D3 slides (Satoshi Kume)

リポカリン型プロスタグランジンD合成酵素の脂溶性低分子に対する系統的相互作用解析
Systematic Interaction Analysis of Human Lipocalin-type Prostaglandin D Synthase with Small Lipophilic Ligands

Transcript

1. ϦϙΧϦϯܕϓϩελάϥϯδϯD߹੒߬ૉͷ ࢷ༹ੑ௿෼ࢠʹର͢Δܥ౷త૬ޓ࡞༻ղੳ ੜ໋؀ڥՊֶݚڀՊɹԠ༻ੜ໋Պֶઐ߈ ੜମߴ෼ࢠػೳֶݚڀࣨ ٱถܛ࢚ Systematic Interaction Analysis of Human Lipocalin-type Prostaglandin D Synthase with Small Lipophilic Ligands 2013.02.06
2. ϦϙΧϦϯܕϓϩελάϥϯδϯD߹੒߬ૉ (L-PGDS) ҟੑԽ߬ૉ PGD2 PGH2 L-PGDS O COOH OH OH O O OH COOH ਭ຾༠ൃ࡞༻ ೴੸਷ӷதʹ͓͍ͯΞϧϒϛϯʹ͍࣍Ͱ̎൪໨ʹଟ͘ଘ ࡏ͢ΔλϯύΫ࣭Ͱ͋Δ(໿12 µg/ml)ɻ ਖ਼ৗѹਫ಄঱ɺ੸ப؅ڱࡥ঱ɺΫϞບԼग़݂ͷΑ͏ͳ ਆܦ࣬ױͷ਍அϚʔΧʔͱͯ͠༗༻Ͱ͋Δɻ
3. ੜମ಺༌ૹλϯύΫ࣭ ʢϦϙΧϦϯϑΝϛϦʔʣ Ξϛϊࢎ࢒ج਺140ʙ200ఔ౓ͷ෼ൻܕλϯύΫ࣭Ͱ ͋ΓɺϏλϛϯA༠ಋମ΍ࢷ๱ࢎͷΑ͏ͳࢷ༹ੑ௿ ෼ࢠΛ݁߹ɾ༌ૹ͢Δɻ ϔϜ୅ँ࢈෺ɺ ϏλϛϯA༠ಋମɺߕঢ়થϗϧϞϯ ͳͲͷࢷ༹ੑ௿෼ࢠͱ݁߹͢Δɻ Ϛ΢ε༝དྷL-PGDS ϦϙΧϦϯܕϓϩελάϥϯδϯD߹੒߬ૉ (L-PGDS)
4. ݚڀͷ໨త ࢷ༹ੑ௿෼ࢠʹର͢Δώτ༝དྷL-PGDSͷ݁߹ಛੑ ಛʹ݁߹ൺɺ਌࿨ੑɺ೤ྗֶతۦಈྗΛ໌Β͔ʹ͢Δ ܬޫফޫޮՌΛ༻͍ͨL-PGDSͷ༷ʑͳࢷ༹ੑ௿෼ࢠ ʹର͢Δ૬ޓ࡞༻ղੳ 1. ༠ىԁภޫೋ৭ੑ෼ޫ๏ʢICDʣɺ͓Αͼ౳Թణఆ ܕ೤ଌఆ๏ʢITCʣΛ༻͍ͨL-PGDSͱϏϦϕϧδϯ ͱͷ૬ޓ࡞༻ղੳ 2. ITCΛ༻͍ͨL-PGDSͱ༷ʑͳࢷ༹ੑ௿෼ࢠͱͷ ೤ྗֶత૬ޓ࡞༻ղੳ 3.
5. ݚڀͷ໨త ࢷ༹ੑ௿෼ࢠʹର͢Δώτ༝དྷL-PGDSͷ݁߹ಛੑ ಛʹ݁߹ൺɺ਌࿨ੑɺ೤ྗֶతۦಈྗΛ໌Β͔ʹ͢Δ ܬޫফޫޮՌΛ༻͍ͨL-PGDSͷ༷ʑͳࢷ༹ੑ௿෼ࢠ ʹର͢Δ૬ޓ࡞༻ղੳ 1. ༠ىԁภޫೋ৭ੑ෼ޫ๏ʢICDʣɺ͓Αͼ౳Թణఆ ܕ೤ଌఆ๏ʢITCʣΛ༻͍ͨL-PGDSͱϏϦϕϧδϯ ͱͷ૬ޓ࡞༻ղੳ 2. ITCΛ༻͍ͨL-PGDSͱ༷ʑͳࢷ༹ੑ௿෼ࢠͱͷ ೤ྗֶత૬ޓ࡞༻ղੳ 3.
