ALS Ⓨ⑕ཎᅉࢱࣥࣃࢡ㉁ SOD1 ࡢᵓ㐀ࡢ୙Ᏻᐃ໬࡟

A

ALS Ⓨ⑕ཎᅉࢱࣥࣃࢡ㉁ SOD1 ࡢᵓ㐀ࡢ୙Ᏻᐃ໬࡟

క࠺࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂᶵᵓ࡟㛵ࡍࡿ◊✲

2016 ᖺ 1 ᭶

஭⏣ᫀᏕ

␎ ␎ྕ

ALS㸸amyotrophic lateral sclerosis API㸸Achromobacter lyticus protease I Apo-㸸Cu and Zn metal free form CD㸸circular dichroism

Cy3㸸Cy3 maleimide DTT㸸dithiothreitol

fALS㸸familial form of amyotrophic lateral sclerosis FITC㸸fluorescein 5-isothiocyanate

FM㸸fluorescence microscopy Gdn-HCl㸸guanidine hydrochloride

G93ASOD1㸸SOD1 with mutation of G93A Holo-㸸Cu and Zn metal bound form

MALDI-TOF㸸matrix-assisted laser desorption ionization time-of-flight PK㸸proteinase K

Red-㸸reduced form of intramolecular disulfide bond between Cys57 and Cys146 sALS㸸sporadic form of amyotrophic lateral sclerosis

SOD1㸸Cu, Zn-superoxide dismutase TCEP㸸tris(2-carboxyethyl)phosphine TEM㸸transmission electron microscopy ThioT㸸Thioflavin T

1,5-IAEDANS㸸N-(iodoacetyl)-N’-(5-sulfo-l-naphthyl)-ethylenediamine ICP㸸Inductively Coupled Plasma

2SCAM-SOD1㸸SOD1 derivative where the free thiols of Cys6 and Cys111 are carboxyamidated

2SCAMRed-SOD1㸸SOD1 derivative in which the free thiols of Cys6 and Cys111 have

been carboxyamidated and the intra-disulfide bond of Cys57-Cys146 has been reduced 4SCAM-SOD1㸸SOD1 derivative where all of the thiols in Cys6, Cys57, Cys111 and Cys146 have been carboxyamidated

┠ ┠ḟ

➨1❶ ᗎㄽ………...1

1-1㸬ࡣࡌࡵ࡟………..……….1

1-2㸬ࢱࣥࣃࢡ㉁ࡢᵓ㐀␗ᖖ࡟ࡼࡾ⏕ࡌࡿ࢔࣑ࣟ࢖ࢻ⥺⥔………..……….2

1-3㸬ALSⓎ⑕ࡢཎᅉࢱࣥࣃࢡ㉁ࡢSOD1………...……....4

1-4㸬ᮏㄽᩥࡢ┠ⓗ࠾ࡼࡧᵓᡂ………..……….6

➨2❶ 㔝⏕ᆺSOD1ࢆ⏝࠸ࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ……….………...7

2-1㸬⥴ゝ………..……….7

2-2㸬ᮦᩱ࡜᪉ἲ……….……….8

2-2-1㸬㔝⏕ᆺSOD1ࡢᇵ㣴………...……….8

2-2-2㸬㔝⏕ᆺSOD1ࡢ⢭〇………...……….8

2-2-3㸬ࢱࣥࣃࢡ㉁ࡢᐃ㔞㸦⣸እ྾཰ἲ㸧..………11

2-2-4㸬Cu㸪Znࡢᐃ㔞㸦ㄏᑟ⤖ྜࣉࣛࢬ࣐ศᯒ㸧……….11

2-2-5㸬ྛ✀㔝⏕ᆺSOD1ࡢస〇᪉ἲ………..……11

2-2-6㸬Gdn-HClࢆ⏝࠸ࡓ㔝⏕ᆺSOD1ࡢᵓ㐀Ᏻᐃᛶホ౯………..……..12

2-2-7㸬㔝⏕ᆺSOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ………..……13

2-2-8㸬0.4 M Gdn-HClᏑᅾୗ࡛ࡢ㔝⏕ᆺSOD1࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ………...…..14

2-3㸬⤖ᯝ……….16

2-3-1㸬ྛ✀㔝⏕ᆺSOD1ࡢ஧ḟᵓ㐀࡜ᵓ㐀Ᏻᐃᛶࡢゎᯒ………..16

2-3-2㸬㔝⏕ᆺSOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ………..……...19

2-3-3㸬0.4 M Gdn-HClᏑᅾୗ࡛ࡢ㔝⏕ᆺSOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ…………...…..22

2-4㸬⪃ᐹ……….25

➨

➨3❶ 㔝⏕ᆺSOD1࡟ࡼࡿ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆ⏝࠸ࡓ᰾㒊఩ࡢྠᐃ………..27

3-1㸬⥴ゝ……….………27

3-2㸬ᮦᩱ࡜᪉ἲ……….……29

3-2-1㸬Red-SOD1, 4SCAM-SOD1࡟ࡼࡾᙧᡂࡉࢀࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢ㓝⣲ᾘ໬……29

3-2-2㸬㏫┦HPLC࡟ࡼࡿࢧࣥࣉࣝࡢศྲྀ……….29

3-2-3㸬MALDI-TOF mass spectrometer࡟ࡼࡿศᏊ㔞ࡢỴᐃ……….30

3-2-4㸬ࣉࣟࢸ࢖ࣥࢩ࣮ࢡ࢚ࣥࢧ࣮࡟ࡼࡿศᯒ……….30

3-2-5㸬᰾㒊఩㡿ᇦࡢྠᐃ……….30

3-2-6㸬Red-SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᾘ໬ᚋSeedᐇ㦂………30

3-3㸬⤖ᯝ……….32

3-3-1㸬Red-SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆ⏝࠸ࡓ᰾㒊఩ྠᐃ……….32

3-3-2㸬4SCAM-SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆ⏝࠸ࡓ᰾㒊఩ྠᐃ………..35

3-3-3㸬Red-SOD1࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᾘ໬ᚋࡢSeedຠᯝࡢホ౯………38

3-4㸬⪃ᐹ……….40

➨4❶ ᰾㒊఩ྜᡂ࣌ࣉࢳࢻࢆ⏝࠸ࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ……….…………...43

4-1㸬⥴ゝ……….43

4-2㸬ᮦᩱ࡜᪉ἲ……….44

4-2-1㸬ྜᡂ࣌ࣉࢳࢻࡢỴᐃ࡜⃰ᗘᐃ㔞……….44

4-2-2㸬ྜᡂ࣌ࣉࢳࢻࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ……….46

4-2-3㸬⺯ගヨ⸆࡛ࣛ࣋ࣝࡋࡓྜᡂ࣌ࣉࢳࢻࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ……….46

4-2-3-1㸬ྜᡂ࣌ࣉࢳࢻࡢ༢⊂࡛ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ………..47

4-2-3-2㸬ྜᡂ࣌ࣉࢳࢻࡢ」ᩘ࡛ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ………..47

4-3㸬⤖ᯝ……….48

➨

➨5❶ fALSኚ␗యG93ASOD1ࢆ⏝࠸ࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ………...58

5-1㸬⥴ゝ……….58

5-2㸬ᮦᩱ࡜᪉ἲ……….61

5-2-1㸬G93ASOD1ࡢᇵ㣴………61

5-2-2㸬G93ASOD1ࡢ⢭〇………61

5-2-3㸬ࢱࣥࣃࢡ㉁ࡢᐃ㔞……….62

5-2-4㸬Cu㸪Znࡢᐃ㔞㸦ㄏᑟ⤖ྜࣉࣛࢬ࣐ศᯒ㸧……….………63

5-2-5㸬ྛ✀G93ASOD1ࡢస〇᪉ἲ………...………63

5-2-6㸬Gdn-HClࢆ⏝࠸ࡓG93ASOD1ࡢᵓ㐀Ᏻᐃᛶホ౯………..63

5-2-7㸬2 mM DTTᏑᅾୗ࡟࠾ࡅࡿ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ……….64

5-2-8㸬␗࡞ࡿDTT⃰ᗘᏑᅾୗ࡟࠾ࡅࡿ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ...……….64

5-2-9㸬SOD1ࡢSHᇶᐃ㔞………...………64

5-3㸬⤖ᯝ……….67

5-3-1㸬G93ASOD1ࡢ஧ḟᵓ㐀࡜ᵓ㐀Ᏻᐃᛶࡢゎᯒ………67

5-3-2㸬㔝⏕ᆺSOD1㸪G93ASOD1ࡢ㑏ඖ๣Ꮡᅾୗ࡛ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ…………..69

5-3-3㸬㑏ඖ๣ࡢ⃰ᗘࡀ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࡟୚࠼ࡿᙳ㡪……….71

5-4㸬⪃ᐹ……….73

➨6❶ ⥲ྜ⪃ᐹ………75

6-1㸬SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ࡟࠾ࡅࡿ㔠ᒓ࢖࢜ࣥࡢᙺ๭………..76

6-2㸬SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ࡟࠾ࡅࡿศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢᙺ๭………..78

6-3㸬㑏ඖᆺSOD1ࡢᵓ㐀࡜࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࡜ࡢ㛵ಀ……….79

6-4㸬SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ࡟࠾ࡅࡿ᰾㒊఩ࡢ┦஫స⏝………..80

➨7❶ ⤖ㄒ……….………82

ᘬ⏝ᩥ⊩……….……….83

ㅰ㎡………..……….93

➨

➨ 1 ❶ ᗎㄽ

1-1 㸬ࡣࡌࡵ࡟

ࢱࣥࣃࢡ㉁ࡣ 20✀㢮ࡢ࢔࣑ࣀ㓟ࡀ㙐≧࡟ࡘ࡞ࡀࡗࡓ㧗ศᏊ࡛࠶ࡾ㸪DNA ࡢ㑇ఏ᝟ሗࡣ

㌿෗࡜⩻ヂ࡟ࡼࡾ㸪ࣜ࣎ࢯ࣮࣒ୖ࡛࣏ࣜ࣌ࣉࢳࢻ㙐ࡢ࢔࣑ࣀ㓟㓄ิ࡟ኚ᥮ࡉࢀࡿࠋࢱࣥࣃ

ࢡ㉁ࡣ⏕⌮ⓗ᮲௳ୗ࡟࠾࠸࡚㸪࢔࣑ࣀ㓟㓄ิ࡟ᛂࡌ࡚ᵝࠎ࡞❧యᵓ㐀ࢆ࡜ࡾ㸪ᛶ㉁ࡶࡲࡓ

኱ࡁࡃ␗࡞ࡿࠋ1960ᖺ௦࡟Anfinsenࡽࡣࣜ࣎ࢾࢡࣞ࢔࣮ࢮAࡢᕳࡁᡠࡋᐇ㦂࡟ࡼࡾ㸪᏶

඲࡟❧యᵓ㐀ࢆኻࡗࡓ≧ែ࠿ࡽ⮬Ⓨⓗ࡟ࡲࡁᡠࡿࡇ࡜ࢆ♧ࡋ㸪ࢱࣥࣃࢡ㉁ࡢኳ↛❧యᵓ㐀 ࡣࡑࡢ࢔࣑ࣀ㓟ࡢ୍ḟ㓄ิ࡟ࡼࡾỴᐃ࡙ࡅࡽࢀࡿ࡜࠸࠺Anfinsenࡢࢻࢢ࣐ࢆᥦၐࡋࡓࠋࡋ

࠿ࡋ࡞ࡀࡽ㸪⢭〇ࡋࡓࢱࣥࣃࢡ㉁ࡣin vitro࡟࠾࠸࡚ඖࡢኳ↛≧ែ࡟ࡲࡁᡠࡿࡇ࡜࡞ࡃ㸪จ 㞟ࢆ⏕ࡌࡿࡇ࡜ࡀ࠶ࡿࠋࡑࢀࡣ࡯࡝ࡅࡓ࣏ࣜ࣌ࣉࢳࢻ㙐ࡀ␯Ỉᛶ࢔࣑ࣀ㓟ࡢഃ㙐ࡀ⾲㠃࡟

㟢ฟࡉࢀ㸪஫࠸࡟ศᏊ㛫ࡸศᏊෆ࡛఍ྜࡍࡿࡇ࡜࡛⏕ࡌࡿ࡜ࡉࢀࡿࠋࡇࢀࡽࡢࢱࣥࣃࢡ㉁

ࡢจ㞟ࡣ㸪 ᗘࢆୗࡆࡿ㸪refoldingࢱࣥࣃࢡ㉁ࡢ⃰ᗘࢆపࡃࡍࡿ࡞࡝᮲௳ࢆኚ᭦ࡍࡿࡇ࡜

࡛ᅇ㑊ࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋIn vitro࡛ࢱࣥࣃࢡ㉁ࡢจ㞟ࡀぢࡘ࠿ࡗࡓᙜึࡣ㸪refoldingࡀ

࠺ࡲࡃ⾜࠼࡞࠿ࡗࡓࡓࡵ࡜⪃࠼ࡽࢀ㸪⣽⬊ෆ࡛㉳ࡇࡿ⌧㇟࡜ࡣ␗࡞ࡿࡶࡢࡉࢀࡓࠋࡋ࠿ࡋ㸪

⣽⬊ෆ࡛ࡢfolding୰㛫యࡢจ㞟ࡸ࡯࡜ࢇ࡝ࡍ࡭࡚ࡢ⏕≀ࡢ⇕ࢩࣙࢵࢡࢱࣥࣃࢡ㉁ࡣࢱࣥࣃ

ࢡ㉁ࡢfoldingࢆຓࡅࡿࢩࣕ࣌ࣟࢽࣥࢆᣢࡘࡇ࡜㸪ࡲࡓࢱࣥࣃࢡ㉁ࡢfoldingࡣࢱࣥࣃࢡ㉁

ࡢᶵ⬟ࡸ㓝⣲άᛶ࡞࡝ࢆⓎ᥹ࡍࡿࡓࡵᚲせ࡛࠶ࡿࡇ࡜ࡀศ࠿ࡾ㸪ࢱࣥࣃࢡ㉁ࡢᵓ㐀ࡸจ㞟

࡟ὀ┠ࡉࢀࡿࡼ࠺࡟࡞ࡗࡓ[1, 2]ࠋࢱࣥࣃࢡ㉁ࡣືⓗᖹ⾮࡟࠶ࡾ㸪⣽⬊ෆ࡛ࡣࢫࢺࣞࢫ࡞࡝

ࡢ⎔ቃࡢኚ໬࡟ࡼࡾ㸪unfoldingࡸ୙㐺ษ࡟foldingࡋࡓࢱࣥࣃࢡ㉁ࡀ⏕ࡌࡿࠋᑠ⬊యࡣࡇ ࡢࡼ࠺࡞␗ᖖ࡞ࢱࣥࣃࢡ㉁ࢆㄆ㆑ࡋ࡚㸪ࢱࣥࣃࢡ㉁ࡢရ㉁ࢆ⟶⌮ࡍࡿാࡁࢆࡋ࡚࠸ࡿ[3]ࠋ

␗ᖖࢱࣥࣃࢡ㉁ࡀ⵳✚ࡍࡿࡇ࡜࡛⏕ࡌࡿᑠ⬊యࢫࢺࣞࢫ࡟ࡼࡾ㸪࠺ࡲࡃရ㉁⟶⌮ࡀᶵ⬟ࡋ

࡞ࡃ࡞ࡗࡓሙྜ㸪⣽⬊⮬㌟ࡣ᪂⏕ࢱࣥࣃࢡ㉁ࡢ⩻ヂࡢࢫࢺࢵࣉࡸmisfoldࡋࡓࢱࣥࣃࢡ㉁ࢆ

ศᏊࢩࣕ࣌ࣟࣥ࡟ࡼࡿಟ᚟㸪misfold ࡋࡓࢱࣥࣃࢡ㉁ࢆ㑅ᢥⓗ࡟ศゎࡍࡿ࡜࠸࠺ಟṇᶵ⬟

㸦ᑠ⬊యࢫࢺࣞࢫᛂ⟅㸸UPR㸪unfolded protein response㸧ࢆᣢࡗ࡚࠸ࡿࠋᣢ⥆ⓗ࡞ࢫࢺ

ࣞࢫࡸUPRࡢᶵ⬟୙඲࡞࡝࡟ࡼࡗ࡚ᑠ⬊యෆ࡟୙Ⰻࢱࣥࣃࢡ㉁ࡀ㐣๫࡟⵳✚ࡋ㸪ᑠ⬊యࡢ

ᜏᖖᛶࢆ⥔ᣢ࡛ࡁ࡞࠸ሙྜ࡟ࡣ࢔࣏ࢺ࣮ࢩࢫࡀㄏᑟࡉࢀࡿ[4]ࠋࢱࣥࣃࢡ㉁ࡢᵓ㐀␗ᖖ࡟ࡼ

ࡿ఍ྜయࡣ⏕యෆ࡛ศゎࡉࢀࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࡀ㸪୰࡟ࡣศゎࡉࢀࡿࡇ࡜࡞ࡃ㸪⤌⧊࡟

