䜢๻ⓗ䛻ኚ໬䛥䛫䚸᪂つ䝦䝮௦ㅰ⏘≀ 䠄䝬䜲䝁䝡䝸䞁䚸ᅗ 4䠅䜢୚䛘䜛䛣䛸䜢♧䛧䛯䚹ᮏᖺ䛿 MhuD ཯ᛂ䛾䛥䜙䛺䜛ゎᯒ䛸䚸཯ᛂ୰㛫య䛾ྠᐃ䜢㐍䜑䛯䚹 MhuD ཯ᛂ䜢ヲ⣽䛻ゎᯒ䛩䜛䛸䚸O2 ⤖ྜᆺ䝦䝮䠄ᅗ 4, oxyheme䠅䛸ᛮ䜟䜜䜛୰㛫య䛜᳨ฟ䛥䜜䛯䚹䛭䛣䛷㓟 ໬ᆺ䝦䝮–MhuD 」ྜయ䜢䜾䝻䞊䝤䝪䝑䜽䝇୰䛷㑏ඖ䛧䚸㑏ඖ๣䜢㝖ཤ䛧䛯ᚋ䛻㓟⣲䛸཯ᛂ䛥䛫䛯䛸䛣䜝䚸ྠ➼ 䛾྾཰䝇䝨䜽䝖䝹䜢♧䛩୰㛫య䜢༢㞳䛩䜛䛣䛸䛻ᡂຌ䛧䛯䚹䛣䛾୰㛫య䛾ඹ㬆䝷䝬䞁䝇䝨䜽䝖䝹䜢 ᐃ䛧䛯䛸䛣䜝䚸ྠ఩య䝅䝣䝖䜢♧䛩୕✀䛾䝅䜾䝘䝹䛜ᚓ䜙䜜䛯䠄ᅗ 5䠅䚹䛣䜜䜙䛿 Fe–O, O–O ఙ⦰᣺ື䛚䜘䜃 Fe–O–O ᒅ᭤ ᣺ື䛸⪃䛘䜙䜜䚸ඛ䛾ྠᐃ䛿☜ᐇ䛺䜒䛾䛸䛺䛳䛯䚹䜎䛯 ពእ䛺䛣䛸䛻䚸MhuD 䛸 HO 䛾ྛ᣺ື䝰䞊䝗䛿䜋䜌ྠ䛨 ᣺ືᩘ䛻ほ䛥䜜䚸䝦䝮䛾ṍ䜏䛜㓟⣲䛻୚䛘䜛ᙳ㡪䛿 ᑠ䛥䛔䛣䛸䛿᫂䜙䛛䛷䛒䜛䚹䜘䛳䛶䚸㕲ୖ䛻⏕ᡂ䛩䜛䛷䛒䜝䛖άᛶ㓟⣲✀䛻䜒኱䛝䛺ᕪ䛿⪃䛘䛻䛟䛟䚸MhuD 䛾≉ ␗䛺཯ᛂ䛿ṍ䜏䛻䜘䜛䝫䝹䝣䜱䝸䞁⎔䛾㟁Ꮚ≧ែ䛾ኚ ໬䚸䜎䛯䛿❧యⓗ䛺㏆᥋ຠᯝ䛻౫䜛䛸䛣䜝䛜኱䛝䛔䛸ண ᝿䛥䜜䜛䚹. ᅗ 㻡㻌 oxyMhuD 䛾ඹ㬆䝷䝬䞁䝇䝨䜽䝖䝹. ୍᪉䚸MhuD ཯ᛂゎᯒ䛾኱䛝䛺㞀ᐖ䛸䛺䛳䛶䛔䛯䛾䛿䚸⏕ᡂ≀䛾཰⋡䛾ప䛥䛷䛒䜛䚹ᚑ᮶䛾཯ᛂ᮲௳䛷䛿䚸䝬䜲䝁䝡䝸䞁䛜䛥䜙䛺䜛௦ㅰ཯ᛂ䛺䛹䛾䛯䜑䛻୙Ᏻᐃ䛷䛒䛳䛯䛜䚸཯ᛂ᮲௳䛾᭱㐺໬䛻䜘䜚䚸䝬䜲䝁䝡䝸䞁䛸ఝ䛯 ྾཰≉ᛶ䜢♧䛩᪂つ⏕ᡂ≀䜢䚸Ᏻᐃ䛛䛴㧗཰⋡䛷ᚓ䜛䛣䛸䛻ᡂຌ䛧䛯䚹䛣䛾᪂つ⏕ᡂ≀䛾ᵓ㐀䜢㉁㔞ศᯒ䛺䛹䛷᳨ウ䛧䛯䛸䛣䜝䚸䝬䜲䝁䝡䝸䞁䛛䜙䝢䝻䞊䝹⎔䛾䠍䛴䛜⬺㞳䛧䛯 biotripyrrin䠄ᅗ 4䠅䛸ண᝿䛥䜜䚸ᮏ᮶䛾᭱⤊⏕ ᡂ≀䛸♧၀䛥䜜䛯䚹௒ᚋ䚸NMR 䛺䛹䛾 ᐃ䛷᭱⤊⏕ᡂ≀䛾ᵓ㐀䜢Ỵᐃ䛧䚸ᐃ㔞ⓗ䛺䝯䜹䝙䝈䝮ゎ᫂䛻ྲྀ䜚⤌ 䜐ணᐃ䛷䛒䜛䚹䛺䛚䚸biotripyrrin 䛿䝠䝖䛾㓟໬䝇䝖䝺䝇䝬䞊䜹䞊䛾䠍䛴䛸䛧䛶▱䜙䜜䛶䛔䜛䚹ᚑ᮶䛿䝡䝸䝧䝹䝆䞁䛾㓟໬⏕ᡂ≀䛸⪃䛘䜙䜜䛶䛝䛯䛜䚸௒ᅇ䛾⤖ᯝ䛿ᚤ⏕≀䛻䜘䜛⏘⏕䛾ྍ⬟ᛶ䜒♧၀䛧䛶䛚䜚䚸⏕≀Ꮫⓗ䛻䜒 ⯆࿡῝䛔䚹.

(20) ◊㻌 ✲㻌 ά㻌 ື㻌 ሗ㻌 ࿌ 研究活動報告. ࠙◊✲άືሗ࿌ࠚ ᩍ ຓ ◊ ✲ ኱Ꮫ㝔㻌. ⏕≀ศᏊᶵ⬟ィ ◊✲ศ㔝㸦2013㸬1㹼2013㸬12㸧㻌 ᤵ㸸▼ᓥ⛅ᙪ ᩍ㸸஭ୖ⿱୍⚟ᒸ๰ ⏕㸸బ⸨ᨻ⛅ ⏕㸸బᕝ㈗ᚿศ㒊ᐶ㐨㻌. ᮏ◊✲ศ㔝䛷䛿䠈⏕యศᏊ䝰䞊䝍䞊䛾ືస䝯䜹䝙䝈䝮䛻䛴䛔䛶◊✲άື䜢⾜䛳䛶䛔䜛䠊2013 ᖺ䛾◊✲άື 䛸䛧䛶䛿䠈௨ୗ䛾䜘䛖䛻ᴫᣓ䛥䜜䜛䠊㻌㻝㻚㻌 ኱⭠⳦ෆ」ᩘ䜉䜣ẟ䝰䞊䝍䞊䛾ᅇ㌿䛾ྠ᫬ィ䛻䜘䜛⣽⬊ෆ᝟ሗఏ㐩䛾ᵝᏊ䛾ィ㻌䝞䜽䝔䝸䜰䛿እ⏺䛾⎔ቃ䜢ᴟ䛻Ꮡᅾ䛩䜛ཷᐜయ䛷ཷ䛡ྲྀ䜚䠈䛭䛾᝟ሗ䜢⣽⬊ෆ䛷ఏ㐩䛧䠈䝰䞊䝍䞊䛿᫬ィ ᅇ䜚䠄CW䠅䠈཯᫬ィᅇ䜚䠄CCW䠅䛸ᅇ㌿᪉ྥ䜢ษ䜚᭰䛘䛶䠈ዲ䜎䛧䛔⎔ቃ䛻ྥ䛛䛳䛶䛔䛟䠊኱⭠⳦䛿ᚷ㑊≀㉁䛜 ཷᐜయ䛻⤖ྜ䛩䜛䛸䠈⣽⬊ෆ䛾 CheY 䝍䞁䝟䜽㉁䛜䝸䞁㓟໬䛧䠄CheY-P䠅䠈䛣䛾䝸䞁㓟໬ CheY 䛜䝰䞊䝍䞊ᇶ㒊 