6. ಺ҼੑTrp࢒جͷܬޫফޫޮՌʹΑΔ ݁߹਌࿨ੑଌఆ Trp43 Trp54 Trp112 ώτ༝དྷL-PGDSͷཱମߏ଄ (PDB ID: 3O2Y) 290 nmͷྭىޫʹର͢Δ 334 nmͷTrpܬޫΛଌఆ ̍ݸͷ݁߹෦ҐΛ࣋ͭϞσϧ ղ཭ఆ਺(Kd)ΛٻΊͨ L-PGDS + Ligand L-PGDS/Ligand Kd ⌦
7. ૬ରܬޫڧ౓ (%) L-PGDSͱϔϜ୅ँ࢈෺ͱͷ૬ޓ࡞༻ղੳ ϏϦϕϧδϯ (෼ࢠྔ: 583) L-PGDSೱ౓: 1.5 µM, ؇িӷ: 5 mM Tris-HCl (pH 8.0) / 5% DMSO ྭى೾௕: 290 nm, ܬޫ೾௕: 334 nm, Թ౓: 25 °C, ଌఆػث: F-7000ܬޫ෼ޫܭ (೔ཱ) [ϏϦϕϧδϯ]/[L-PGDS]
8. Kd = 20 nM ૬ରܬޫڧ౓ (%) L-PGDSͱϔϜ୅ँ࢈෺ͱͷ૬ޓ࡞༻ղੳ ϏϦϕϧδϯ (෼ࢠྔ: 583) [ϏϦϕϧδϯ]/[L-PGDS] L-PGDSೱ౓: 1.5 µM, ؇িӷ: 5 mM Tris-HCl (pH 8.0) / 5% DMSO ྭى೾௕: 290 nm, ܬޫ೾௕: 334 nm, Թ౓: 25 °C, ଌఆػث: F-7000ܬޫ෼ޫܭ (೔ཱ)
9. ࢷ༹ੑ௿෼ࢠʹର͢ΔL-PGDSͷ݁߹਌࿨ੑ ϦΨϯυ (෼ࢠྔ) Խֶߏ଄ࣜ Kd ϦΨϯυ (෼ࢠྔ) Խֶߏ଄ࣜ Kd ϔϜ୅ँ࢈෺ ϏλϛϯA༠ಋମ ߕঢ়થϗϧϞϯ L-αΠϩΩγϯ (777) 335 nM ϏϦϕϧδϯ (583) 20 nM ԫମϗϧϞϯ ϓϩήεςϩϯ (314) H H H O H O 2.3 µM ϨνϊΠϯࢎ (300) OH O 308 nM ২෺ϗϧϞϯ 11.3 µM ήχεςΠϯ (270)
10. ࢷ༹ੑ௿෼ࢠʹର͢ΔL-PGDSͷ݁߹਌࿨ੑ ϦΨϯυ (෼ࢠྔ) Խֶߏ଄ࣜ Kd ϦΨϯυ (෼ࢠྔ) Խֶߏ଄ࣜ Kd ϔϜ୅ँ࢈෺ ϏλϛϯA༠ಋମ ߕঢ়થϗϧϞϯ L-αΠϩΩγϯ (777) 335 nM ϏϦϕϧδϯ (583) 20 nM ԫମϗϧϞϯ ϓϩήεςϩϯ (314) H H H O H O 2.3 µM ϨνϊΠϯࢎ (300) OH O 308 nM ২෺ϗϧϞϯ 11.3 µM ήχεςΠϯ (270)
11. ΫϞບԼग़݂ ೴੸਷ӷ ϏϦϕϧδϯͱ݁߹ L-PGDSೱ౓্ঢ (໿12 → 26 µg/ml) ϏϦϕϧδϯʹର͢ΔL-PGDSͷߴ͍݁߹਌࿨ੑ͸ɺ ੜཧతͳػೳͱؔ܎͕͋Δɻ ΫϞບԼग़݂࣌ʹ͓͚ΔL-PGDSʹΑΔ ϏϦϕϧδϯ݁߹ͷੜཧతҙٛ
12. ݚڀͷ໨త ࢷ༹ੑ௿෼ࢠʹର͢Δώτ༝དྷL-PGDSͷ݁߹ಛੑ ಛʹ݁߹ൺɺ਌࿨ੑɺ೤ྗֶతۦಈྗΛ໌Β͔ʹ͢Δ ܬޫফޫޮՌΛ༻͍ͨL-PGDSͷ༷ʑͳࢷ༹ੑ௿෼ࢠ ʹର͢Δ૬ޓ࡞༻ղੳ 1. ༠ىԁภޫೋ৭ੑ෼ޫ๏ʢICDʣɺ͓Αͼ౳Թణఆ ܕ೤ଌఆ๏ʢITCʣΛ༻͍ͨL-PGDSͱϏϦϕϧδϯ ͱͷ૬ޓ࡞༻ղੳ 2. ITCΛ༻͍ͨL-PGDSͱ༷ʑͳࢷ༹ੑ௿෼ࢠͱͷ ೤ྗֶత૬ޓ࡞༻ղੳ 3.