⵳✚ࡍࡿ࢔࣑ࣟ࢖ࢻ⥺⥔࡜࿧ࡤࢀࡿ୙⁐ᛶ⥺⥔ࢆᙧᡂࡍࡿࡇ࡜ࡶሗ࿌ࡉࢀ࡚࠸ࡿ[2]ࠋࡇࡢ

ࡼ࠺࡟㸪in vitroࡸin vivo࡟㛵ࢃࡽࡎ㸪⏕࿨⌧㇟࡟࡜ࡗ࡚࡜࡚ࡶ኱ษ࡞ᙺ๭ࢆᯝࡓࡋ࡚࠸

ࡿࢱࣥࣃࢡ㉁ࡢfoldingࡸࢱࣥࣃࢡ㉁ࡢᵓ㐀␗ᖖ࡟ᑐࡍࡿ⣽⬊ᛂ⟅ᶵᵓ࡟ࡘ࠸࡚◊✲ࡀ㐍ࡵ

ࡽࢀ࡚࠸ࡿࠋ

11-2 㸬ࢱࣥࣃࢡ㉁ࡢᵓ㐀␗ᖖ࡟ࡼࡾ⏕ࡌࡿ࢔࣑ࣟ࢖ࢻ⥺⥔

㏆ᖺ㸪࢔ࣝࢶࣁ࢖࣐࣮⑓㸪ࣉࣜ࢜ࣥ⑓㸪ࣃ࣮࢟ࣥࢯࣥ⑓࡞࡝ࡢ⚄⤒ኚᛶ⑌ᝈ࡟࠾࠸࡚≉

ᐃࡢཎᅉࢱࣥࣃࢡ㉁ࡀ࢔࣑ࣟ࢖ࢻ⥺⥔࡜࿧ࡤࢀࡿจ㞟యࢆᙧᡂࡋ࡚⤌⧊࡟ỿ╔ࡍࡿࡇ࡜ࡀ ሗ࿌ࡉࢀ࡚࠸ࡿࠋ1854ᖺ࡟Rudolph Virchow࡟ࡼࡾ⤌⧊࠿ࡽྲྀࡾฟࡋࡓจ㞟యࡀࣚ࢘⣲࡛

ࢇ࡫ࢇ཯ᛂࢆ♧ࡋ㸪࢔࣑ࣟ࢖ࢻ࡜⤂௓ࡉࢀࡓࡇ࡜ࢆጞࡵ࡟㸪1959ᖺ࡟ࡣ㟁Ꮚ㢧ᚤ㙾ࡢほᐹ

࡟ࡼࡾ⤌⧊࡟⥺⥔ࡀᏑᅾࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉࢀࡓ[5]ࠋࡇࢀࡲ࡛࡟㸪40✀㢮ࡶࡢࢱࣥࣃࢡ

㉁ࡀ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ[6]ࠋ୍᪉࡛㸪⑓Ẽ࡜㛵ಀࡢ࡞࠸ࢱࣥ

ࣃࢡ㉁ࡸ࣌ࣉࢳࢻ࡛ࡶ in vitro ࡢ≉ᐃ᮲௳ୗ࡛࢔࣑ࣟ࢖ࢻ⥺⥔ࡀᙧᡂࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ

࡚࠸ࡿ[7]ࠋࡑࡢࡓࡵ㸪࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂࡣࢱࣥࣃࢡ㉁ࡢ≉ᛶࡢ୍ࡘ࡛࠶ࡾ㸪ከࡃࡢࢱ

ࣥࣃࢡ㉁ࡣ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿ⬟ຊࢆ᭷ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿ[8]ࠋࡲࡓ㸪࢔࣑ࣟ࢖

ࢻ⥺⥔࡟ࡼࡾ⣽⬊ẘᛶࡸ⅖⑕཯ᛂࡀ⏕ࡌࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ[9, 10]㸪࢔࣑ࣟ࢖ࢻ⥺⥔

ࡢᙧᡂึᮇ࡟ぢࡽࢀࡿྍ⁐ᛶࡢከ㔞య㸦࢜ࣜࢦ࣐࣮㸧࡟ᙉ࠸⣽⬊ẘᛶࢆ♧ࡍሗ࿌ࡶ࠶ࡾ[11, 12]㸪ẘᛶࡢ⋓ᚓࡣ᭱⤊ᙧែࡢ࢔࣑ࣟ࢖ࢻ⥺⥔࡟ࡼࡿࡶࡢ࡞ࡢ࠿㸪ࡑࡢ๓㥑య࡟ࡼࡿࡶࡢ࡞

ࡢ࠿ࡣ㆟ㄽࡉࢀ࡚࠸ࡿࠋ

࢔࣑ࣟ࢖ࢻ⥺⥔ࡣᵓ㐀ࡸᛶ㉁࡟࠸ࡃࡘ࠿ࡢඹ㏻Ⅼࡀぢࡽࢀ㸪Congo Red㸪Thioflavin T࡞

࡝ࡢⰍ⣲ࢆ⏝࠸ࡿࡇ࡜࡛ほᐹࡉࢀ㸪┤ᚄࡣ10-20 nm⛬ᗘࡢᯞศ࠿ࢀࡢ࡞࠸⥺⥔ᵓ㐀ࢆ࡜ࡾ㸪

ぢࡽࢀࡿ୍᪉࡛㸪ཎᅉࢱࣥࣃࢡ㉁࡟ࡼࡗ࡚ࡣ㔝⏕ᆺ㸪ኚ␗య㸪truncated proteinࡢ≧ែ࡛

࠶ࡗ࡚ࡶ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿࡓࡵ[19]㸪࢔࣑ࣀ㓟㓄ิ࡟࠾ࡅࡿỴᐃⓗ࡞ඹ㏻Ⅼࡣᮍࡔ ぢࡘ࠿ࡗ࡚࠸࡞࠸ࠋࡋ࠿ࡋ࡞ࡀࡽ㸪ࡇࢀࡲ࡛࡟ᮏ◊✲ᐊ࡛ࡶⰾ㤶᪘࢔࣑ࣀ㓟ࡢࣇ࢙ࢽࣝ࢔

ࣛࢽࣥ[15]ࡸࢳࣟࢩࣥ[18]࡜࢔࣑ࣟ࢖ࢻ⥺⥔࡜ࡢ㛵ࢃࡾࢆ♧ࡋ࡚ࡁࡓࠋⰾ㤶᪘࢔࣑ࣀ㓟࡟ࡼ

ࡿS-S┦஫స⏝ࡣ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࡟ࡘ࡞ࡀࡿศᏊㄆ㆑ࡸ⮬ᕫจ㞟࡬ࡢᙳ㡪ࡀ♧၀ࡉ

ࢀ࡚࠸ࡿࡇ࡜࠿ࡽ[20]㸪࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࣓࢝ࢽࢬ࣒ࡢゎ᫂࡬ࡢᡭࡀ࠿ࡾ࡜࡞ࡾᚓࡿ࠿

ࡶࡋࢀ࡞࠸ࠋ

࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࣓࢝ࢽࢬ࣒ࡣཎᅉࢱࣥࣃࢡ㉁࡟ࡼࡗ࡚␗࡞ࡾ㸪Rebecca Nelsonࡽ

ࡣ኱ࡁࡃศࡅ࡚3ࡘࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᵓ㐀ࣔࢹࣝࢆᥦၐࡋ࡚࠸ࡿࠋ1ࡘ┠࡟㸪Refoldingࣔ

ࢹ࡛ࣝ࠶ࡿࠋInsulinࡸPrion protein࡟ࡳࡽࢀࡿࡼ࠺࡟㸪nativeᵓ㐀ࢆᣢࡘࢱࣥࣃࢡ㉁ࡀ୍

᪦unfoldࡋ㸪E-sheetᵓ㐀࡟ᐩࢇࡔᵓ㐀࡟refoldingࡍࡿࠋ2ࡘ┠࡟Natively disorderedࣔࢹ

࡛ࣝ࠶ࡿࠋSup35ࡸE-amyloid polypeptide㸦AE㸧࡟௦⾲ࡉࢀࡿࡼ࠺࡟㸪ࡶ࡜ࡶ࡜ᵓ㐀ࢆ࡜

ࡽ࡞࠸࣏ࣜ࣌ࣉࢳࢻ㙐ࡀ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿ㐣⛬࡛cross-E spineࡢᵓ㐀ࢆ⋓ᚓࡍࡿࠋ 3ࡘ┠࡟Gain-of-interactionࣔࢹࣝࡀᣲࡆࡽࢀࡿࠋᵓ㐀ኚ໬࡟ࡼࡾ≉ᐃࡢ㡿ᇦࡀ㟢ฟࡋ㸪┦

஫స⏝ࡍࡿࡇ࡜࡛࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿࠋGain-of-interactionࣔࢹࣝࡣDirect stacking

ࣔࢹࣝ࡞࡝ࡉࡽ࡟⣽ศ໬ࡉࢀࡿ[21]ࠋࡇࢀࡼࡾ㸪ࢱࣥࣃࢡ㉁ࡢᵓ㐀ኚ໬ࡣ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢ ᙧᡂ࣓࢝ࢽࢬ࣒ࢆ▱ࡿୖ࡛㔜せ࡞ᅉᏊ࡛࠶ࡿࡇ࡜ࡀศ࠿ࡿࠋ

ᅗ1-1㸬ࢡࣟࢫEᵓ㐀࠿ࡽ࡞ࡿ࢔࣑ࣟ࢖ࢻ⥺⥔[22]

11-3 㸬 ALS Ⓨ⑕ࡢཎᅉࢱࣥࣃࢡ㉁ࡢ SOD1

➽ⴎ⦰ᛶഃ⣴◳໬⑕㸦amyotrophic lateral sclerosis; ALS㸧ࡣ⚄⤒ኚᛶ⑌ᝈࡢ୍ࡘ࡛࠶ࡾ㸪 㐠ືࢽ࣮ࣗࣟࣥࡀᦆയࡉࢀ㸪ᴟࡵ࡚㐍⾜ࡀ᪩ࡃ㸪Ⓨ⑕ᚋ3ᖺ࠿ࡽ5ᖺ࡛Ṛ࡟⮳ࡿ࡜ࡉࢀࡿࠋ

⣙90%ࡣᏙⓎᛶALS㸦sALS㸧࡛࠶ࡿ[23]ࠋࡲࡓ㸪⣙10㸣ࡣᐙ᪘ᛶALS㸦fALS㸧࡛࠶ࡾ㸪 ࡑࡢ࠺ࡕ⣙20㸣ࡀCu, Zn-superoxide dismutase㸦SOD1㸧ࡢኚ␗࡟ࡼࡿࡶࡢ࡜ࡉࢀࡿࠋ1993 ᖺ࡟SOD1ࡀALSࡢཎᅉ㑇ఏᏊ࡜ࡋ࡚ぢࡘ࠿ࡗ࡚௨᮶[24]㸪⌧ᅾࡲ࡛࡟fALS࡟㛵ࡋ࡚160

✀㢮௨ୖࡢኚ␗యࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ㸦http://alsod.iop.kcl.ac.uk/㸧[25]㸪ᮍࡔ࡟ཎᅉゎ᫂

࡟ࡣ⮳ࡗ࡚࠸࡞࠸ࠋ

SOD1ࡣ153ṧᇶࡢ࣏ࣜ࣌ࣉࢳࢻ㙐࠿ࡽ࡞ࡿ2㔞యࢆᙧᡂࡋ࡚࠸ࡿ㸦ᅗ1-2㸧[26]ࠋSOD1 ࡣ8ࡘࡢEࢫࢺࣛࣥࢻ㸪7ࡘࡢ࣮ࣝࣉ࡜1ࡘࡢ▷࠸D࡬ࣜࢵࢡࢫ࠿ࡽ࡞ࡿ[27]ࠋྛࢧࣈࣘࢽࢵ

ࢺ࡟㖡࢖࢜ࣥ㸪ள㖄࢖࢜ࣥࢆ1ࡘࡎࡘ㓄఩ࡋ㸪4ࡘࡢCys㸦Cys6㸪57㸪111㸪146㸧ࢆᣢࡗ

࡚࠸ࡿࠋ㖡࢖࢜ࣥࡣ㓝⣲άᛶ࡟㛵୚ࡋ㸪ள㖄࢖࢜ࣥࡣᵓ㐀Ᏻᐃᛶ࡟㛵୚ࡋ࡚࠸ࡿ[28-30]ࠋ Cys6㸪Cys111 ࡣࣇ࣮ࣜࡢࢳ࣮࢜ࣝࡢ≧ែ࡛Ꮡᅾࡍࡿ୍᪉࡛㸪Cys57㸪Cys146 ࡣศᏊෆࢪ ࢫࣝࣇ࢕ࢻ⤖ྜࢆᙧᡂࡋ࡚࠸ࡿ[31]ࠋ㑏ඖ᮲௳ୗ࡛࠶ࡿ⣽⬊㉁ෆ࡟࠾࠸࡚ࢪࢫࣝࣇ࢕ࢻ⤖ྜ

ࢆᙧᡂࡋ࡚࠸ࡿࢱࣥࣃࢡ㉁ࡣ⛥࡛࠶ࡿࠋSOD1 ࡣ㖡࢖࢜ࣥࢆᑟධࡍࡿാࡁࢆᣢࡘ CCS 㸦Copper Chaperone for SOD1㸧࡟ࡼࡾ㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡣᑟධࡉࢀࡿ[32]ࠋSOD1 ࡣᵝࠎ࡞⑌ᝈࡸ⪁໬ࢆᘬࡁ㉳ࡇࡍ࡜ࡉࢀࡿάᛶ㓟⣲✀ࢫ࣮ࣃ࣮࢜࢟ࢩࢻࣛࢪ࢝ࣝ࡟ᑐࡋ࡚

㜵ᚚⓗ࡟ാࡁ㸪㓟⣲ࡸ㐣㓟໬Ỉ⣲࡟⨨᥮ࡍࡿᢠ㓟໬㓝⣲࡛࠶ࡿ[33]ࠋSOD1ࡢኚ␗ࡀྠᐃࡉ

ࢀࡓᙜึ㸪ኚ␗࡟ࡼࡾ㓝⣲ᶵ⬟ࡀపୗࡋ㸪ALS ࡢⓎ⑕࡟ࡘ࡞ࡀࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡓࠋࡋ࠿

ࡋ࡞ࡀࡽ㸪ከࡃࡢSOD1ኚ␗యࡣṇᖖ࡞㓝⣲άᛶࢆ♧ࡋ[34, 35]㸪ࡲࡓ㸪SOD1㑇ఏᏊࢆࣀ

ࢵࢡ࢔࢘ࢺࡋࡓ࣐࢘ࢫࡢ㐠ືࢽ࣮ࣗࣟࣥࡣṇᖖ࡛࠶ࡗࡓ[36]ࠋ୍᪉࡛㸪fALS ኚ␗ࢆⓎ⌧ࡋ ࡓࢺࣛࣥࢫࢪ࢙ࢽࢵࢡ࣐࢘ࢫࡣᝈ⪅࡟ࡳࡽࢀࡿ␗ᖖࡀሗ࿌ࡉࢀࡓ[37]ࠋࡇࢀࡼࡾ㸪ALS ࡣ SOD1 ࡢ㓝⣲ᶵ⬟ࡢᦆኻ࡟ࡼࡿࡶࡢ࡛ࡣ࡞ࡃ㸪ኚ␗࡟క࠸᪂ࡓ࡞␗ᖖࡀ⏕ࡌ㸪ALSࡢⓎ⑕

ሗ࿌ࡉࢀ࡚࠸ࡿ[38, 50]ࠋࡉࡽ࡟㸪sALSࡢᝈ⪅࡟ࡳࡽࢀࡿincusion body࡟㔝⏕ᆺSOD1 ࡀぢࡘ࠿ࡾ[38, 39, 51]㸪㔝⏕ᆺSOD1ࢆ㐣๫Ⓨ⌧ࡉࡏࡓࢺࣛࣥࢫࢪ࢙ࢽࢵࢡ࣐࢘ࢫࡣALS