య䛻⤖ྜ䛩䜛䛣䛸䛻䜘䜚䝰䞊䝍䞊䛾ᅇ㌿䛜 CW ᅇ㌿䜈䛸㌿᥮䛩䜛䠊⌧ᅾ䜎䛷䛻䠈䛣䛾᝟ሗఏ㐩䛻㛵୚䛩䜛䝍䞁䝟䜽㉁䛾ྠᐃ䠈䛭䛾⣽⬊ෆ䛻䛚䛡䜛ᒁᅾ䛺䛹䛜◊✲䛥䜜䛶䛝䛯䛜䠈䛹䛾䜘䛖䛺⤒㊰䞉ᡭẁ䛷᝟ሗఏ㐩≀㉁䛜⣽ ⬊ෆ䜢ఏ䜟䛳䛶䛔䛟䛾䛛䠈䝰䞊䝍䞊䛿䛹䛾䜘䛖䛻䛧䛶ᅇ㌿᪉ྥ䜢ኚ᥮䛩䜛䛾䛛䠈䛺䛹䛾␲ၥ䛿ᮍ䛰ゎỴ䛧䛶䛔䛺䛔䠊ᡃ䚻䛿 1 ⣽⬊ෆ䛻䛚䛡䜛」ᩘ䛾䝰䞊䝍䞊䛾ᅇ㌿䜢ྠ᫬䛻ィ䛩䜛䛣䛸䛻䜘䜚䠈䛣䛾ㅦ䜢᫂䜙䛛䛻䛧䜘䛖䛸䛧䛶䛔䜛䠊䝰䞊䝍䞊ᇶ㒊య䜈䛾 CheY-P 䛾⤖ྜ䛾ᵝᏊ䜢☜䛛䜑䜛䛯䜑䛻䠈GFP 䜢⼥ྜ䛥䛫䛯 CheY-P 䜢Ⓨ⌧䛥䛫䠈䝔䝄䞊䝗䝉䝹䛻䛚䛔䛶ᅇ㌿୰ᚰ䛾⺯ගᙉᗘ䛾ኚ໬䛸ᅇ㌿䛾ᵝᏊ䛾ྠ᫬ィ䜢⾜䛳䛯䠊䛭䛾⤖ᯝ䠈CW ᅇ ㌿୰䛻⣽⬊ᅇ㌿୰ᚰ䛻ᙉ䛔⺯ග䝇䝫䝑䝖䛜⌧䜜䠈୍᪉䛷 CCW ୰䛻䛿⺯ග䝇䝫䝑䝖䛿ぢ䜙䜜䛺䛛䛳䛯䠊䛣䛾⤖ ᯝ䛿䠈」ᩘ䛾 CheY-P 䛜䝰䞊䝍䞊ᇶ㒊య䛻ᙉ䛔༠ྠᛶ䜢ᣢ䛳䛶⤖ྜ䞉ゎ㞳䜢⾜䛳䛶䛔䜛䛣䛸䜢♧䛩䠊䜎䛯䠈䛭䛾㝿䛾 CheY-P 䛾ᩘ䛿⣙ 13 䛸ぢ✚䜒䜙䜜䛯䠊䛣䛾⤖ᯝ䛿䠈䝰䞊䝍䞊ෆ䛾 CheY-P ⤖ྜ㒊఩䛷䛒䜛䠈FliM 䛾ᩘ䠈34 䛻ẚ䜉䛶ᑡ䛺䛔್䛷䛒䜛䠊䜎䛯䠈⺯ගᙉᗘ䛾ୖ᪼䠈⌧㇟䛻ᚲせ䛺᫬㛫䜢ぢ✚䜒䛳䛯䛸䛣䜝䠈ῶᑡ䛻䛚䛔䛶䛿ᣑ ᩓ䛻క䛖 CheY-P ⃰ᗘኚ໬䜘䜚䜒▷䛔᫬㛫䛷䛾ኚ໬䛷䛒䛳䛯䠊䛣䛾䛣䛸䛿ᑡ䛺䛟䛸䜒 CheY-P 䛾䝰䞊䝍䞊䛛䜙䛾ゎ㞳䛻㛵䛧䛶䛿䠈㈇䛾༠ྠᛶ䛜ാ䛔䛶䛔䜛䛣䛸䜢♧၀䛩䜛䠊CheY-P 䛾⤖ྜ䛻㛵䛧䛶䛿䠈ᣑᩓ䛻䜘䜛⃰ᗘୖ᪼䛜 ᪩䛔䛾䛷☜ㄆ䛷䛝䛺䛛䛳䛯䠊⌧≧䛷䛿䠈⺯ග⏬ീ䛾ྲྀᚓ䛾䛯䜑䠈1 䝣䝺䞊䝮䛒䛯䜚䛾᫬㛫䛜ẚ㍑ⓗ㛗䛔䠄20 frames/s䠅䛷䛒䜛䛯䜑䠈䜘䜚ヲ⣽䜢᳨ウ䛩䜛䛯䜑䠈㧗䛔᫬㛫ศゎ⬟䜢᭷䛩䜛ィ ⣔䛾㛤Ⓨ䛻ྲྀ䜚⤌䜣䛷䛔䜛䠊㻌㻞㻚㻌䜿䞊䝆䝗໬ྜ≀䜢⏝䛔䛯኱⭠⳦⣽⬊ఏ㐩䛾ᵝᏊ䛾ィ㻌 ኱⭠⳦䛜䛔䛛䛻䛧䛶እ⏺䛾่⃭䛻཯ᛂ䛩䜛䛛䜢ㄪ䜉䜛䛯䜑䛻䠈ᒁᡤⓗ่⃭䛻䜘䜛⣽⬊ᛂ⟅䛾ィ䜢⾜䛺䛳䛯䠊䛭䛾䛯䜑䛻䜿䞊䝆䝗໬ྜ≀䛾䠍䛴䛷䛒䜛䜿䞊䝆䝗䝉䝸䞁䜢⏝䛔䛯◊✲䛻ᑟධ䛧䛯䠊䜿䞊䝆䝗䝉䝸䞁䛿⣸Ⰽ䛾 ග䜢↷ᑕ䛩䜛䛣䛸䛷䠈䝉䝸䞁䛸ಖㆤᇶ䛻ගゎ⿣䛩䜛䠊䜿䞊䝆䝗䝉䝸䞁䛛䜙኱⭠⳦䛾ㄏᘬ≀㉁䠄䝉䝸䞁䠅䜢䠈ᶆⓗ䛸䛩䜛⣽⬊䛾࿘ᅖ䛻䛾䜏䛻Ⓨ⏕䛥䛫䠈䝉䝸䞁⃰ᗘ䛾ୖ᪼䛚䜘䜃䝉䝸䞁⃰ᗘ䛾ῶᑡ䛻ᑐ䛩䜛኱⭠⳦䛾ᛂ⟅䜢ィ䛧䛯䠊ㄏᘬ≀㉁䛷䛒䜛䝉䝸䞁௨እ䛻䠈ᚷ㑊≀㉁䛷䛒䜛䝞䝸䞁䠈䝻䜲䝅䞁䠈䝙䝑䜿䝹䛺䛹䛾⣽⬊ᛂ⟅䛾ィ䜢⾜䛳䛶䛔䜛䠊䛥䜙䛻䠈኱⭠⳦䛾䜉䜣ẟ䝰䞊䝍䞊䛾ᅇ㌿᪉ྥ䜢䝸䜰䝹䝍䜲䝮䛻䝰䝙䝍䞊䛧䠈䜿䞊䝆䝗䝉䝸䞁䜢ගゎ⿣䛥䛫䜛䛯䜑䛾䝺䞊䝄䞊↷ᑕ䛾䝍䜲䝭䞁䜾䜢⮬ື໬䛩䜛䝥䝻䜾䝷䝮䜢㛤Ⓨ䛧䛯䠊ᮏィ䝅䝇䝔䝮䛷䛿䠈CCW 䛛䜙 CW ᪉ྥ䜈䛾ᅇ㌿ኚ᥮䛻ᛂ䛨䛶 60 ms 㛫䝺䞊䝄䞊䜢↷ᑕ䛩䜛䜘䛖䛻タᐃ䛧䛯䠊䛭䛾⤖ᯝ䠈䝉䝸䞁ᢞ୚䛻䜘䜛 CW ᅇ ㌿ᣢ⥆᫬㛫䛾᫂☜䛺▷⦰䜢ぢ䜛䛣䛸䛜䛷䛝䛯䠊䛣䛾䛣䛸䛿䠈䜿䞊䝆䝗䝉䝸䞁䛛䜙䛾䝉䝸䞁ᨺฟ䛻䜘䜚⣽⬊䛜ㄏᅉᛂ ⟅䛧䛯䛣䛸䜢♧䛧䛶䛔䜛䠊䛥䜙䛻䠈䛭䛾ᣢ⥆᫬㛫䛾ᴟ䠉䝰䞊䝍䞊㛫䛾㊥㞳౫Ꮡᛶ䜢ㄪ䜉䜛䛸䠈㊥㞳౫Ꮡᛶ䛜ぢ. 12.