13. ٵޫ౓ ೾௕ (nm) ϏϦϕϧδϯͷٵऩεϖΫτϧ ϏϦϕϧδϯೱ౓: 20 µM, ؇িӷ: 20 mM Tris-HCl (pH 8.0) / 5% DMSO, ηϧ௕: 10 mm ଌఆػث: DU® 800 spectrophotometer (Beckman coulter) ༡཭ܕϏϦϕϧδϯ L-PGDS/ϏϦϕϧδϯෳ߹ମ ICD
14. L-PGDS/ϏϦϕϧδϯෳ߹ମͷICDεϖΫτϧ L-PGDSೱ౓: 5 µM, ؇িӷ: 20 mM Tris-HCl (pH 8.0) / 5% DMSO, Թ౓: 25 °C, ଌఆػث: ԁೋ৭ੑ෼ࢄܭJ-820 (JASCO) ਺ࣈ: Ϟϧೱ౓ൺ ([ϏϦϕϧδϯ]/[L-PGDS]) 0.2 0.4 0.8 1.0 ೾௕ (nm) IICD (mdeg)
15. L-PGDSೱ౓: 5 µM, ؇িӷ: 20 mM Tris-HCl (pH 8.0) / 5% DMSO, Թ౓: 25 °C, ଌఆػث: ԁೋ৭ੑ෼ࢄܭJ-820 (JASCO) 0.2 0.4 0.8 1.0 1.2 1.4 1.8 2.1 2.7 5.2 ೾௕ (nm) IICD (mdeg) ਺ࣈ: Ϟϧೱ౓ൺ ([ϏϦϕϧδϯ]/[L-PGDS]) L-PGDS/ϏϦϕϧδϯෳ߹ମͷICDεϖΫτϧ
16. 0.2 0.4 0.8 1.0 1.2 1.4 1.8 2.1 2.7 5.2 L-PGDSೱ౓: 5 µM, ؇িӷ: 20 mM Tris-HCl (pH 8.0) / 5% DMSO, Թ౓: 25 °C, ଌఆػث: ԁೋ৭ੑ෼ࢄܭJ-820 (JASCO) ೾௕ (nm) IICD (mdeg) ਺ࣈ: Ϟϧೱ౓ൺ ([ϏϦϕϧδϯ]/[L-PGDS]) L-PGDS/ϏϦϕϧδϯෳ߹ମͷICDεϖΫτϧ
17. [ϏϦϕϧδϯ]/[L-PGDS] IICD (mdeg) L-PGDSೱ౓: 5 µM, ؇িӷ: 20 mM Tris-HCl (pH 8.0) / 5% DMSO, Թ౓: 25 °C, ଌఆػث: ԁೋ৭ੑ෼ࢄܭJ-820 (JASCO) L-PGDS/ϏϦϕϧδϯෳ߹ମͷICDεϖΫτϧ
18. 2ݸͷಠཱͳ݁߹෦ҐΛ࣋ͭϞσϧ L-PGDS + Ligand L-PGDS/Ligand L-PGDS + Ligand L-PGDS/Ligand Kd2 Kd1 € I ICD = n 1 [L f ] [L f ]+ K d1 ICD 1 + n 2 [L f ] [L f ]+ K d2 ICD 2 [L f ] = 2 3 a2 − 3b cos 1 3 arccos −2a3 + 9ab − 27c 2 a2 − 3b ( )3 # $ % % % & ' ( ( ( − a 3 a = K d1 + K d2 +[P t ] n 1 + n 2 ( )−[L t ] b =[P t ] n 1 K d1 + n 2 K d2 ( )− K d1 + K d2 ( )[L t ]+ K d1 K d2 c = −K d1 K d2 [L t ] # $ % & % IICD: ؍࡯͞ΕͨICDڧ౓ ICD1 & ICD2: ֤݁߹ʹΑΔICDڧ౓ n1 & n2: ݁߹ൺ Kd1 & Kd2: ղ཭ఆ਺ [Pt]: શλϯύΫ࣭ೱ౓ [Lt]: શϦΨϯυೱ౓ [Lf]: ༡཭ܕϦΨϯυೱ౓ ղੳࣜ ̎ݸͷಠཱͳ݁߹෦ҐϞσϧ (֤෦Ґʹ͓͍ͯ1:n݁߹ΛԾఆ) ⌦ ⌦
19. 2ݸͷಠཱͳ݁߹෦ҐΛ࣋ͭϞσϧ L-PGDS + Ligand L-PGDS/Ligand L-PGDS + Ligand L-PGDS/Ligand ⌦ ⌦ Kd2 = 710 nM Kd1 = 2.8 nM [ϏϦϕϧδϯ]/[L-PGDS] IICD (mdeg) L-PGDS/ϏϦϕϧδϯෳ߹ମͷICDεϖΫτϧ
20. ɾGibbs ΤωϧΪʔมԽ (ΔG) ɾΤϯλϧϐʔมԽ (ΔH) ɾΤϯτϩϐʔมԽ (ΔS) ΔG = RT ln Kd ΔG = ΔH – TΔS ೤ྗֶํఔࣜ L-PGDS (500 µM) ϏϦϕϧδϯ (40 µM) ΔT Sample Cell Reference Cell (H2O) ITCΛ༻͍ͨL-PGDSͱϏϦϕϧδϯͱͷ૬ޓ࡞༻ղੳ
21. ITCΛ༻͍ͨL-PGDSͱϏϦϕϧδϯͱͷ૬ޓ࡞༻ղੳ µJ/sec ణఆຖΤϯλϧϐʔ (kJ/mol) ࣌ؒ (෼) [L-PGDS]/[ϏϦϕϧδϯ] L-PGDSೱ౓: 500 µM, ϏϦϕϧδϯೱ౓: 40 µM, ؇িӷ: 50 mM Tris-HCl (pH 8.0) / 5% DMSO,Թ౓: 25 °C, ଌఆػث: MicroCal VP-ITC 2ݸͷಠཱͳ݁߹෦ҐΛ࣋ͭϞσϧ L-PGDS + Ligand L-PGDS/Ligand L-PGDS + Ligand L-PGDS/Ligand Kd2 Kd1 ⌦ ⌦
22. L-PGDS + Ligand L-PGDS/Ligand L-PGDS + Ligand L-PGDS/Ligand ⌦ ⌦ ITCΛ༻͍ͨL-PGDSͱϏϦϕϧδϯͱͷ૬ޓ࡞༻ղੳ Kd2 = 1200 nM Kd1 = 6.9 nM [L-PGDS]/[ϏϦϕϧδϯ] µJ/sec ࣌ؒ (෼) 2ݸͷಠཱͳ݁߹෦ҐΛ࣋ͭϞσϧ L-PGDSೱ౓: 500 µM, ϏϦϕϧδϯೱ౓: 40 µM, ؇িӷ: 50 mM Tris-HCl (pH 8.0) / 5% DMSO,Թ౓: 25 °C, ଌఆػث: MicroCal VP-ITC ణఆຖΤϯλϧϐʔ (kJ/mol)