࡟㢮ఝࡋࡓ⑕≧ࢆ♧ࡋ㸪⬨㧊ࡸ⬻࡟จ㞟యࡀ☜ㄆࡉࢀࡓ[52]ࠋࡇࢀࡼࡾ㸪fALS ኚ␗య㸪㔝

⏕ᆺSOD1࡜ࡶ࡟จ㞟యࡢᙧᡂ࡜ALSࡢⓎ⑕࡜ࡢ㛵ಀᛶࡀ♧၀ࡉࢀࡿࠋࡑࡢࡓࡵ㸪จ㞟య ࡀ⏕ࡌࡿ࣓࢝ࢽࢬ࣒ࢆㄪ࡭ࡿࡇ࡜࡛㸪fALSࡔࡅ࡛࡞ࡃsALS ࡟ࡘ࠸࡚ࡶALSⓎ⑕ཎᅉࡢ ゎ᫂࡟ࡘ࡞ࡀࡿ࡜ᮇᚅࡉࢀࡿࠋࡲࡓ㸪SOD1ࡢจ㞟యࡣ࢔ࣝࢶࣁ࢖࣐࣮⑓ࡸࣃ࣮࢟ࣥࢯࣥ⑓

ࡢࡼ࠺࡞⚄⤒ኚᛶ⑌ᝈࡢᝈ⪅ࡢ⤌⧊࡟ࡳࡽࢀࡿ࢔࣑ࣟ࢖ࢻ⥺⥔[53, 54]࡜ඹ㏻ࡋࡓ≉ᚩࢆᣢ ࡘࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ[42, 55]ࠋ୰࡟ࡣGdn-HCl ࡸ᭷ᶵ⁐፹ࢆ⏝࠸࡚ពᅗⓗ࡟ኚᛶࡋࡓ SOD1 ࡣ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿ[56-58]ࠋࡋ࠿ࡋ㸪ࡇࢀࡽࡢ᮲௳ࡣ

⣽⬊ෆ⎔ቃ࡜ࡣ␗࡞ࡗ࡚࠸ࡿࠋ㏻ᖖ㸪SOD1ࡣ㑏ඖ≧ែ࡟࠶ࡿ⣽⬊㉁ෆ࡟Ꮡᅾࡍࡿ[59]ࠋࡑ ࡢࡓࡵ㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢ㑏ඖࡣin vivo࡛㉳ࡇࡾ࠺ࡿࠋᐇ㝿࡟㸪fALSኚ␗ࢆᑟ ධࡋࡓࢺࣛࣥࢫࢪ࢙ࢽࢵࢡ࣐࢘ࢫ࡛ࡣศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡀ㑏ඖࡉࢀࡓSOD1 ࡀ☜ㄆ ࡉࢀ࡚࠸ࡿ[44, 46]ࠋࡇࡢࡼ࠺࡟㸪⏕⌮ⓗ᮲௳ୗ࡛ᵓ㐀ࡢ≉ᚩࡸᵓ㐀Ᏻᐃᛶࢆホ౯ࡍࡿࡇ࡜

ࡀ㔜せ࡛࠶ࡿࠋࡇࢀࡽࡢሗ࿌ࡼࡾ㸪SOD1ࡢmisfoldingࡸจ㞟ࡀ⏕ࡌࡿ࣓࢝ࢽࢬ࣒ࡢゎ᫂

ࡣALSࡢⓎ⑕ࢆ▱ࡿୖ࡛ࡣ㔜せ࡞ㄢ㢟࡛࠶ࡿ࡜ゝ࠼ࡿࠋ

Cu

Zn Cys111

Cys6

Cys146-Cys57

C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C Cu u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u u Cu

Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Zn n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n Zn C

C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C

Cy y y y y y y y y y y y y y y y y y y y y y y y y y y ys y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y s s s s s s s s s s s s s s s1 s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 111 Cys111 C

C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C

Cy y ys y y y y y y y y y y y y y y y y y y y y y y y y y y y y s s s s s s s s s s s s s s s s6 s s s s s s s s s s s s s s s s s s s s 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Cys6

C 146 C 57 Cys146-C C Cys5 C C C C C C C C C C C C C C C C C C C C C C C 57 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 Cys146-Cys57

ᅗ1-2㸬SOD1ࡢᵓ㐀

11-4 㸬ᮏㄽᩥࡢ┠ⓗ࠾ࡼࡧᵓᡂ

ᮏ◊✲࡛ࡣ㔝⏕ᆺSOD1ࡲࡓfALSࡢኚ␗య࡛࠶ࡿG93ASOD1ࢆ⏝࠸࡚SOD1ࡢ࢔࣑

ࣟ࢖ࢻ⥺⥔ᙧᡂ࣓࢝ࢽࢬ࣒ࡢゎ᫂ࢆ┠ⓗ࡜ࡋࡓࠋ ᮏᏛ఩ㄽᩥࡣ㸪7❶࡛ᵓᡂࡉࢀࡿࠋ

ࠕ➨ 1❶ ᗎㄽ࡛ࠖࡣ㸪ᮏ◊✲ࡢ⫼ᬒ࡜ࡋ࡚࢔࣑ࣟ࢖ࢻ⥺⥔ࡸ ALS Ⓨ⑕ࡢཎᅉࢱࣥࣃࢡ

㉁࡛࠶ࡿSOD1㸪ࡲࡓᮏㄽᩥࡢ┠ⓗ࠾ࡼࡧᵓᡂ࡟ࡘ࠸࡚㏙࡭ࡓࠋ

ࠕ➨2❶ 㔝⏕ᆺSOD1ࢆ⏝࠸ࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂࡢホ౯࡛ࠖࡣ㸪㔝⏕ᆺ SOD1ࢆ⏝

࠸࡚㔠ᒓ࢖࢜ࣥࡢ㝖ཤ㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜ㸦Cys57-Cys146㸧ࡢ㑏ඖ࡟ࡼࡾ⏕ࡌࡿᵓ 㐀Ᏻᐃᛶ࡟ࡘ࠸࡚ホ౯ࡍࡿࡇ࡜࡛㸪ᵓ㐀୙Ᏻᐃ໬ࡀ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࡟࡝ࡢࡼ࠺࡟ᙳ 㡪ࢆ୚࠼࡚࠸ࡿࡢ࠿ࢆㄪ࡭ࡓࠋ

ࠕ➨ 3 ❶ 㔝⏕ᆺ SOD1 ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆ⏝࠸ࡓ᰾㒊఩ࡢྠᐃ࡛ࠖࡣ㸪㔝⏕ᆺ SOD1

ࢆ⏝࠸࡚㸪࡝ࡢ㡿ᇦࡀస⏝ࡋ࡚⥺⥔᰾ࢆᙧᡂࡋ࡚࠸ࡿࡢ࠿ࢆㄪ࡭ࡓࠋ

ࠕ➨4❶ ᰾㒊఩ྜᡂ࣌ࣉࢳࢻࢆ⏝࠸ࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ⬟ࡢホ౯࡛ࠖࡣ㸪➨2❶㸪➨

3❶ࡼࡾSOD1ࡣ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿ㐣⛬࡛unfoldࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࡓࡵ㸪ྠ

ᐃࡉࢀࡓ᰾㒊఩ࡀ┦஫స⏝ࡋ㸪࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿ࠿࡝࠺࠿ࢆㄪ࡭ࡓࠋ

ࠕ➨ 5 ❶ fALS ኚ␗య G93ASOD1 ࢆ⏝࠸ࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂࡢホ౯࡛ࠖࡣ㸪

G93ASOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ⬟ࢆㄪ࡭ࡿ࡜࡜ࡶ࡟㸪㔝⏕ᆺSOD1ࢆ⏝࠸࡚࢔࣑ࣟ࢖ࢻ

⥺⥔ࡢᙧᡂࡢ㐪࠸࡟ࡘ࠸࡚ホ౯ࡋࡓࠋ

ࠕ➨6❶ ⥲ྜ⪃ᐹ࡛ࠖࡣ㸪➨2❶㹼➨5❶࡟ࡼࡗ࡚♧ࡉࢀࡓ㔝⏕ᆺSOD1࡜G93ASOD1 ࡢ⤖ᯝࢆ⥲ྜ⪃ᐹࡍࡿ࡜࡜ࡶ࡟㸪SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ࣓࢝ࢽࢬ࣒ࢆ♧ࡋࡓࠋ

ࠕ➨7❶ ⤖ㄒ࡛ࠖࡣ㸪ᮏ◊✲ࡢࡲ࡜ࡵࢆ㏙࡭ࡓࠋ

➨

➨ 2 ❶ 㔝⏕ᆺ SOD1 ࢆ⏝࠸ࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ

2-1 㸬⥴ゝ

ࢱࣥࣃࢡ㉁ࡣᶵ⬟ࢆ⥔ᣢࡍࡿࡓࡵ࡟Ᏻᐃࡋࡓ㧗ḟᵓ㐀ࢆ࡜ࡗ࡚࠸ࡿࠋᵓ㐀ࡢᏳᐃ໬࡟ࡣ㸪 ࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢࡼ࠺࡞ඹ᭷⤖ྜࡸ࢖࢜ࣥ⤖ྜ㸪Ỉ⣲⤖ྜ㸪ࣇ࢓ࣥࢹ࣮ࣝ࣡ࣝࢫຊ㸪␯

Ỉ⤖ྜࡢࡼ࠺࡞㠀ඹ᭷⤖ྜ࡞࡝ࡀస⏝ࡋ࡚࠸ࡿࠋࡋ࠿ࡋ࡞ࡀࡽ㸪 ᗘ㸪pH㸪ሷ⃰ᗘ㸪⏺㠃 άᛶ๣㸪ኚᛶ๣࡞࡝ࡢእⓗ࡞せᅉ࡟ࡼࡾኚ໬ࡍࡿࡇ࡜࡛㸪ࢱࣥࣃࢡ㉁ࡢᵓ㐀୙Ᏻᐃ໬ࡋ㸪 ኚᛶࡀ⏕ࡌࡿࠋ➨1❶࡛㏙࡭ࡓࡼ࠺࡟㸪ࢱࣥࣃࢡ㉁ࡢᵓ㐀Ᏻᐃᛶ࡜࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࡟

ࡣᐦ᥋࡞㛵ಀᛶࡀぢࡽࢀࡿࠋ㔠ᒓ࢖࢜ࣥࡣࢱࣥࣃࢡ㉁࡟㓄఩ࡍࡿࡇ࡜࡛㸪ᵓ㐀ࢆᏳᐃ໬ࡉ ࡏࡿാࡁࢆᣢࡗ࡚࠸ࡿࠋࡲࡓ㸪ࢪࢫࣝࣇ࢕ࢻ⤖ྜ࡟ࡘ࠸࡚ࡶᵓ㐀Ᏻᐃᛶ࡟኱ࡁࡃᐤ୚ࡍࡿࠋ Insulin[60]ࡸhen egg white lysozyme㸦HEWL㸧[61]ࡣࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢ㑏ඖ࡟ࡼࡗ࡚㸪 ᵓ㐀ࡢ୙Ᏻᐃ໬ࡀ⏕ࡌ㸪࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋ୍᪉࡛㸪 E2-microgobulin࡛ࡣࢪࢫࣝࣇ࢕ࢻ⤖ྜࢆ㑏ඖࡍࡿ࡜࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂࡀᢚไࡉࢀࡿ

[62]ࠋࡑࡢࡓࡵ㸪ࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢ㑏ඖࡣᵓ㐀ࡢ୙Ᏻᐃ໬ࡸᦂࡽࡂ࡟ᙳ㡪ࢆ୚࠼ࡿࡀ㸪ࢱ

ࣥࣃࢡ㉁ࡢ✀㢮࡟ࡼࡗ࡚࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࡟୚࠼ࡿᙳ㡪ࡣ␗࡞ࡿࡇ࡜ࡀ♧၀ࡉࢀࡿࠋ

SOD1ࡢሙྜ㸪㖡࢖࢜ࣥ࡜ள㖄࢖࢜ࣥࢆ㓄఩ࡋ㸪Cys57࡜Cys146࡛ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜ

ࢆᙧᡂࡋ࡚࠸ࡿࠋࡑࡇ࡛㸪ᮏ❶࡛ࡣ㸪ᵓ㐀ࡢᏳᐃ໬࡟ᐤ୚ࡍࡿ㔠ᒓ࢖࢜ࣥ㸪ศᏊෆࢪࢫࣝ

ࣇ࢕ࢻ⤖ྜ࡟ὀ┠ࡋ㸪㔝⏕ᆺSOD1ࢆ⏝࠸࡚㔠ᒓ࢖࢜ࣥࡢ㝖ཤ㸦apo໬㸧㸪ศᏊෆࢪࢫࣝࣇ࢕

ࢻ⤖ྜࡢ㑏ඖ࡟ࡼࡾ⏕ࡌࡿᵓ㐀Ᏻᐃᛶࡢኚ໬ࢆㄪ࡭㸪ᵓ㐀ࡢ୙Ᏻᐃ໬࡟క࠺࢔࣑ࣟ࢖ࢻ⥺

⥔ᙧᡂ࡟ࡘ࠸࡚ホ౯ࡋࡓࠋࡑࡇ࡛㸪SOD1ࡢᵓ㐀Ᏻᐃᛶ࡜࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂࡢ㛵ಀࢆ㆟ㄽ ࡍࡿࠋ

22-2 㸬ᮦᩱ࡜᪉ἲ

2-2-1㸬㔝⏕ᆺSOD1ࡢᇵ㣴

E. coli BLR (DE3) ࡟0.5 PLࡢⓎ⌧⣔ࣉࣛࢫ࣑ࢻpET23a-SOD1ࢆධࢀ㸪30 minị୰࡟

࠾ࡁ㸪ᜏ ᵴ࡛42°C㸪45 secࣄ࣮ࢺࢩࣙࢵࢡࡋ㸪ị୰࡛2 min࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋࡑࡋ

࡚㸪඲㔞1 mL࡟࡞ࡿࡼ࠺SOCᇵᆅࢆධࢀ㸪37°C㸪170 min^-1㸦TAITEC, Personal-11 Water Bath Shaker㸧࡟࡚᣺࡜࠺ᇵ㣴ࢆ1 h⾜࠸㸪LBᇵᆅ࡟ࡲࡁ㸪37°C㸪18 h㸦EYELA, Soft incubator SLI-450N㸧㟼⨨ᇵ㣴ࡋࡓࠋࡑࡢᚋ㸪ヨ㦂⟶࡟LBᇵᆅࢆධࢀ㸪᳜⳦ࡋ㸪37°C㸪 170 min^-1࡛17 h᣺࡜࠺ࡋ㸪ࣉࣞᇵ㣴ࢆ⾜ࡗࡓࠋ950 mLᇵᆅࡢධࡗࡓᆏཱྀࣇࣛࢫࢥ࡟㸪⤊

⃰ᗘ50 Pg/mLࡢ࢔ࣥࣆࢩࣜࣥࢆධࢀ㸪ࣉࣞᇵ㣴ࡋࡓ⳦యࢆ1 mLࢆῧຍࡋࡓࠋ8.55 Lࡢᇵ ᆅࢆᆏཱྀࣇࣛࢫࢥ9ᮏ⏝࠸࡚㸪37°C࡛24 h᣺࡜࠺ᇵ㣴ࡋࡓࠋᇵ㣴ᚋ㸪㐲ᚰศ㞳㸦4°C㸪8,000 rpm㸪20 min㸧ࡋ㸪⳦యࢆᅇ཰ࡋ㸪-85°C࡛ಖᏑࡋࡓࠋ

2-2-2㸬㔝⏕ᆺSOD1ࡢ⢭〇

E. coli BLR (DE3)/ pET23a-SOD1࡟ᑐࡋ࡚5ಸ㔞ࡢLysis buffer㸦50 mM Tris-HCl pH 8.0㸪150 mM KCl㸪0.1 mM EDTA㸪1 mM DTT㸧ࢆຍ࠼ᠱ⃮ࡉࡏ㸪ị୰࡟࡚㉸㡢Ἴฎ⌮

㸦Amplotude 60㸪Pulser 1㸪90 min㸧ࢆ⾜࠸㸪⳦యࢆ◚○ࡋࡓࠋ◚○ᚋ㸪㐲ᚰศ㞳㸦14,000 rpm㸪30 min㸪4°C㸧࡟ࡼࡾ㸪ỿẊ≀ࢆ㝖ཤࡋࡓࠋᚓࡽࢀࡓୖΎ⏬ศ࡟ị୰࡟࡚⤊⃰ᗘ2.5%

࡟࡞ࡿࡼ࠺࡟ࢫࢺࣞࣉࢺ࣐࢖ࢩࣥ⁐ᾮࢆࡺࡗࡃࡾ࡜⁲ୗࡋ 30 min ᧠ᢾࡋࡓᚋ㸪㐲ᚰศ㞳 㸦14,000 rpm㸪30 min㸪4°C㸧࡟ࡼࡾ㸪ୖΎ⏬ศࢆᅇ཰ࡋ㸪᰾㓟ࢆ㝖ཤࡋࡓࠋୖΎࢆị୰

࡛᧠ᢾࡋ࡞ࡀࡽ⤊⃰ᗘࡀ55%࡟࡞ࡿࡲ࡛ࡺࡗࡃࡾ࡜◲㓟࢔ࣥࣔࢽ࣒࢘ࢆຍ࠼㸪30 min᧠ᢾ ࡋࡓࠋࡑࡢᚋ㸪㐲ᚰศ㞳㸦14,000 rpm㸪30 min㸪4°C㸧ࡋ㸪ࡉࡽ࡟㸪ࡑࡢୖΎ࡟ị୰࡟࡚

᧠ᢾࡋ࡞ࡀࡽ⤊⃰ᗘ65%࡟࡞ࡿࡼ࠺࡟ࡺࡗࡃࡾ࡜◲㓟࢔ࣥࣔࢽ࣒࢘ࢆຍ࠼㸪30 min᧠ᢾࡋ ࡓᚋ㸪㐲ᚰศ㞳㸦14,000 rpm㸪30 min㸪4°C㸧ࡋ㸪ୖΎ⏬ศࢆᅇ཰ࡋࡓࠋQ-Sepharose column

SDS-PAGE࡟ࡼࡾ┠ⓗࡢࢱࣥࣃࢡ㉁ࡀྵࡲࢀ࡚࠸ࡿ⏬ศࢆ☜ㄆࡋࡓࠋᅇ཰ࡋࡓ⏬ศ࡟⤊⃰

ᗘࡀ2 M࡟࡞ࡿࡼ࠺࡟◲㓟࢔ࣥࣔࢽ࣒࢘ࢆῧຍࡋ㸪ᐊ ࡟࡚Phenyl-Toyopearl 650␯Ỉᛶ

┦஫స⏝ࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮࡟࢔ࣉࣛ࢖ࡋ㸪2࠿ࡽ0 M ◲㓟࢔ࣥࣔࢽ࣒࢘ࡢ⠊ᅖ࡛ࢢࣛࢪ

࢚ࣥࢺࢆ⾜࠸㸪ࢱࣥࣃࢡ㉁ࢆ⁐ฟࡉࡏࡓࠋ280 nmࡢ྾ගᗘ࡜SDS-PAGE࡟ࡼࡾ┠ⓗࡢࢱ

ࣥࣃࢡ㉁ࡀྵࡲࢀ࡚࠸ࡿ⏬ศࢆ☜ㄆࡋࡓࠋᅇ཰ࡋࡓ⏬ศ࡟2.5 mM 㔜Ⅳ㓟࢔ࣥࣔࢽ࣒࢘ࢆ

⏝࠸࡚㏱ᯒࡋ㸪⬺ሷࢆ⾜࠸㸪EYELA ෾⤖஝⇱ᶵFD-50ࢆ⏝࠸࡚෾⤖஝⇱ࡋࡓࠋ஝⇱ࡋࡓ 㔝⏕ᆺ SOD1ࡣ4°C࡛ಖᏑࡋࡓࠋ௨ୖࡢ⢭〇㐣⛬ࢆᅗ2-1࡟♧ࡋࡓࠋ

22-2-3㸬ࢱࣥࣃࢡ㉁ࡢᐃ㔞㸦⣸እ྾཰ἲ㸧

㔝⏕ᆺSOD1ࡢ280 nm࡟࠾ࡅࡿࣔࣝ྾ගᐃᩘ10,800 M^-1cm^-1ࢆ⏝࠸࡚[63]㸪྾ගᗘᘧ

(1)ࡼࡾ㸪ィ⟬್࡟ࡼࡿSOD1ࡢࢱࣥࣃࢡ㉁⃰ᗘࢆᐃ㔞ࡋࡓࠋ

(1): A=H412 nm lc

㸦H྾ගᐃᩘ (M^-1cm^-1)㸪l: ග㊰㛗 (cm)㸪c: ⃰ᗘ (mol/l)㸧

2-2-4㸬Cu㸪Znࡢᐃ㔞㸦ㄏᑟ⤖ྜࣉࣛࢬ࣐ศᯒ㸧

Cu㸪Znࡢ⤖ྜ㔞ࢆṇ☜࡟☜ㄆࡍࡿࡓࡵ㸪ICPศᯒࢆ⾜ࡗࡓࠋ ᐃࡣ㫽ྲྀ኱Ꮫ࣋ࣥࢳ࣮࣭ࣕ

ࣛ࣎ࣛࢺ࣮ࣜࡢ᳃ᮏඛ⏕࡟౫㢗ࡋࡓࠋ

2-2-5㸬ྛ✀㔝⏕ᆺSOD1 ࡢస〇᪉ἲ

ྛ✀㔝⏕ᆺSOD1ࡣ௨ୗࡢࡼ࠺࡟స〇ࡋࡓࠋ

Holo-SOD1ࡣ⢭〇ᚋࡢ㔝⏕ᆺSOD1ࢆࢱࣥࣃࢡ㉁⃰ᗘࡀ1 mg/mL࡟࡞ࡿࡼ࠺࡟buffer 㸦50 mM HEPES-KOH pH 7.0 at 25°C㸪6 M urea㸪0.1 mM CuCl2࣭2H2O㸪0.1 mM Zn(CH3COO)2࣭2H2O)㸧࡛⁐ゎࡉࡏ㸪25°C ࡟࡚㸪11 h ࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋࡑࡢᚋ㸪㐲 ᚰศ㞳㸦12,000 rpm㸪4°C㸪5 min㸧ࢆ⾜࠸㸪PD-10ࢆ⏝࠸࡚Milli-QỈ࡟⨨᥮ࡋ㸪෾⤖஝

⇱ࡋࡓࠋApo-SOD1ࡣ⢭〇ᚋࡢ㔝⏕ᆺSOD1ࢆࢱࣥࣃࢡ㉁⃰ᗘࡀ1 mg/mL࡟࡞ࡿࡼ࠺࡟

buffer㸦50 mM HEPES-KOH pH 7.0 at 25°C㸪10 mM EDTA࣭2Na㸪3 M Gdn-HCl࡛⁐

ゎࡉࡏ㸪25°C࡟࡚1 h࢖࣮ࣥ࢟ࣗ࣋ࢺࡋࡓࠋࡑࡢᚋ㸪PD-10ࢆ⏝࠸࡚Milli-QỈ࡟⨨᥮ࡋ㸪

෾⤖஝⇱ࡋࡓࠋ2SCAM-SOD1㸦Cys6㸪Cys111ࢆ࢔ࣝ࢟ࣝ໬㸧ࡣApo-SOD1࡟⤊⃰ᗘ2 M Gdn-HCl㸪10 mM ICH2CONH2ࢆῧຍࡋ㸪㐽ගࡋ࡞ࡀࡽᐊ ࡟࡚30 min㟼⨨ࡋ㸪ኚᛶ࠿

ࡘ࢔ࣝ࢟ࣝ໬཯ᛂࢆࡉࡏࡓࠋࡑࡢᚋ㸪PD-10ࢆ⏝࠸࡚Milli-QỈ࡟⨨᥮ࡋ㸪෾⤖஝⇱ࡋࡓࠋ 2SCAMRed-SOD1㸦apo ໬㸪Cys6㸪Cys111 ࢆ࢔ࣝ࢟ࣝ໬ࡉࡏ㸪㑏ඖ๣Ꮡᅾୗ࡛ศᏊෆࢪ ࢫࣝࣇ࢕ࢻ⤖ྜࡣ㑏ඖࡉࢀࡓ≧ែࢆ⥔ᣢ㸧ࡣApo-SOD1࡟⤊⃰ᗘ2 M Gdn-HCl㸪10 mM ICH2CONH2ࢆῧຍࡋ㸪㐽ගࡋ࡞ࡀࡽᐊ ࡟࡚30 min㟼⨨ࡋ㸪ኚᛶ࠿ࡘ࢔ࣝ࢟ࣝ໬཯ᛂࢆ

ࡉࡏࡓࠋࡑࡢᚋ㸪PD-10ࢆ⏝࠸࡚50 mM Tris-HCl pH 7.0 at 25°C㸪150 mM NaCl࡟⨨᥮

ࡋ㸪⤊⃰ᗘ5 mM࡟࡞ࡿࡼ࠺࡟ DTTࢆῧຍࡋ㸪30 min㟼⨨ࡋ㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜ

ࢆ㑏ඖࡋࡓࠋࡑࡢᚋ㸪50 mM Tris-HCl pH 7.0 at 37°C㸪150 mM NaCl㸪1mM TCEP࡟⨨

᥮ࡋࡓࠋ4SCAM-SOD1㸦apo໬㸪Cys57㸪146ࡢࢪࢫࣝࣇ࢕ࢻ⤖ྜࢆ㑏ඖࡋ㸪ࡍ࡭࡚ࡢCys

ࢆ࢔ࣝ࢟ࣝ໬㸧ࡣApo-SOD1࡟⤊⃰ᗘ2 M Gdn-HCl㸪5 mM DTTࢆῧຍࡋ㸪30 minᐊ 

࡛㟼⨨ࡋ㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࢆ㑏ඖࡉࡏࡓࠋࡉࡽ࡟㸪10 mM ICH2CONH2ࢆῧຍࡋ㸪 30 minᐊ ࡛㐽ගࡋ࡞ࡀࡽ㟼⨨ࡋ㸪࢔ࣝ࢟ࣝ໬཯ᛂࢆࡉࡏࡓࠋࡑࡢᚋ㸪PD-10ࢆ⏝࠸࡚50 mM Tris-HCl pH 7.0 at 37°C㸪150 mM NaCl࡟⨨᥮ࡋࡓࠋRed-SOD1㸦apo໬㸪㑏ඖ๣Ꮡ ᅾୗ࡛ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢ㑏ඖ≧ែࢆ⥔ᣢ㸧ࡣApo-SOD1࡟⤊⃰ᗘ2 M Gdn-HCl㸪

5 mM DTTࢆῧຍࡋ㸪30 minᐊ ࡛㟼⨨ࡋ㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࢆ㑏ඖࡉࡏࡓࠋࡉࡽ

࡟㸪PD-10ࢆ⏝࠸࡚50 mM Tris-HCl pH 7.0 at 37°C㸪150 mM NaCl㸪1 mM TCEP࡟⨨

᥮ࡋࡓࠋ

22-2-6㸬Gdn-HClࢆ⏝࠸ࡓ㔝⏕ᆺSOD1ࡢᵓ㐀Ᏻᐃᛶホ౯

Holo-SOD1㸪Apo-SOD1㸪Red-SOD1ࡢᵓ㐀Ᏻᐃᛶࢆㄪ࡭ࡿࡓࡵ㸪ኚᛶ๣࡜ࡋ࡚Gdn-HCl

ࢆ⏝࠸࡚ᵓ㐀Ᏻᐃᛶࢆホ౯ࡋࡓࠋ

Holo-SOD1㸪Apo-SOD1ࡣ0.15 mg/mL㸦50 mM Tris-HCl pH 7.0 at 25°C㸪150 mM NaCl㸪 0㹼5.7 M Gdn-HCl㸧㸪Red-SOD1ࡣ0.15 mg/mL㸦50 mM Tris-HCl pH 7.0 at 25°C㸪150 mM NaCl㸪1 mM TCEP㸪0㹼5.7 M Gdn-HCl㸧ࡢ⤊⃰ᗘ࡟࡞ࡿࡼ࠺࡟ㄪ〇ࡋࡓࠋࡑࡢᚋ㸪25°C

୍࡛ᬌ㟼⨨ࡋ㸪ᵓ㐀ࡢኚ໬ࢆホ౯ࡍࡿࡓࡵ㸪CDࢫ࣌ࢡࢺࣝ ᐃ㸪Tryptophan⺯ගࢫ࣌ࢡ ࢺࣝ ᐃࢆ⾜ࡗࡓࠋ

SOD1ࡢ஧ḟᵓ㐀ኚ໬ࢆㄪ࡭ࡿࡓࡵ㸪JASCO J-720 SpectrophotometerࡲࡓࡣJASCO J-820 Spectrophotometerࢆ⏝࠸㸪෇஧Ⰽᛶ㸦CD㸧ࢫ࣌ࢡࢺࣝ ᐃࡋࡓࠋ

ㄪ〇ࡋࡓࢧࣥࣉࣝࡣ㸪ග㊰㛗1 mmࡢ▼ⱥࢭࣝࢆ⏝࠸㸪❅⣲࢞ࢫὶ㏿2 L/min㸪25°C࡛ ௨ୗ࡟♧ࡍ ᐃ᮲௳࡛ ᐃࡋࡓࠋ

㸦 ᐃ᮲௳㸧ឤᗘ: Low㸦1000 mdeg㸧㸪㛤ጞἼ㛗: 250 nm㸪⤊஢Ἴ㛗: 200 nm㸪ࢹ࣮ࢱྲྀ

Tyrosineࡣ280 nm࡛ບ㉳ࡉࡏࡿ࡜310 nm௜㏆࡛᭱኱⺯ගἼ㛗ࢆ♧ࡍࠋSOD1ࡣTyrosine

ࢆᣢࡓ࡞࠸ࡓࡵ㸪Tryptophan ௜㏆ࡢ⎔ቃࢆㄪ࡭ࡿࡇ࡜࡛㸪SOD1 ࡢኚᛶࡢᵝᏊࢆㄪ࡭ࡓࠋ Tryptophan⺯ගࢫ࣌ࢡࢺࣝ ᐃࡣ㸪JASCO FP-6300 Fluorescence Spectrophotometer㸪 ග㊰㛗1 cmࡢ▼ⱥࢭࣝࢆ⏝࠸࡚㸪௨ୗࡢ ᐃ᮲௳࡛⾜ࡗࡓࠋ

㸦 ᐃ᮲௳㸧 ᐃ࣮ࣔࢻ: Emission㸪ບ㉳ࣂࣥࢻᖜ: 5 nm㸪⺯ගࣂࣥࢻᖜ: 5 nm㸪ࣞࢫ࣏ࣥ

ࢫ: Medium㸪ឤᗘ: Medium㸪ບ㉳Ἴ㛗: 295 nm㸪㛤ጞἼ㛗: 300 nm㸪⤊஢Ἴ㛗: 450 nm㸪

ྲྀ㎸㛫㝸: 0.5 nm㸪㏿ࡉ: 50 nm/min㸪✚⟬࡞ࡋ

ゎᯒࡣCDࢫ࣌ࢡࢺࣝ ᐃࡢ216 nm㸪Trypthophan⺯ගࢫ࣌ࢡࢺࣝ ᐃࡢ295 nmບ㉳

᫬ࡢ350 nmࡢ ᐃࢹ࣮ࢱࢆ⏝࠸ࡓࠋኚᛶ᭤⥺ࡣḟࡢᘧ (2) ࢆ⏝࠸࡚⟬ฟࡋࡓࠋࡉࡽ࡟㸪

Cm (ኚᛶ୰Ⅼ)ࡣfD=0.5ࡢCDࢫ࣌ࢡࢺࣝ ᐃ࡜Tryptophan ⺯ගࢫ࣌ࢡࢺࣝ ᐃ࡟ࡼࡾ

ᚓࡽࢀࡓGdn-HCl⃰ᗘࡢᖹᆒ್ࡼࡾ⟬ฟࡋࡓࠋ

(2): fD=(N-Nl)/(Nu-Nl)

㸦uࡣୖ㒊୍࡛ᐃ࡟࡞ࡿ㡿ᇦ㸪lࡣୗ㒊୍࡛ᐃ࡟࡞ࡿ㡿ᇦࢆᣦࡋ㸪Nࡣ ᐃࡋࡓGdn-HCl

࡛ࡢ ᐃࢹ࣮ࢱࢆᣦࡍࠋ㸧

22-2-7㸬㔝⏕ᆺSOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ

ྛ✀㔝⏕ᆺSOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ⬟ࢆㄪ࡭ࡓࠋホ౯᪉ἲࡣThioflavin T assay㸪 CDࢫ࣌ࢡࢺࣝ ᐃ㸪㏱㐣ᆺ㟁Ꮚ㢧ᚤ㙾㸦TEM㸧ほᐹࢆ⏝࠸ࡓࠋ

࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂࢧࣥࣉࣝࡣ㸪Holo-SOD1㸪Apo-SOD1㸪2SCAM-SOD1㸪4SCAM-SOD1 ࡣ50 mM Tris-HCl pH 7.0 at 37°C㸪150 mM NaCl㸪ࡲࡓ㸪Red-SOD1㸪2SCAMRed-SOD1 ࡣ50 mM Tris-HCl pH 7.0 at 37°C㸪150 mM NaCl㸪1 mM TCEPࡢbufferࢆ⏝࠸࡚SOD1