23. ࣌ؒ (෼) µJ/sec (kJ/mol) ΔH° ΔΔH° ߴ਌࿨݁߹ –12.6 ௿਌࿨݁߹ –23.9 11.3
24. L-PGDS ϦΨϯυ ߴ਌࿨෦Ґ ௿਌࿨෦Ґ ա৒ͳ༡཭ϦΨϯυଘࡏԼ ൃ೤൓Ԡ (kJ/mol) ΔH° ΔΔH° ߴ਌࿨݁߹ ௿਌࿨݁߹ –12.6 –23.9 11.3 ࣌ؒ (෼) µJ/sec
25. ٵ೤൓Ԡ ߴ਌࿨෦Ґ ௿਌࿨෦Ґ (kJ/mol) ΔH° ΔΔH° ߴ਌࿨݁߹ ௿਌࿨݁߹ L-PGDS ༡཭ϦΨϯυඇଘࡏԼ –12.6 –23.9 11.3 ࣌ؒ (෼) µJ/sec ϦΨϯυ
26. ΔG° –TΔS° ΔH° (kJ/mol) ܬޫফޫ ICD ITC (nM) ૬ޓ࡞༻ͷ೤ྗֶύϥϝʔλ ߴ਌࿨݁߹ 19 2.8 6.9 ௿਌࿨݁߹ 710.0 1200.0 ߴ਌࿨݁߹ –46.6 –12.6 ௿਌࿨݁߹ –34.0 –9.9 –23.9 –33.8 ֤ଌఆ๏ʹΑΓಘΒΕͨϏϦϕϧδϯͷKd ஋ͷൺֱ
27. ΔG° –TΔS° ΔH° (kJ/mol) ܬޫফޫ ICD ITC (nM) ߴ਌࿨݁߹ 19 2.8 6.9 ௿਌࿨݁߹ 710.0 1200.0 ߴ਌࿨݁߹ –46.6 –12.6 ௿਌࿨݁߹ –34.0 –9.9 –23.9 –33.8 ֤ଌఆ๏ʹΑΓಘΒΕͨϏϦϕϧδϯͷKd ஋ͷൺֱ ૬ޓ࡞༻ͷ೤ྗֶύϥϝʔλ
28. ΔG° –TΔS° ΔH° (kJ/mol) ܬޫফޫ ICD ITC (nM) ߴ਌࿨݁߹ 19 2.8 6.9 ௿਌࿨݁߹ 710.0 1200.0 ߴ਌࿨݁߹ –46.6 –12.6 ௿਌࿨݁߹ –34.0 –9.9 –23.9 –33.8 ֤ଌఆ๏ʹΑΓಘΒΕͨϏϦϕϧδϯͷKd ஋ͷൺֱ ૬ޓ࡞༻ͷ೤ྗֶύϥϝʔλ
29. ΔG° –TΔS° ΔH° (kJ/mol) ܬޫফޫ ICD ITC (nM) ߴ਌࿨݁߹ 19 2.8 6.9 ௿਌࿨݁߹ 710.0 1200.0 ߴ਌࿨݁߹ –46.6 –12.6 ௿਌࿨݁߹ –34.0 –9.9 –23.9 –33.8 ֤ଌఆ๏ʹΑΓಘΒΕͨϏϦϕϧδϯͷKd ஋ͷൺֱ ૬ޓ࡞༻ͷ೤ྗֶύϥϝʔλ
30. ΔG° –TΔS° ΔH° (kJ/mol) ܬޫফޫ ICD ITC (nM) ߴ਌࿨݁߹ 19 2.8 6.9 ௿਌࿨݁߹ 710.0 1200.0 ߴ਌࿨݁߹ –46.6 –12.6 ௿਌࿨݁߹ –34.0 –9.9 –23.9 –33.8 ֤ଌఆ๏ʹΑΓಘΒΕͨϏϦϕϧδϯͷKd ஋ͷൺֱ ૬ޓ࡞༻ͷ೤ྗֶύϥϝʔλ