⃰ᗘ1 mg/mL㸦62.5 PM㸧㸪1 mL࡟ㄪ〇ࡋ㸪I10 mmࡢࢸࢫࢺࢳ࣮ࣗࣈ࡟ධࢀ㸪ࣃࣛࣇ࢕

࣒ࣝ࡟࡚ᐦᑒࡋ㸪37°C㸪170 min^-1㸦TAITEC, Personal-11 Water Bath Shaker㸧࡟࡚᣺࡜

࠺ࡋࡓࠋThioflavin Tࡣ࢔࣑ࣟ࢖ࢻ⥺⥔࡜≉␗ⓗ࡟⤖ྜࡋ㸪440 nm࡛ບ㉳ࡉࡏࡿ࡜480 nm ࡢ᭱኱⺯ගἼ㛗ࢆほᐹࡍࡿࡇ࡜ࡀ࡛ࡁࡿ࡜࠸࠺≉ᛶࢆᣢࡘࡇ࡜ࢆ฼⏝ࡋ㸪᣺࡜࠺୰ࡢࢧࣥ

ࣉࣝࢆ୍㒊ᢤࡁྲྀࡾ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢ⤒᫬ኚ໬ ᐃࢆ⾜࠺ࡓࡵ࡟Thioflavin T assayࢆ⾜

ࡗࡓࠋ ᐃ᪉ἲࡣThioflavin T Buffer㸦5 mM Sodium phosphate㸪150 mM NaCl㸪25 PM

Thioflavin T㸧ࢆ2 mL࡟ᑐࡋ㸪࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂࢧࣥࣉࣝ15 PL㸦SOD1⤊⃰ᗘ0.0075 mg/mL㸧ῧຍࡋ㸪30 sec࣎ࣝࢸࢵࢡࢫࡋ㸪ග㊰㛗1 cmࡢ▼ⱥࢭࣝ࡟ධࢀ㸪HITACHI F-4500 Fluorescence Spectrophotometerࢆ⏝࠸࡚ ᐃࡋࡓࠋ ᐃ᮲௳ࡣ௨ୗࡢࡼ࠺࡟⾜ࡗࡓࠋࢹ

࣮ࢱࡣ60 sec ᐃࡋ㸪ࡑࡢᖹᆒ್ࢆ⏝࠸ࡓࠋ

㸦 ᐃ᮲௳タᐃ㸧ࢹ࣮ࢱ࣮ࣔࢻ: ⺯ග㸪᫬㛫༢఩: sec㸪ບ㉳Ἴ㛗: 440 nm㸪⺯ගἼ㛗: 480 nm㸪  ᐃ᫬㛫: 60 sec㸪ึᮇᚅࡕ᫬㛫: 0

㸦ගᗘィ᮲௳㸧ບ㉳ഃࢫࣜࢵࢺ: 5 nm㸪⺯ගഃࢫࣜࢵࢺ 5 nm㸪࣍ࢺ࣐ࣝ㟁ᅽ(V) : 700㸪ࣞ

ࢫ࣏ࣥࢫ(sec) : 0.1㸪ࢩࣕࢵࢱไᚚ: ON

CDࢫ࣌ࢡࢺࣝ ᐃࡣ᣺࡜࠺๓ᚋ࡟ࢧࣥࣉࣝࢆ᥇ྲྀࡋ㸪SOD1⃰ᗘ0.1 mg/mL࡟࡞ࡿࡼ

࠺࡟ྛ✀⁐፹࡟࡚ᕼ㔘ࡋࡓᚋ㸪2-2-6ࡢ᪉ἲ࡜ྠᵝ࡟ ᐃࡋࡓࠋ᣺࡜࠺ᚋࡢࢧࣥࣉࣝࡣỿẊ ࡢᵝᏊࢆ☜ㄆࡋ࡞ࡀࡽ㉸㡢Ἴฎ⌮㸦Amplitude 10㹼20㸪Pulser 1㸪1㹼10 sec㸧ࢆ⾜࠸㸪  ᐃࡋࡓࠋ

TEMほᐹ⏝ࡢࢧࣥࣉࣝࡣ᣺࡜࠺ᚋࡢࢧࣥࣉࣝࢆMilli-QỈ࡛5-10ಸᕼ㔘ࡋ㸪ࢥࣟࢪ࢜ࣥ

⭷ᙇ௜࣓ࢵࢩࣗ࡟ࢧࣥࣉࣝࢆ5ࡲࡓࡣ10 PLࡢࡏ࡚㸪60~90 sec㟼⨨ࡋࡓࠋࢁ⣬࡛ࢧࣥࣉࣝ

ࢆ྾࠸ྲྀࡗࡓᚋ㸪2%࢘ࣛࢽࣝ㓑㓟ࢆ5 PLࡢࡏ࡚30~50 sec㟼⨨ࡋ㸪ࢁ⣬࡛྾࠸ྲྀࡾ㸦ྛ㟼

⨨᫬㛫ࡣࢧࣥࣉࣝ࡟ࡼࡗ࡚ㄪᩚࡋࡓ㸧㸪㢼஝ࡉࡏࡓࠋTEM ほᐹࡣ㸪80 kV ࡛సືࡉࡏࡓ JEOL-100CXࡲࡓࡣJEOL-1400 plus Transmission Electron Microscopeࢆ⏝࠸ࡓࠋಸ⋡

ࡣ27,000㹼50,000ಸ࡛⾜ࡗࡓࠋࡲࡓ㸪TEM⏬ീࡼࡾ⥺⥔ࡢ3Ⅼࡢᖜࢆ ࡾ㸪ࡑࡢᖹᆒࡢ 㛗ࡉࢆ⥺⥔ᖜ࡜ࡋࡓࠋ

22-2-8㸬0.4 M Gdn-HClᏑᅾୗ࡛ࡢ㔝⏕ᆺSOD1࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ

Red-SOD1ࡢኚᛶ୰Ⅼ࡛࠶ࡿ0.4 M Gdn-HClᏑᅾୗ࡛࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂᐇ㦂ࢆ⾜࠺ࡇ

࡜࡛㸪ᵓ㐀ࡀ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࡟୚࠼ࡿᙳ㡪ࢆㄪ࡭ࡓࠋ

࢔ ࣑ ࣟ ࢖ ࢻ ⥺ ⥔ ᙧ ᡂ ⏝ ࢧ ࣥ ࣉ ࣝ ࡣ 㸪Holo-SOD1㸪Apo-SOD1㸪2SCAM-SOD1㸪

࡟ㄪ〇ࡋ㸪I10 mmࡢࢸࢫࢺࢳ࣮ࣗࣈ࡟ධࢀ㸪ࣃࣛࣇ࢕࣒ࣝ࡟࡚ᐦᑒࡋ㸪37°C㸪170 min^-1 ࡢ᮲௳࡛TAITEC, Personal-11 Water Bath Shaker࡟࡚᣺࡜࠺ࡋࡓࠋ2-2-7࡜ྠᵝ࡟ࡋ࡚㸪 Thioflavin T assay㸪CDࢫ࣌ࢡࢺࣝ ᐃࢆ⾜ࡗࡓࠋ

22-3 㸬⤖ᯝ

2-3-1㸬ྛ✀㔝⏕ᆺSOD1 ࡢ஧ḟᵓ㐀࡜ᵓ㐀Ᏻᐃᛶࡢゎᯒ

ࡲ ࡎ 㸪Holo-SOD1㸪Apo-SOD1㸪Red-SOD1㸪2SCAM-SOD1㸪2SCAMRed-SOD1㸪

4SCAM-SOD1ࢆ⏝࠸࡚㔠ᒓ࢖࢜ࣥ࡜ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡀ SOD1ࡢ஧ḟᵓ㐀࡟୚࠼

ࡿᙳ㡪ࢆCDࢫ࣌ࢡࢺࣝ ᐃ࡟࡚ㄪ࡭ࡓࠋ

ᅗ2-2࡟ྛ✀㔝⏕ᆺSOD1ࡢ஧ḟᵓ㐀ࢆ♧ࡋࡓࠋHolo-SOD1ࡣEࢩ࣮ࢺᵓ㐀≉᭷ࡢ208 nm௜㏆࡟㈇ࡢᴟ኱ࡀぢࡽࢀࡓ[64]ࠋࡲࡓ㸪Apo-SOD1࡜Red-SOD1ࡣ࡜ࡶ࡟Holo-SOD1

࡜㢮ఝࡋࡓ஧ḟᵓ㐀࡛࠶ࡗࡓࠋࡘࡲࡾ㸪㔝⏕ᆺSOD1ࡣapo໬㸪ࡉࡽ࡟ศᏊෆࢪࢫࣝࣇ࢕

ࢻ⤖ྜࡀ㑏ඖࡉࢀ࡚࠸࡚ࡶ㸪Holo-SOD1࡜㢮ఝࡋࡓ஧ḟᵓ㐀ࢆ⥔ᣢࡋ࡚࠸ࡿࡇ࡜ࡀ♧ࡉࢀ

ࡓࠋ୍᪉࡛㸪ࡍ࡭࡚ࡢCysࢆ࢔ࣝ࢟ࣝ໬ࡋࡓ4SCAM-SOD1ࡣ᏶඲࡟ኚᛶࡋ㸪ࡲࡓCys6㸪 Cys111ࢆ࢔ࣝ࢟ࣝ໬ࡋࡓ2SACM-SOD1࡜2SCAMRed-SOD1ࡣ㒊ศⓗ࡟ኚᛶࡋ࡚࠾ࡾ㸪 2SCAMRed-SOD1ࡣ2SACM-SOD1࡟ẚ࡭㸪ࢃࡎ࠿࡟ኚᛶࡢ⛬ᗘࡀ኱ࡁ࠿ࡗࡓࠋಟ㣭㒊఩

ࡣᵓ㐀ୖ㞀ᐖ࡜࡞ࡿࡓࡵ㸪≉࡟4SCAM-SOD1ࡣCys57࡜Cys146ࡢಟ㣭㒊఩ࡢᙳ㡪ࢆཷ

ࡅ㸪᏶඲࡟ኚᛶࡋࡓ࡜⪃࠼ࡽࢀࡿࠋ

ࡉࡽ࡟㸪஧ḟᵓ㐀ࢆ⥔ᣢࡋ࡚࠸ࡓHolo-SOD1㸪Apo-SOD1㸪Red-SOD1ࢆ⏝࠸࡚Gdn-HCl

࡟ᑐࡍࡿᵓ㐀Ᏻᐃᛶࢆホ౯ࡋࡓࠋ0㹼5.7 M Gdn-HClᏑᅾୗ࡟࠾ࡅࡿCDࢫ࣌ࢡࢺࣝ ᐃ࡜

Tryptophan⺯ගࢫ࣌ࢡࢺࣝ ᐃࢆ⾜ࡗࡓࠋ

Gdn-HCl⃰ᗘ࡟ࡼࡿᵓ㐀Ᏻᐃᛶࡢ⤖ᯝࢆᅗ2-3࡟♧ࡋࡓࠋࡲࡓ㸪⾲2-1ࡣᅗ2-3ࡼࡾ⟬

ฟࡉࢀࡓኚᛶ୰Ⅼ㸦Cm㸧ࢆ♧ࡋࡓࠋHolo-SOD1㸪Apo-SOD1㸪Red-SOD1ࡢCmࡣࡑࢀࡒ

ࢀ4.37 M㸪0.90 M㸪0.42 M Gdn-HCl࡛࠶ࡗࡓࠋHolo-SOD1ࡣ᭱ࡶᏳᐃࡋ࡚࠾ࡾ㸪apo໬

ࡋࡓApo-SOD1ࡣⴭࡋࡃᵓ㐀ࡀ୙Ᏻᐃ໬ࡋ࡚࠸ࡓࠋࡉࡽ࡟㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࢆ㑏

ඖࡋࡓRed-SOD1ࡣࡉࡽ࡟ᵓ㐀ࡀ୙Ᏻᐃ໬ࡋࡓࠋኚᛶ᭤⥺ࡼࡾ㸪Red-SOD1࡜Apo-SOD1

ࡣ0 M Gdn-HCl୰࡛ࡣᏳᐃᛶࢆ⥔ᣢࡋ࡚࠸ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋ௒ᅇࡣ2≧ែ㌿⛣࠶ࡿ࠸ࡣ

ᅗ2-2㸬ྛ✀㔝⏕ᆺSOD1㸦Holo-SOD1㸪Apo-SOD1㸪Red-SOD1㸪2SCAM-SOD1㸪 2SCAMRed-SOD1㸪4SCAM-SOD1㸧ࡢCDࢫ࣌ࢡࢺࣝ ᐃ

ᅗ2-3㸬Holo-SOD1㸪Apo-SOD1㸪Red-SOD1ࡢGdn-HCl࡟ᑐࡍࡿᵓ㐀Ᏻᐃᛶࡢホ౯ CDࢫ࣌ࢡࢺࣝ ᐃࡣ216 nm࡟࠾ࡅࡿ ᐃࢹ࣮ࢱ㸪Trypthophan⺯ගࢫ࣌ࢡࢺࣝ ᐃࡣ 295 nmບ㉳᫬ࡢ350 nmࡢ ᐃࢹ࣮ࢱࢆfD࡟⨨᥮ࡋ㸪Gdn-HCl⃰ᗘ࡟ᑐࡋ࡚ࣉࣟࢵࢺࡋ

ࡓࠋ(ە)ࡣCDࢫ࣌ࢡࢺࣝ ᐃ㸪(ی)ࡣ⺯ගࢫ࣌ࢡࢺࣝ ᐃࢆ♧ࡍࠋ

⾲2-1㸬Holo-SOD1㸪Apo-SOD1㸪Red-SOD1ࡢኚᛶ୰Ⅼ㸦Cm㸧*

Holo- (M) Apo- (M) Red- (M) 㔝⏕ᆺ SOD1 4.37 0.90 0.42

*Cmࡣᅗ2-3ࡢኚᛶ᭤⥺ࡼࡾ⟬ฟࡋ࡚࠸ࡿࠋ

22-3-2㸬㔝⏕ᆺSOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ

㔝⏕ᆺSOD1ࡢapo໬ࡸศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢ㑏ඖ࡟ࡼࡾ⏕ࡌࡓᵓ㐀ࡢ୙Ᏻᐃ໬ࡀ㸪

࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࡟࡝ࡢࡼ࠺࡟ᙳ㡪ࡍࡿࡢ࠿ࢆㄪ࡭ࡿࡓࡵ㸪Holo-SOD1㸪Apo-SOD1㸪 Red-SOD1㸪2SCAM-SOD1㸪2SCAMRed-SOD1㸪4SCAM-SOD1ࢆ⏝࠸࡚࢔࣑ࣟ࢖ࢻ⥺⥔

ᙧᡂࢆThioflavin T assay㸪CDࢫ࣌ࢡࢺࣝ ᐃ㸪TEMほᐹ࡟࡚ホ౯ࡋࡓࠋ

ᅗ 2-4 ࡟ Thioflavin T assay ࡢ⤖ᯝࢆ♧ࡋࡓࠋ4SCAM-SOD1㸪2SCAMRed-SOD1㸪

Red-SOD1 ࡣⴭࡋ࠸⺯ගᙉᗘࡢቑຍࡀぢࡽࢀࡓࠋ୍᪉࡛㸪Holo-SOD1㸪Apo-SOD1㸪

2SCAM-SOD1ࡣ300 h᣺࡜࠺ᐇ㦂ࢆ⾜ࡗࡓࡀ㸪⺯ගᙉᗘࡢቑຍࡀぢࡽࢀ࡞࠿ࡗࡓࠋ㠃ⓑ࠸

ࡇ࡜࡟㸪๓⪅ࡢ⺯ගᙉᗘࡢቑຍࡀぢࡽࢀࡓSOD1ࡣapo໬ࡋ㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡀ ษ᩿ࡉࢀ㸪ᚋ⪅ࡣศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࢆᙧᡂࡋ࡚࠸ࡿSOD1 ࡛࠶ࡗࡓࠋࡲࡓ㸪⺯ගᙉ ᗘࡢቑຍࡀぢࡽࢀࡓSOD1ࡢ୰࡛ࡶ㸪4SCAM-SOD1㸪2SCAMRed-SOD1㸪Red-SOD1ࡢ 㡰࡟⺯ගᙉᗘࡢቑຍࡢ❧ࡕୖࡀࡾࡢ᪩࠸ࡇ࡜ࡀศ࠿ࡗࡓࠋࡉࡽ࡟㸪Thioflavin T assay࡟࡚