31. L-PGDS/ϏϦϕϧδϯෳ߹ମͷϞσϧߏ଄ ιϑτ΢ΣΞ: ౷߹ܭࢉԽֶγεςϜ (MOE), ΞϧΰϦζϜ: ASEDock ߴ਌࿨݁߹ ௿਌࿨݁߹
32. ΫϞບԼग़݂࣌ʹ͓͚ΔL-PGDSʹΑΔ ϔϜ୅ँ࢈෺݁߹ͷੜཧతҙٛ ϔϜͷ୅ँ෼ղܦ࿏ ϏϦϕϧδϯ ϔϜ (ϔϛϯ) ϏϦϧϏϯ ϔϛϯɺ͓ΑͼϏϦϧϏϯ͸ɺਆܦࡉ๔ʹରͯ͠Ξϙτʔγε Λհ͢Δࡉ๔ࢮΛ༠ൃ͢Δɻ
33. ΫϞບԼग़݂࣌ʹ͓͚ΔL-PGDSʹΑΔ ϔϜ୅ँ࢈෺݁߹ͷੜཧతҙٛ ϔϜͷ୅ँ෼ղܦ࿏ ϏϦϕϧδϯ ϔϜ (ϔϛϯ) ϏϦϧϏϯ L-PGDS͸ϔϜ୅ँ࢈෺ͷεΧϕϯδϟʔͰ͋Δ ϔϛϯɺ͓ΑͼϏϦϧϏϯ͸ɺਆܦࡉ๔ʹରͯ͠Ξϙτʔγε Λհ͢Δࡉ๔ࢮΛ༠ൃ͢Δɻ
34. L-PGDSʹΑΔϔϜ୅ँ࢈෺ͷ݁߹Ϟσϧ ϏϦϕϧδϯͷٸܹͳೱ౓্ঢʹ൐͍ɺ̎෼ࢠͷϏϦϕϧδϯ Λัଊ͢Δɻ L-PGDSͷೱ౓্ঢʹ൐͍ɺΤωϧΪʔతʹΑΓ҆ఆͳϏϦϕϧ δϯ̍෼ࢠෳ߹ମͷܗ੒͕ى͜Γɺ෼ղɾഉग़͕ଅਐ͞ΕΔɻ
35. ϏϦϕϧδϯͷٸܹͳೱ౓্ঢʹ൐͍ɺ̎෼ࢠͷϏϦϕϧδϯ Λัଊ͢Δɻ L-PGDSʹΑΔϔϜ୅ँ࢈෺ͷ݁߹Ϟσϧ L-PGDSͷೱ౓্ঢʹ൐͍ɺΤωϧΪʔతʹΑΓ҆ఆͳϏϦϕϧ δϯ̍෼ࢠෳ߹ମͷܗ੒͕ى͜Γɺ෼ղɾഉग़͕ଅਐ͞ΕΔɻ
36. ϏϦϕϧδϯͷٸܹͳೱ౓্ঢʹ൐͍ɺ̎෼ࢠͷϏϦϕϧδϯ Λัଊ͢Δɻ L-PGDSʹΑΔϔϜ୅ँ࢈෺ͷ݁߹Ϟσϧ L-PGDSͷೱ౓্ঢʹ൐͍ɺΤωϧΪʔతʹΑΓ҆ఆͳϏϦϕϧ δϯ̍෼ࢠෳ߹ମͷܗ੒͕ى͜Γɺ෼ղɾഉग़͕ଅਐ͞ΕΔɻ
37. ·ͱΊ ώτ༝དྷL-PGDS͸ɼ̎෼ࢠͷϏϦϕϧδϯΛߴ਌࿨ɺ͓ Αͼ௿਌࿨ੑͰ݁߹͢Δɻ ϏϦϕϧδϯʹର͢ΔL-PGDSͷߴ͍݁߹਌࿨ੑʹ͸ɺ ຊλϯύΫ࣭ͷૄਫੑΩϟϏςΟ͔Βͷਫ෼ࢠͷ୤ਫ࿨ ʹΑΔΔSརಘ͕େ͖͘د༩͢Δɻ L-PGDSͷߴ਌࿨ɺ͓Αͼ௿਌࿨݁߹ʹΑΔϔϜ୅ँ࢈෺ ͷ݁߹ϞσϧΛఏҊͨ͠ɻ Kume, S., et al. Biochem. J. 446, 279-289 (2012)
38. ݚڀͷ໨త ࢷ༹ੑ௿෼ࢠʹର͢Δώτ༝དྷL-PGDSͷ݁߹ಛੑ ಛʹ݁߹ൺɺ਌࿨ੑɺ೤ྗֶతۦಈྗΛ໌Β͔ʹ͢Δ ܬޫফޫޮՌΛ༻͍ͨL-PGDSͷ༷ʑͳࢷ༹ੑ௿෼ࢠ ʹର͢Δ૬ޓ࡞༻ղੳ 1. ༠ىԁภޫೋ৭ੑ෼ޫ๏ʢICDʣɺ͓Αͼ౳Թణఆ ܕ೤ଌఆ๏ʢITCʣΛ༻͍ͨL-PGDSͱϏϦϕϧδϯ ͱͷ૬ޓ࡞༻ղੳ 2. ITCΛ༻͍ͨL-PGDSͱ༷ʑͳࢷ༹ੑ௿෼ࢠͱͷ ೤ྗֶత૬ޓ࡞༻ղੳ 3.