⺯ගᙉᗘࡢቑຍࡀぢࡽࢀ࡞ࡃ࡞ࡗࡓ࡜ࡇࢁ࡛㸪CDࢫ࣌ࢡࢺࣝ ᐃ㸪TEMほᐹࢆ⾜ࡗࡓࠋ ᅗ2-5࡟4SCAM-SOD1㸪2SCAMRed-SOD1㸪Red-SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂᐇ㦂๓ᚋ ࡢCDࢫ࣌ࢡࢺࣝ ᐃࡢẚ㍑࡜࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂᚋࡢTEMほᐹࡢ⤖ᯝࢆ♧ࡋࡓࠋ࢔࣑ࣟ

࢖ࢻ⥺⥔ᙧᡂᐇ㦂ᚋ㸪CDࢫ࣌ࢡࢺࣝࡣ218 nm௜㏆࡟㈇ࡢࣆ࣮ࢡࡀほᐹࡉࢀ㸪඾ᆺⓗ࡞E ࢩ࣮ࢺᵓ㐀ࢆ♧ࡋࡓࠋࡲࡓ㸪TEMほᐹࡼࡾ㸪ᯞศ࠿ࢀࡢ࡞࠸⥺⥔ᵓ㐀ࡀ☜ㄆࡉࢀࡓࠋ⥺⥔

ᖜࡣࡑࢀࡒࢀ㸪4SCAM-SOD1 ࡣ 13.2±0.6 nm㸪2SCAMRed-SOD1 ࡣ 13.6±2.4 nm㸪 Red-SOD1 ࡣ 10.9±0.6 nm ࡛࠶ࡗࡓ㸦 ⾲ 2-2㸧ࠋ௨ୖࡢ⤖ᯝࡼࡾ㸪4SCAM-SOD1㸪

2SCAMRed-SOD1㸪Red-SOD1ࡣ඾ᆺⓗ࡞࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡋ࡚࠸ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋ

ᅗ2-4㸬ྛ✀㔝⏕ᆺSOD1ࡢThioflavin T assay

(ڧ)ࡣHolo-SOD1㸪(ۑ)ࡣApo-SOD1㸪(ە) ࡣRed-SOD1㸪(ۍ)ࡣ2SCAM-SOD1㸪 (ی)ࡣ2SCAMRed-SOD1㸪(ڦ)ࡣ4SCAM-SOD1ࢆ♧ࡋ㸪

ᖹᆒ್ࢆࣉࣟࢵࢺࡋ㸪࢚࣮ࣛࣂ࣮ࡣ3ᅇ ᐃࡢᶆ‽೫ᕪࢆ♧ࡍࠋ

⾲2-2㸬࢔࣑ࣟ࢖ࢻ⥺⥔ᖜ

⥺⥔ᖜ* (nm) 4SCAM-SOD1 13.2±0.6 2SCAMRed-SOD1 13.6±2.4

Red-SOD1 10.9±0.6

*⥺⥔ᖜࡣᅗ2-5ࡢTEM⏬ീࡼࡾ㸪⥺⥔ࡢ3࠿ᡤ࡟࠾ࡅࡿᖹᆒ್±ᶆ‽೫ᕪࢆ♧ࡍࠋ

ᅗ2-5㸬4SCAM-SOD1㸪2SCAMRed-SOD1㸪Red-SOD1ࡢ CDࡢࢫ࣌ࢡࢺࣝ ᐃ࡜TEMほᐹ

Aࡣ4SCAM-SOD1㸪Bࡣ2SCAMRed-SOD1㸪CࡣRed-SOD1ࢆ♧ࡋ㸪ᕥᅗࡣCDࢫ࣌ࢡ ࢺࣝ ᐃ㸪ྑᅗࡣTEM⏬ീࢆ♧ࡍࠋࡲࡓ㸪CDࢫ࣌ࢡࢺࣝ ᐃࡢ㯮Ꮠࡣ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧ ᡂᐇ㦂๓㸪㉥Ꮠࡣ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂᐇ㦂ᚋࡢ ᐃ⤖ᯝࢆ♧ࡍࠋTEMほᐹࡢࢫࢣ࣮ࣝࣂ࣮

ࡣ200 nmࢆ♧ࡍࠋ

22-3-3㸬0.4 M Gdn-HClᏑᅾୗ࡛ࡢ㔝⏕ᆺSOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ

apo໬㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢ㑏ඖᚋ㸪ᵓ㐀ࡢ୙Ᏻᐃ໬ࡀ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࡟୚

࠼ ࡿ ᙳ 㡪 ࢆ ㄪ ࡭ ࡿ ࡓ ࡵ 㸪Red-SOD1㸪2SCAMRed-SOD1㸪4SCAM-SOD1 ࢆ ⏝ ࠸ ࡚ Red-SOD1ࡢCm࡛࠶ࡿ0.4 M Gdn-HClᏑᅾୗ࡛ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂࢆThoflavin T

assay㸪CDࢫ࣌ࢡࢺࣝ ᐃ࡟࡚ホ౯ࡋࡓࠋ

ᅗ 2-6 ࡟ Thioflavin T assay ࡢ⤖ᯝࢆ♧ࡋࡓࠋ4SCAM-SOD1㸪2SCAMRed-SOD1㸪 Red-SOD1 ࡣ ⴭ ࡋ ࠸ ⺯ ග ᙉ ᗘ ࡢ ቑ ຍ ࡀ ぢ ࡽ ࢀ ࡓ ࠋ ࡉ ࡽ ࡟ 㸪4SCAM-SOD1㸪 2SCAMRed-SOD1㸪Red-SOD1ࡢ⺯ගᙉᗘࡢቑຍࡀ18 h࡛ぢࡽࢀࡓࠋኚᛶ๣ࢆ⏝࠸࡞࠸

⤖ᯝ࡟ẚ࡭㸦ᅗ2-4㸧㸪0.4 M Gdn-HCl୰࡛ࡢRed-SOD1࡜2SCAMRed-SOD1ࡢ࢔࣑ࣟ࢖

ࢻ⥺⥔ࢆᙧᡂࢫࣆ࣮ࢻࡣ᪩ࡃ࡞ࡗࡓࠋThioflavin T assay࡛⺯ගᙉᗘࡢቑຍࡀぢࡽࢀ࡞ࡃ࡞

ࡗࡓ࡜ࡇࢁ࡛㸪CDࢫ࣌ࢡࢺࣝ ᐃࢆ⾜ࡗࡓࠋᅗ2-7࡟4SCAM-SOD1㸪2SCAMRed-SOD1㸪

Red-SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂᐇ㦂๓ᚋࡢ CD ࢫ࣌ࢡࢺࣝ ᐃࡢẚ㍑ࡢ⤖ᯝࢆ♧ࡋࡓࠋ

0.4 M Gdn-HClᏑᅾୗ࡛ࡣRed-SOD1ࡣ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂᐇ㦂๓࡟ኚᛶࡋ࡚࠸ࡓࡀ㸪ᐇ

㦂ᚋ࡟ࡣ 218 nm ௜㏆࡟㈇ࡢࣆ࣮ࢡࡀほᐹࡉࢀ㸪඾ᆺⓗ࡞Eࢩ࣮ࢺᵓ㐀ࢆ♧ࡋࡓࠋ

4SCAM-SOD1㸪2SCAMRed-SOD1 ࡟ࡘ࠸࡚ࡶྠᵝ࡞⤖ᯝ࡛࠶ࡗࡓࠋ௨ୖࡢ⤖ᯝ࠿ࡽ㸪

Red-SOD1ࡣᵓ㐀ࡀኚᛶࡍࡿ࡜㸪㏿ࡸ࠿࡟࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋ

ᅗ2-6㸬0.4 M Gdn-HClᏑᅾୗ࡛ࡢRed-SOD1㸪2SCAMRed-SOD1㸪4SCAM-SOD1ࡢ Thioflavin T assay

(ە)ࡣRed-SOD1㸪(ی)ࡣ2SCAMRed-SOD1㸪(ڦ)ࡣ4SCAM-SOD1ࢆ♧ࡍࠋᖹᆒ್ࢆࣉࣟ

ࢵࢺࡋ㸪࢚࣮ࣛࣂ࣮ࡣ3ᅇ ᐃࡢᶆ‽೫ᕪࢆ♧ࡍࠋ

ᅗ2-7㸬0.4 M Gdn-HClᏑᅾୗ࡛ࡢ4SCAM-SOD1㸪2SCAMRed-SOD1㸪Red-SOD1ࡢ CDࢫ࣌ࢡࢺࣝ ᐃ

Aࡣ4SCAM-SOD1㸪Bࡣ2SCAMRed-SOD1㸪CࡣRed-SOD1ࢆ♧ࡍࠋࡲࡓ㸪㯮Ꮠࡣ࢔࣑

ࣟ࢖ࢻ⥺⥔ᙧᡂᐇ㦂๓㸪㉥Ꮠࡣ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂᐇ㦂ᚋࡢ ᐃ⤖ᯝࢆ♧ࡍࠋ

22-4 㸬⪃ᐹ

ᮏ❶࡛ࡣ㸪㔝⏕ᆺ SOD1 ࢆ⏝࠸࡚㸪apo ໬ࡸศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢ㑏ඖ࡟ࡼࡾ⏕ࡌ

ࡿᵓ㐀Ᏻᐃᛶ࡜࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂࢆホ౯ࡋࡓࠋ

apo ໬࡟ࡼࡾ㔝⏕ᆺSOD1 ࡣᵓ㐀ࡢ୙Ᏻᐃ໬ࡀ☜ㄆࡉࢀࡓ㸦ᅗ2-3㸪⾲2-1㸧ࠋࡇࢀࡲ࡛

࡟㸪SOD1ࡣapo໬࡟ࡼࡾ㸪ள㖄࢖࢜ࣥࡢࣜ࢞ࣥࢻࡀ఩⨨ࡍࡿloop Phe50-Gly82࡟ᦂࡽࡂ ࡀ⏕ࡌࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ[65, 66]ࠋࡋ࠿ࡋ࡞ࡀࡽ㸪apo໬ࡢࡳ࡛ࡣ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆ

ᙧᡂࡋ࡞࠿ࡗࡓࠋࡑࡇ࡛㸪ࡉࡽ࡟ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࢆ㑏ඖࡋࡓSOD1 ࢆస〇ࡋࡓࠋ Red-SOD1ࡣApo-SOD1࡟ẚ࡭㸪ࡉࡽ࡟ᵓ㐀ࡀ୙Ᏻᐃ໬ࡋ㸪37°C࡛࢖࣮ࣥ࢟ࣗ࣋ࢺࡋ࡞ࡀ

ࡽ᣺࡜࠺ࡍࡿ࡜඾ᆺⓗ࡞࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡋࡓࠋศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡣ㔠ᒓ࢖࢜

ࣥ࡟ẚ࡭㸪ᵓ㐀Ᏻᐃᛶ࡟ᐤ୚ࡋ࡚࠸࡞࠸ࡀ[30, 65]㸪apo໬㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢ㑏 ඖࡀ࡜ࡶ࡟⏕ࡌࡿࡇ࡜࡛ࢲ࢖࣐࣮ࡢゎ㞳[29, 65, 67]ࡸCys57ࡢ㟢ฟ[68]ࡀ⏕ࡌࡿࡇ࡜ࡀሗ

࿌ࡉࢀ࡚࠸ࡿࠋࡇࢀࡣศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡀloop Glu49-Asn53ࢆᅛᐃࡋ࡚࠾ࡾ㸪apo

໬ࡸศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢ㑏ඖ࡟ࡼࡾloop Phe50-Gly82ࡢᦂࡽࡂࡀࡉࡽ࡟኱ࡁࡃ࡞ࡿ

ࡇ࡜ࡀᙳ㡪ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿ[65]ࠋࡘࡲࡾ㸪㔝⏕ᆺ SOD1 ࡢ㔠ᒓ࢖࢜ࣥ࡜ศᏊෆࢪࢫ

ࣝࣇ࢕ࢻ⤖ྜࡣᵓ㐀ࡢᦂࡽࡂ࡟኱ࡁࡃ㛵୚ࡋ㸪࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࡟㔜せ࡞ᙺ๭ࢆᣢࡗ

࡚࠸ࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋࡉࡽ࡟㸪apo໬㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢ㑏ඖࡀ⏕ࡌࡓᚋ㸪 SOD1ࡢᵓ㐀ࡀ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂ࡟୚࠼ࡿᙳ㡪ࢆホ౯ࡋࡓࠋThioflavin T assayࡼࡾ㸪 4SCAM-SOD1㸪2SCAMRed-SOD1㸪Red-SOD1 ࡢ㡰࡟࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿࢫࣆ࣮

ࢻࡀ᪩࠿ࡗࡓ㸦ᅗ 2-4㸧ࠋࡇࡢ㡰␒ࡣ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿ๓ࡢ஧ḟᵓ㐀ࡢኚᛶࡢ⛬ᗘ

࡜୍⮴ࡋࡓ㸦ᅗ2-2㸧ࠋࡉࡽ࡟㸪0.4 M Gdn-HClᏑᅾୗ࡛Red-SOD1ࢆពᅗⓗ࡟ኚᛶࡉࡏࡿ

࡜㸪࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂࡀⴭࡋࡃ᪩ࡃ࡞ࡾ㸪᏶඲ኚᛶࡋ࡚࠸ࡿ 4SCAM-SOD1 ࡜ྠᵝ࡞

⥺⥔ᙧᡂࢫࣆ࣮ࢻࢆ♧ࡋࡓ㸦ᅗ2-6㸧ࠋࡘࡲࡾ㸪Red-SOD1 ࡣHolo-SOD1 ࡜㢮ఝࡋࡓ஧ḟ ᵓ㐀ࢆ࡜ࡗ࡚࠸ࡿࡀ㸪᏶඲ኚᛶࡋ࡚࠸ࡿ 4SCAM-SOD1 ࡜ྠࡌ⥺⥔ᙧᡂ࣓࢝ࢽࢬ࣒ࢆ࡜ࡿ

࡜⪃࠼ࡽࢀࡿࠋRed-SOD1ࡣᵓ㐀Ᏻᐃᛶࡀప࠸ࡓࡵ㸪ᵓ㐀ࡀᦂࡽࡄࡇ࡜࡛᭱⤊ⓗ࡟unfold ࡋ㸪࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋࡑࡢࡓࡵ㸪➨ 1 ❶࡛㏙࡭ࡓ Rebecca

NelsonࡽࡢᥦၐࡍࡿRefoldingࣔࢹࣝ࡜ྠᵝ࡞࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ࣓࢝ࢽࢬ࣒ࢆ࡜ࡿࡇ࡜

ࡀ♧၀ࡉࢀࡓ[21]ࠋ௨ୖࡼࡾ㸪apo໬㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢ㑏ඖࡀ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧ ᡂࡢ㔜せ࡞ᅉᏊ࡛࠶ࡾ㸪୍᪦2ࡘࡢᅉᏊࡀ㝖࠿ࢀࡿ࡜SOD1ࡢᵓ㐀࡟኱ࡁ࡞ᦂࡽࡂࡀ⏕ࡌ㸪

᭱⤊ⓗ࡟unfoldࡍࡿࡇ࡜࡛࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋ

࢔࣑ࣟ࢖ࢻ⥺⥔ࡣ᰾ᙧᡂ཯ᛂࡀ⏕ࡌ㸪᰾ࢆ㗪ᆺ࡜ࡋ࡚ఙ㛗཯ᛂࡀ⏕ࡌࡿࡇ࡜࡛⥺⥔ࡀᙧ ᡂࡉࢀࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋࡑࡇ࡛㸪SOD1 ࡣᵓ㐀ࡀᦂࡽࡂ㸪unfold ࡋ㸪᰾㒊఩ࡀ㟢ฟ ࡋ㸪┦஫స⏝ࡍࡿࡇ࡜࡛࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿ࡜⪃࠼ࡽࢀࡿࠋࡑࡢࡓࡵ㸪➨ 3 ❶࡛ࡣ 㔝⏕ᆺSOD1ࢆ⏝࠸࡚࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂࡢඖ࡜࡞ࡿ᰾㒊఩㡿ᇦࡢྠᐃࢆ⾜࠺ࠋ

➨

➨ 3 ❶ 㔝⏕ᆺ SOD1 ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆ⏝࠸ࡓ᰾㒊

఩ࡢྠᐃ

3-1 㸬⥴ゝ

➨2❶࡛ࡣ㔝⏕ᆺSOD1ࡣ㔠ᒓ࢖࢜ࣥࡢ㝖ཤ㸦apo໬㸧㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡢ㑏ඖ

࡟ࡼࡾ㸪ᵓ㐀ࡀ୙Ᏻᐃ໬ࡋ㸪࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿࡇ࡜ࢆ♧ࡋࡓࠋ୍⯡ⓗ࡟㸪࢔࣑ࣟ