39. ຊݚڀʹ͓͍ͯ࢖༻ͨ͠ࢷ༹ੑϦΨϯυ ϏλϛϯA༠ಋମ ߕঢ়થϗϧϞϯ PGH2 Ξφϩά εςϩΠυϗϧϞϯ ২෺ϗϧϞϯ ܬޫϓϩʔϒ L-αΠϩΩγϯ (T4) (777 Da) ϓϩήεςϩϯ (314 Da) All-rans ϨνϊΠϯࢎ (300 Da) ήχεςΠϯ (270 Da) 9-cis ϨνϊΠϯࢎ (300 Da) L-αΠϩχϯ (T3) (651 Da) U-46619 (350 Da) ςετεςϩϯ (288 Da) φϦϯήχϯ (272 Da) μΠθΠϯ (254 Da) TNS (335 Da) ίϧνίεςϩϯ (346 Da)
40. ITCΛ༻͍ͨL-PGDSͱL-αΠϩΩγϯ(T4) ͱͷ૬ޓ࡞༻ղੳ µJ/sec ణఆຖΤϯλϧϐʔ (kJ/mol) ࣌ؒ (෼) [L-PGDS]/[T4] T4 (777 Da) K d (µM) n
    ΔH°
    ΔG°
    –TΔS°
    (kJ/mol) 1.0
    2.1
    –63.8
    –32.4
    31.4
    L-PGDSೱ౓: 500 µM, T4ೱ౓: 30 µM, ؇িӷ: 50 mM Tris-HCl (pH 8.0) / 5% DMSO,Թ౓: 25 °C, ଌఆػث: MicroCal VP-ITC
41. L-PGDSͱࢷ༹ੑϦΨϯυͷ૬ޓ࡞༻ܥʹ͓͚Δ ΤϯλϧϐʔɾΤϯτϩϐʔิঈ ΔH° (kJ/mol) –TΔS° (kJ/mol) ϔϛϯ (ߴ਌࿨) ϔϛϯ (௿਌࿨) ϏϦϕϧδϯ (௿਌࿨) ϏϦϕϧδϯ (ߴ਌࿨) ϏϦϧϏϯ (௿਌࿨) ϏϦϧϏϯ (ߴ਌࿨) All-trans ϨνϊΠϯࢎ 9-cis ϨνϊΠϯࢎ T4 T3 ϓϩήεςϩϯ ςετεςϩϯ ίϧνίεςϩϯ ήχεςΠϯ φϦϯήχϯ μΠθΠϯ U-46619 TNS
42. λϯύΫ࣭/ϦΨϯυෳ߹ମ༧ଌΞϧΰϦζϜΛ༻͍ͨ ೤ྗֶύϥϝʔλͷ༧ଌ ਫૉ݁߹ʹΑΔد༩ (kJ/mol) ૄਫੑ݁߹਺ ΔH° (kJ/mol) –TΔS° (kJ/mol) ιϑτ΢ΣΞ: AutoDock4ɺΞϧΰϦζϜ: Lamarckian genetic algorithms λϯύΫ࣭: ώτ༝དྷL-PGDS (PDB ID: 3O2Y) TNS ϔϛϯ ϏϦϕϧδϯ ϏϦϧϏϯ All-trans ϨνϊΠϯࢎ 9-cis ϨνϊΠϯࢎ T4 T3 ϓϩήεςϩϯ ςετεςϩϯ ίϧνίεςϩϯ ήχεςΠϯ φϦϯήχϯ μΠθΠϯ U-46619 TNS
43. ·ͱΊ ώτ༝དྷL-PGDS͸ɺϔϜ୅ँ࢈෺΍ߕঢ়થϗϧϞϯͳͲ ͷ༷ʑͳࢷ༹ੑ௿෼ࢠΛ݁߹͢Δɻ L-PGDSͱࢷ༹ੑ௿෼ࢠͱͷ૬ޓ࡞༻͸ɺਫૉ݁߹ͷ࠶ߏ ੒ʹىҼ͢ΔΤϯλϧϐʔɾΤϯτϩϐʔิঈΛࣔ͢ɻ L-PGDSͷ෯޿͍݁߹બ୒ੑ͸ɺΤϯλϧϐʔมԽɺ͓Α ͼΤϯτϩϐʔมԽ྆ํͷόϥϯεʹΑͬͯ೤ྗֶతʹ࠷ దԽ͞ΕΔɻ