࢖ࢻ⥺⥔ࡣ᰾ᙧᡂ཯ᛂ㸪ఙ㛗཯ᛂࡢ2ࡘࡢ㐣⛬࡛ᙧᡂࡉࢀࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ㸦ᅗ3-1㸧ࠋ

᰾ᙧᡂ཯ᛂࡣ⮬Ⓨⓗ࡟⏕ࡌ࡟ࡃࡃ㸪㓟ᛶ᮲௳ୗࡸ➨2❶ࡢࡼ࠺࡟ኚᛶ๣ࡢῧຍ࡞࡝㸪native ᵓ㐀ࡢ୙Ᏻᐃ໬ࡸ㸪㒊ศⓗ࡟unfoldࡍࡿࡇ࡜࡛㏿ࡸ࠿࡟᰾ࡀᙧᡂࡉࢀࡿࠋ࢔࣑ࣟ࢖ࢻ⥺⥔

ࡣ᰾ᙧᡂ཯ᛂࡀᚊ㏿࡜࡞ࡗ࡚࠸ࡿࡀ㸪୍᪦㸪᰾ࡀᙧᡂࡉࢀࡿ࡜㸪ࡑࢀࢆ㗪ᆺ࡜ࡋ࡚ఙ㛗ࡍ

ࡿࠋࡲࡓ㸪᰾࡜࡞ࡿSeed㸦㉸㡢Ἴฎ⌮࡟ࡼࡾ㸪࢔࣑ࣟ࢖ࢻ⥺⥔ࢆ᩿∦໬ࡉࡏࡓ⥺⥔࡞࡝㸧 ࡢῧຍ࡟ࡼࡾ㸪᰾ᙧᡂ㐣⛬ࢆ⤒ࡎ࡟㸪ఙ㛗཯ᛂࡀ⏕ࡌࡿࡓࡵ㸪࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂࢫࣆ

࣮ࢻࡀⴭࡋࡃ᪩ࡃ࡞ࡿ㸦Seed ຠᯝ㸧[69]ࠋࡘࡲࡾ㸪᰾㒊఩㡿ᇦࡣ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂึ

ᮇ࡟㛵ࢃࡿ㠀ᖖ࡟㔜せ࡞㡿ᇦ࡛࠶ࡿࡇ࡜ࡀ♧၀ࡉࢀࡿࠋ⥺⥔᰾ࡢᵓ㐀ࡢ≉ᚩ࡜ࡋ࡚㸪ࢡࣟ

ࢫEᵓ㐀ࢆᙧᡂࡋ࡚࠾ࡾ㸪ሀࡃᏳᐃࡋ࡚࠸ࡿࡓࡵ㸪ࣉࣟࢸ࢔࣮ࢮ⪏ᛶࢆᣢࡗ࡚࠸ࡿࠋࡇࢀࡲ

࡛࡟㸪⥺⥔᰾ࡀࣉࣟࢸ࢔࣮ࢮ⪏ᛶࢆᣢࡘ࡜࠸࠺≉ᚩࢆ฼⏝ࡋ㸪D-synuclein[15]㸪 AE (1−40) [70]㸪E2-microglobulin[71]ࡢࡼ࠺࡞⑓Ẽࡢཎᅉࢱࣥࣃࢡ㉁࡟ࡼࡾᙧᡂࡉࢀࡿ࢔࣑ࣟ࢖ࢻ⥺

⥔ࡔࡅ࡛࡞ࡃ㸪GroES[7]ࡢࡼ࠺࡞┤᥋⑓Ẽ࡜㛵ಀࡢ࡞࠸ࢱࣥࣃࢡ㉁࡟ࡼࡾᙧᡂࡉࢀࡿ࢔࣑

ࣟ࢖ࢻ⥺⥔࡛ࡶ᰾㒊఩ࡢྠᐃࡀ⾜ࢃࢀ࡚࠸ࡿࠋ᰾࡜࡞ࡿ㡿ᇦࢆ᥈⣴ࡍࡿࡇ࡜ࡣ⥺⥔ᙧᡂ࣓

࢝ࢽࢬ࣒ࢆ⌮ゎࡍࡿୖ࡛࡜࡚ࡶ㔜せ࡞ࡇ࡜࡛࠶ࡿࠋ㏆ᖺ࡛ࡣ㸪᰾㒊఩ࢆண ࡍࡿࣉࣟࢢࣛ

࣒ࡶ㛤Ⓨࡉࢀ࡚࠾ࡾ㸪Waltz㸦http://waltz.switchlab.org/㸧ࡀ▱ࡽࢀ࡚࠸ࡿࠋ

ᮏ❶࡛ࡣ㸪㔝⏕ᆺSOD1ࡢRed-SOD1ࡼࡾᙧᡂࡉࢀࡓ࢔࣑ࣟ࢖ࢻ⥺⥔࡟ᑐࡋ࡚㸪ࣜࢩࣥ

ṧᇶࢆ≉␗ⓗ࡟ษ᩿ࡍࡿAchromobacter lyticus Protease I㸦API㸧࡜␯Ỉᛶࡢ㧗࠸⬡⫫᪘

࠾ࡼࡧⰾ㤶᪘࢔࣑ࣀ㓟ࢆศゎࡍࡿproteinase K㸦PK㸧ࡢ2✀㢮ࡢ㓝⣲ࢆ⏝࠸࡚㸪᰾㒊఩ࡢ

ྠᐃࢆ⾜ࡗࡓࠋࡲࡓ㸪᏶඲ኚᛶࣔࢹࣝࡢ 4SCAM-SOD1 ࡟ࡘ࠸࡚ࡶྠᵝ࡟᰾㒊఩ࡢྠᐃࢆ

⾜࠸㸪Red-SOD1ࡢ᰾㒊఩࡜ᑐẚࡋ㸪㔝⏕ᆺSOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ࣓࢝ࢽࢬ࣒࡟ࡘ

࠸࡚㆟ㄽࡍࡿࠋ

ᅗ3-1㸬࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂࣔࢹࣝ

᰾ࡀᙧᡂࡋ㸪ࡑࢀࢆ㗪ᆺ࡜ࡋ࡚ఙ㛗ࡍࡿࡇ࡜࡛࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿࠋ

33-2 㸬ᮦᩱ࡜᪉ἲ

3-2-1㸬Red-SOD1㸪4SCAM-SOD1 ࡟ࡼࡾᙧᡂࡉࢀࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢ㓝⣲ᾘ

໬

➨2❶ࡢ2-2-7࡜ྠᵝ࡟Red-SOD1㸪4SCAM-SOD1ࢆ⏝࠸࡚1 mg/mL㸪1 mLࢆࢸࢫࢺ ࢳ࣮ࣗࣈ࡟ධࢀ㸪37°C㸪170 min^-1࡟࡚᣺࡜࠺ࡋ㸪࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡉࡏࡓࠋHITACHI himac CS 150 GXL Micro Ultracentrifugeࢆ⏝࠸࡚㸪ᙧᡂࡋࡓ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆ㉸㐲ᚰศ 㞳㸦4°C㸪150,000™g㸪1 h㸧ࡋ㸪ỿẊࢆᅇ཰ࡋࡓࠋࡲࡓ㸪ᅇ཰ࡋࡓỿẊࡣ㸪buffer㸦25 mM Tris-HCl pH 7.5 at 37°C㸧ࢆ200 PLຍ࠼㸪෌ࡧ㉸㐲ᚰศ㞳㸦4°C㸪150,000™g㸪1 h㸧ࡋ㸪 ỿẊࡢὙίࢆ⾜ࡗࡓࠋࡑࡢᚋ㸪ࢱࣥࣃࢡ㉁⃰ᗘࡀ10 mg/mL࡟࡞ࡿࡼ࠺࡟buffer㸦25 mM Tris-HCl pH 7.5 at 37°C㸧࡛ᠱ⃮ࡉࡏ㸪2~3 sec㉸㡢Ἴฎ⌮㸦Amplitude 20㸪Pulser 1㸧࡟

ࡼࡾ㸪ỿẊࢆ࡯ࡄࡋࡓࠋ㉸㡢Ἴฎ⌮ᚋࡢ⁐ᾮࢆ࢚ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈ࡟200 PLࡎࡘศὀ ࡋࡓࠋศὀࡋࡓྛࠎࡢ࢚ࢵ࣌ࣥ࡟㸪1 mg/mL࡟ㄪ〇ࡋࡓAchromobactor lyticus Protease I 㸦API㸧㸪Proteinase K㸦PK㸧ࢆࣔࣝẚ࡛1/100࡟࡞ࡿࡼ࠺࡟ῧຍࡋࡓࠋࡑࡢᚋ㸪37°C࡛㸪

ྛ᫬㛫࡛ᾘ໬ࡋ㸪཯ᛂࡣࢧࣥࣉࣝ100 PL࡟ᑐࡋ࡚25%㓑㓟4 PLࡢ๭ྜ࡛ΰྜࡋ㸪pH 3

࡟ࡍࡿࡇ࡜࡛೵Ṇࡋࡓࠋ

3-2-2㸬㏫┦HPLC࡟ࡼࡿࢧࣥࣉࣝࡢศྲྀ

ᾘ໬ᚋࢧࣥࣉࣝࢆ㉸㐲ᚰศ㞳㸦4°C㸪233,000™g㸪1 h㸧ࡋ㸪ୖΎ࡜ỿẊࢆᅇ཰ࡋࡓࠋỿ Ẋࡣwash bufferࢆῧຍࡋ㸪㉸㐲ᚰศ㞳㸦4°C㸪233,000™g㸪1 h㸧ࡋ㸪Ὑίࢆ⾜ࡗࡓࠋWash buffer࡜ࡋ࡚25%㓑㓟4 PL࡟ᑐࡋ㸪25 mM Tris-HCl pH 7.5 at 25°C 100 PLࡢ๭ྜ࡛ΰ

ྜࡋࡓࡶࡢࢆ౑⏝ࡋࡓࠋࡑࡢᚋ㸪ୖΎ㸪ࡲࡓ7.5 M Gdn-HCl 50 PL࡛ྍ⁐໬ࡉࡏࡓỿẊࢆ㸪

㏫┦HPLC㸦CAPCELL PAK C18࣒࢝ࣛ: SHISEIDO㸧࡟࢔ࣉࣛ࢖ࡋࡓࠋ᳨ฟࡉࢀࡓࣆ࣮

ࢡ⏬ศࢆ࢚ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈ࡟ᅇ཰ࡋࡓࠋ㏫┦HPLCࡢ᮲௳ࡣ㸪ὶ㏿1 mL/min㸪᳨

ฟUV 215 nm㸪buffer A : 0.05% TFA in Milli-Q㸪buffer B : 0.05% TFA in Acetonitrile࡜ ࡋ㸪Acetonitrile┤⥺ࢢࣛࢪ࢚ࣥࢺ0~80%㸦30 min㸧࡛⾜ࡗࡓࠋ

33-2-3㸬MALDI-TOF mass spectrometer࡟ࡼࡿศᏊ㔞ࡢỴᐃ

㏫┦HPLC࡛ᅇ཰ࡋࡓ࣌ࣉࢳࢻ⁐ᾮࡢAcetonitrile㸪TFA࡞࡝ࡢ⁐፹ࢆ㸪࢚ࣂ࣏࣮ࣞࢱ

࣮ࢆ ⏝࠸࡚㝖࠸ࡓ ࠋ࢚ࢵ࣌ ࣥࢻࣝࣇࢳࣗ ࣮ࣈ࡟ TA buffer㸦0.1% TFA in Milli-Q : Acetonitrile=2 : 1㸧50 PLࢆຍ࠼⁐ゎࡋ㸪ࡇࢀࢆࢧࣥࣉࣝ⁐ᾮ࡜ࡋࡓࠋMass spectrometer ࡢ࢟ࣕࣜࣈ࣮ࣞࢩࣙࣥᶆ‽ヨᩱ࡜ࡋ࡚Peptide Calibration Standard㸪ཬࡧࢧࣥࣉࣝ⁐ᾮࢆ

Matrix (D-Cyano-4-hydroxy-cinnamic acid/ TA) 㣬࿴⁐ᾮ࡜1 : 1ࡢ๭ྜ࡛ΰྜࡉࡏ㸪ࢱ࣮

ࢤࢵࢺୖ࡟ྛࠎ (࢟ࣕࣜࣈ࣮ࣞࢩࣙࣥ0.5 PL㸪ࢧࣥࣉࣝ1 PLࡎࡘ) ࡢࡏ㸪ᖖ ࡛஝⇱ࡉࡏ ࡓࠋ ᐃࡣࣉࣟࢺࢥ࣮ࣝ࡟ᚑ࠸㸪ࢧࣥࣉࣝศᏊ㔞ࡢ ᐃࢆBRUKER DALTONICS autoflex MALDI-TOF mass spectrometerࢆ⏝࠸㸪ᚓࡽࢀࡓศᏊ㔞࠿ࡽSOD1ࡢ࢔࣑ࣀ㓟㓄ิ࡟┦

ᙜࡍࡿ㡿ᇦࢆゎᯒࡋࡓࠋ

3-2-4㸬ࣉࣟࢸ࢖ࣥࢩ࣮ࢡ࢚ࣥࢧ࣮࡟ࡼࡿศᯒ

㏫┦HPLC࡛ᅇ཰ࡋࡓࢧࣥࣉࣝࡢAcetonitrile㸪TFA࡞࡝ࡢ⁐፹ࢆ㸪࢚ࣂ࣏࣮ࣞࢱ࣮ࢆ

⏝࠸࡚㝖࠸ࡓᚋ㸪࢚ࢵ࣌ࣥࢻࣝࣇࢳ࣮ࣗࣈ࡟1ᮏᙜࡓࡾ㏫┦HPLCࡢbuffer A㸦0.05% TFA

in Milli-Q㸧30 PL࡟⁐࠿ࡋ㸪ࡇࢀࢆࢧࣥࣉࣝ⁐ᾮ࡜ࡋࡓࠋࡲࡎ㸪࣏ࣜࣈࣞࣥ⁐ᾮࢆ࢞ࣛࢫ

ࣇ࢓࢖ࣂ࣮ࢹ࢕ࢫࢡ࡟30 PLࡢࡏ㸪5ศ㛫஝⇱ࡉࡏࡓᚋ㸪ࣉࣟࢺࢥ࣮ࣝ࡟ᚑ࠸࣏ࣜࣈࣞࣥฎ

⌮ࡋࡓࠋḟ࡟㸪ࢧࣥࣉࣝ⁐ᾮࡶ࢞ࣛࢫࣇ࢓࢖ࣂ࣮ࢹ࢕ࢫࢡ࡟ 30 PLࡎࡘࡢࡏ㸪ࡑࢀࡒࢀ 5 ศ㛫஝⇱ࡉࡏࡓᚋ㸪ࣉࣟࢺࢥ࣮ࣝ࡟ᚑ࠸㸪SHIMADZU PPSQ-10 Protein Sequencerࢆ⏝

࠸ࡓࠋゎᯒࡣྛࠎࡢ࢔࣑ࣀ㓟ṧᇶࡢNᮎ➃࠿ࡽ5ࡲࡓࡣ10ṧᇶࡢỴᐃࢆ⾜ࡗࡓࠋ

3-2-5㸬᰾㒊఩㡿ᇦࡢྠᐃ

ྛࠎࡢ࢔࣑ࣟ࢖ࢻ⥺⥔᰾ᵓ㐀㒊఩࡜⪃࠼ࡽࢀࡿ࢔࣑ࣀ㓟㓄ิࡣ㸪MALDI-TOF mass

spectrometer ࡜ࣉࣟࢸ࢖ࣥࢩ࣮ࢡ࢚ࣥࢧ࣮࡟ࡼࡿศᯒ⤖ᯝ㸪࢔࣑ࣟ࢖ࢻ⥺⥔᰾ண ࣉࣟࢢ

Red-SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔࡟ᑐࡋ㸪ࣔࣝẚ1/100ࡢPKࢆῧຍࡋ㸪10 hᾘ໬ᚋࡢࢧࣥ

ࣉ ࣝ 㸦Red-SOD1-PK㸧㸪 ࣔ ࣝ ẚ 1/100 ࡢ API ࢆ ῧ ຍ ࡋ 㸪20 h ᾘ ໬ ᚋ ࡢ ࢧ ࣥ ࣉ ࣝ 㸦Red-SOD1-API㸧ࢆస〇ࡋ㸪㉸㡢Ἴฎ⌮㸦Amplitude 20㸪Pulser 1㸪10 sec㸧ࡋࡓࠋ୍᪉

࡛㸪㉸㡢Ἴฎ⌮㸦Amplitude 20㸪Pulser 1㸪40 sec㸧ࡋࡓ Red-SOD1 ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔

㸦Red-SOD1-full length㸧ࡶ Seed ຠᯝࢆẚ㍑ࡍࡿࡓࡵ࡟⏝࠸ࡓࠋࡇࢀࡽࡢࢧࣥࣉࣝࢆ

Thioflavin T assay࡟ࡼࡿ⺯ගᙉᗘࢆ⣙20࡟ᕼ㔘ࡋ㸪Seedࢧࣥࣉࣝࢆస〇ࡋࡓࠋࡇࢀࢆ➨

2❶ࡢ2-2-7࡜ྠᵝ࡟1 mg/mL, 1 mL࡟ㄪ〇ࡋࡓRed-SOD1ࢆࢸࢫࢺࢳ࣮ࣗࣈ࡟ධࢀ㸪ㄪ

〇ࡋࡓSeedࢧࣥࣉࣝࢆ1%ࡲࡓࡣ5%ῧຍࡋ㸪37°C㸪170 min^-1࡟࡚᣺࡜࠺ࡋ㸪ࡑࢀࡒࢀࡢ Seedຠᯝ࡟ࡘ࠸࡚ㄪ࡭ࡓࠋ

33-3 㸬⤖ᯝ

3-3-1㸬Red-SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆ⏝࠸ࡓ᰾㒊఩ྠᐃ

Red-SOD1࠿ࡽᙧᡂࡋࡓ࢔࣑ࣟ࢖ࢻ⥺⥔࡟ᑐࡋ࡚㸪APIᾘ໬㸪PKᾘ໬ᚋࡢTEMほᐹࢆ

ᅗ3-2㸪ࡲࡓ㸪⥺⥔ᖜࢆ⾲3-1࡟♧ࡋࡓࠋTEMほᐹࡼࡾ㸪⥺⥔ᖜࡀ⣽ࡃ㸪⥺⥔㛗ࡀ▷ࡃ࡞

ࡗ࡚࠾ࡾ㸪㓝⣲ᾘ໬ࡉࢀ࡚࠸ࡿࡇ࡜ࡀ☜ㄆࡉࢀࡓࠋ

ࡉࡽ࡟㸪ᾘ໬ᚋࢧࣥࣉࣝࢆ7.5 M Gdn-HClࢆ⏝࠸࡚⁐ゎࡋ㸪㏫┦HPLC࡟࡚࣌ࣉࢳࢻ᩿

∦ࡢᅇ཰ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࢆᅗ3-3࡟♧ࡋࡓࠋAPIᾘ໬㸦20 h㸧ᚋ㸪⁐ฟ᫬㛫16-20ศ ࡢ㛫࡟3ࡘࡢPeak㸦Peak 1㸪2㸪3㸧ࢆᚓࡓࠋࡉࡽ࡟㸪ศྲྀࡋࡓPeak 1ࢆMALDI-TOF mass spectrometer࡟࡚ ᐃࡍࡿ࡜㉁㔞ࡣ1587.6࡛࠶ࡗࡓࠋ୍᪉㸪ࣉࣟࢸ࢖ࣥࢩ࣮ࢡ࢚ࣥࢧ࣮࡟

ࡼࡿศᯒࡼࡾ㸪Nᮎ➃㓄ิ࡜ࡋ࡚Thr-Gly-Asn-Ala-GlyࡀᚓࡽࢀࡓࠋSOD1ࡢ࢔࣑ࣀ㓟㓄

ิୖ࡛Nᮎ➃ࡀThr-Gly-Asn-Ala-Glyࡢ㓄ิ࠿ࡘศᏊ㔞ࡀ1587.6࡟┦ᙜࡍࡿ࢔࣑ࣀ㓟㓄

ิࡣ ¹³⁷Thr-Gly-Asn-Ala-Gly-Ser-Arg-Leu-Ala-Cys-Gly-Val-Ile-Gly-Ile-Ala-Gln¹⁵³㸦⌮ㄽ

್1587.7㸧࡛࠶ࡗࡓࠋྠᵝ࡟ゎᯒࢆ⾜࠺࡜㸪Peak 2ࡣ¹⁰Gly-Asp-Gly-Pro-Val-Gln-Gly-Ile- Ile-Asn-Phe-Glu-Gln-Lys²³㸪Peak 3ࡣ⁹²Asp-Gly-Val-Ala-Asp-Val-Ser-Ile-Glu-Asp-Ser- Val-Ile-Ser-Leu-Ser-Gly-Asp-His-Cys-Ile-Ile-Gly-Arg-Thr-Leu-Val-Val-His-Glu-Lys¹²² ࡛

࠶ࡗࡓࠋ୍᪉࡛㸪PKᾘ໬㸦10 h㸧ᚋ㸪༢୍ࡢPeak 4ࢆᚓࡓࠋࡲࡓ㸪PKᾘ໬2 hࢆ⾜࠸㸪 Peak 5ࢆᚓࡓࠋࡇࢀࡽࡢ⤖ᯝ࡟┦ᙜࡍࡿ࢔࣑ࣀ㓟㓄ิࡢPeak 4ࡣ¹⁶Gly-Ile-Ile-Asn-Phe²⁰㸪 Peak 5ࡣ¹Ala-Thr-Lys-Ala-Val-Cys-Val-Leu-Lys-Gly-Asp-Gly-Pro-Val-Gln-Gly-Ile-Ile- Asn-Phe-Glu-Gln-Lys-Glu-Ser-Asn-Gly-Pro-Val-Lys-Val-Trp³²࡜⁸⁹Ala-Asp-Lys-Asp-Gly- Val-Ala-Asp-Val-Ser-Ile-Glu-Asp-Ser-Val-Ile-Ser-Leu-Ser-Gly-Asp-His-Cys-Ile-Ile-Gly- Arg-Thr-Leu-Val-Val-His-Glu-Lys-Ala-Asp-Asp-Leu-Gly-Lys-Gly-Gly¹³⁰ࡢ 2 ࡘࡢ࣌ࣉࢳ ࢻࡀΰྜࡋ࡚࠸ࡓࠋ௨ୖࡢゎᯒࡋࡓ⤖ᯝࢆ㸪⾲3-2࡟♧ࡋࡓࠋ

ᅗ3-2㸬Red-SOD1࢔࣑ࣟ࢖ࢻ⥺⥔ࡢ㓝⣲ᾘ໬๓ᚋࡢTEM⏬ീ

ᕥᅗࡣ㓝⣲ᾘ໬๓ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔㸪୰ኸᅗࡣAPIᾘ໬20 hᾘ໬ᚋ㸪

ྑᅗࡣPKᾘ໬10 hᾘ໬ᚋࡢTEMほᐹࢆ♧ࡍࠋࢫࢣ࣮ࣝࣂ࣮ࡣ200 nmࢆ♧ࡍࠋ

⾲3-1㸬Red-SOD1ࡢ㓝⣲ᾘ໬๓ᚋࡢ⥺⥔ᖜ

⥺⥔ᖜ* (nm) 㓝⣲ᾘ໬๓ 10.9±0.6 APIᾘ໬20 h 5.2±1.0

PKᾘ໬10 h 10.3±0.1

*⥺⥔ᖜࡣᅗ3-2ࡢTEM⏬ീࡼࡾ㸪

⥺⥔ࡢ3࠿ᡤ࡟࠾ࡅࡿᖹᆒ್±ᶆ‽೫ᕪࢆ♧ࡋ࡚࠸ࡿࠋ

ᅗ3-3㸬Red-SOD1ࡢᾘ໬ࢧࣥࣉࣝࡢHPLC⁐ฟࣃࢱ࣮ࣥ

*ࡣ㒊ศⓗ࡟ᾘ໬ࡉࢀࡓ୰㛫యࢆ♧ࡍࠋ

⾲3-2㸬Red-SOD1࢔࣑ࣟ࢖ࢻ⥺⥔ࡢ᰾㒊఩࣌ࣉࢳࢻࡢゎᯒ Peak

No.

Detected

mass number Assignment (⌮ㄽ್) APIᾘ໬ PKᾘ໬

1 1587.6 ¹³⁷TGNAGSRLACGVIGIAQ¹⁵³ (1587.7) 2 1501.6 ¹⁰GDGPVQGIINFEQK²³ (1501.5)

3 3264.4 ⁹²DGVADVSIEDSVISLSGDHCIIGRTLVVHEK¹²² (3264.6) 4 563.7 ¹⁶GIINF²⁰ (562.7)

5 3412.2 ¹ATKAVCVLKGDGPVQGIINFEQKESNGPVKVW³² (3412.8)

4292.4 ⁸⁹ADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGG¹³⁰ (4293.7)

33-3-2㸬4SCAM-SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆ⏝࠸ࡓ᰾㒊఩ྠᐃ

4SCAM-SOD1ࡼࡾᙧᡂࡋࡓ࢔࣑ࣟ࢖ࢻ⥺⥔࡟ᑐࡋ࡚㸪APIᾘ໬㸪PKᾘ໬ᚋࡢTEMほ

ᐹࢆᅗ3-4㸪ࡲࡓ㸪⥺⥔ᖜࢆ⾲3-3࡟♧ࡋࡓࠋTEMほᐹ࡟ࡼࡾ㸪Red-SOD1࡜ྠᵝ࡟⥺⥔

ᖜࡀ⣽ࡃ㸪⥺⥔㛗ࡀ▷ࡃ࡞ࡗ࡚࠾ࡾ㸪㓝⣲ᾘ໬ࡉࢀ࡚࠸ࡿࡇ࡜ࡀ☜ㄆࡉࢀࡓࠋ

ࡉࡽ࡟㸪ᾘ໬ᚋࢧࣥࣉࣝࢆ7.5 M Gdn-HCl࡛⁐ゎࡋ㸪㏫┦HPLC࡟࡚࣌ࣉࢳࢻ᩿∦ࢆᅇ

཰ࡋࡓࠋࡑࡢ⤖ᯝࢆᅗ3-5࡟♧ࡋࡓࠋAPIᾘ໬㸦20 h㸧ᚋ㸪⁐ฟ᫬㛫16-21ศࡢ㛫࡟3ࡘ

ࡢ Peak㸦Peak 6㸪7㸪8㸧ࢆᚓࡓࠋRed-SOD1 ࡜ྠᵝ࡟ࣉࣟࢸ࢖ࣥࢩ࣮ࢡ࢚ࣥࢧ࣮࡜

MALDI-TOF mass spectrometer࡟࡚ᚓࡽࢀࡓศᯒ⤖ᯝࡼࡾSOD1ࡢ࢔࣑ࣀ㓟㓄ิ࡟┦ᙜ ࡍࡿ㓄ิࡢྠᐃࢆ⾜ࡗࡓࠋࡓࡔࡋ㸪4SCAM-SOD1ࡣCysࢆ࢔ࣝ࢟ࣝ໬ࡋ࡚࠸ࡿࡓࡵ㸪Peak

6㸪8㸪10ࡣSOD1ࡢ࢔࣑ࣀ㓟㓄ิࡼࡾ⟬ฟࡉࢀࡿศᏊ㔞࡟57ࢆ௜ຍࡋࡓࠋࡇࢀࡽࡢ⤖ᯝ

࡟┦ᙜࡍࡿ࢔࣑ࣀ㓟㓄ิࡢPeak 6ࡣ¹³⁷Thr-Gly-Asn-Ala-Gly-Ser-Arg-Leu-Ala-Cys-Gly- Val-Ile-Gly-Ile-Ala-Gln¹⁵³㸪Peak 7ࡣ¹⁰Gly-Asp-Gly-Pro-Val-Gln-Gly-Ile-Ile-Asn-Phe-Glu- Gln-Lys²³㸪Peak 8ࡣ⁹²Asp-Gly-Val-Ala-Asp-Val-Ser-Ile-Glu-Asp-Ser-Val-Ile-Ser-Leu-Ser- Gly-Asp-His-Cys-Ile-Ile-Gly-Arg-Thr-Leu-Val-Val-His-Glu-Lys¹²²࡛࠶ࡗࡓࠋ୍᪉࡛㸪PK ᾘ໬㸦15 h㸧ᚋ㸪༢୍ࡢPeak 9ࢆᚓࡓࠋࡲࡓ㸪PKᾘ໬2 hࢆ⾜࠸㸪Peak 9㸪10ࢆᚓࡓࠋ

ࡇࢀࡽࡢ⤖ᯝ࡟┦ᙜࡍࡿ࢔࣑ࣀ㓟㓄ิࡢ Peak 9 ࡣ ¹⁶Gly-Ile-Ile-Asn-Phe²⁰㸪Peak 10 ࡣ

1Ala-Thr-Lys-Ala-Val-Cys-Val-Leu-Lys-Gly-Asp-Gly-Pro-Val-Gln-Gly-Ile-Ile-Asn-Phe- Glu-Gln-Lys-Glu-Ser-Asn-Gly-Pro-Val-Lys-Val-Trp³²࡜⁸⁹Ala-Asp-Lys-Asp-Gly-Val-Ala- Asp-Val-Ser-Ile-Glu-Asp-Ser-Val-Ile-Ser-Leu-Ser-Gly-Asp-His-Cys-Ile-Ile-Gly-Arg-Thr- Leu-Val-Val-His-Glu-Lys-Ala-Asp-Asp-Leu-Gly-Lys-Gly-Gly¹³⁰ࡢ2ࡘࡢ࣌ࣉࢳࢻࡀΰྜࡋ

࡚࠸ࡓࠋ௨ୖࡢゎᯒࡋࡓ⤖ᯝࢆ㸪⾲3-4࡟♧ࡋࡓࠋ

ᅗ3-4㸬4SCAM-SOD1࢔࣑ࣟ࢖ࢻ⥺⥔ࡢ㓝⣲ᾘ໬๓ᚋࡢTEM⏬ീ

ᕥᅗࡣ㓝⣲ᾘ໬๓ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔㸪୰ኸᅗࡣAPIᾘ໬20 hᾘ໬ᚋ㸪

ྑᅗࡣPKᾘ໬15 hᾘ໬ᚋࡢTEMほᐹࢆ♧ࡍࠋࢫࢣ࣮ࣝࣂ࣮ࡣ200 nmࢆ♧ࡍࠋ

⾲3-3㸬4SCAM-SOD1ࡢ㓝⣲ᾘ໬๓ᚋࡢ⥺⥔ᖜ

⥺⥔ᖜ* (nm) 㓝⣲ᾘ໬๓ 13.2±0.6 APIᾘ໬20 h 10.3±0.1 PKᾘ໬15 h 9.2±1.1

*⥺⥔ᖜࡣᅗ3-4ࡢTEM⏬ീࡼࡾ㸪

ᅗ3-5㸬4SCAM-SOD1ࡢHPLC⁐ฟࣃࢱ࣮ࣥ

*ࡣ㒊ศⓗ࡟ᾘ໬ࡉࢀࡓ୰㛫యࢆ♧ࡍࠋ

⾲3-4㸬4SCAM-SOD1࢔࣑ࣟ࢖ࢻ⥺⥔ࡢ᰾㒊఩࣌ࣉࢳࢻࡢゎᯒ Peak

No.

Detected

mass number Assignment (⌮ㄽ್) APIᾘ໬ PKᾘ໬

6 1644.8 ¹³⁷TGNAGSRLACGVIGIAQ¹⁵³ (1644.7) 7 1501.8 ¹⁰GDGPVQGIINFEQK²³ (1501.5)

8 3321.1 ⁹²DGVADVSIEDSVISLSGDHCIIGRTLVVHEK¹²² (3321.6) 9 563.2 ¹⁶GIINF²⁰ (562.7)

10 3470.2 ¹ATKAVCVLKGDGPVQGIINFEQKESNGPVKVW³² (3469.8) 4351.8 ⁸⁹ADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGG¹³⁰

(4350.7)

33-3-3㸬Red-SOD1࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᾘ໬ᚋࡢSeedຠᯝࡢホ౯

API㸪PK㓝⣲ᾘ໬ᚋࢧࣥࣉࣝࡣࡑࢀࡒࢀ␗࡞ࡿ࣌ࣉࢳࢻ᩿∦ࡀྵࡲࢀ࡚࠸ࡓࠋࡑࢀࡽ᩿

∦ࢆ࢔࣑ࣟ࢖ࢻ⥺⥔ᙧᡂ๓ࡢRed-SOD1࡟ῧຍࡋ㸪Thioflavin T assay࡟ࡼࡾSeedຠᯝࢆ

ホ౯ࡋࡓࠋ

⤖ᯝࢆᅗ3-6࡟♧ࡋࡓࠋFull length㸪APIᾘ໬ᚋ㸪PKᾘ໬ᚋࢆSeed࡜ࡋ࡚ῧຍࡍࡿࡇ

࡜࡛㸪no seed࡟ẚ࡭㸪⺯ගᙉᗘࡢቑຍࡀ᪩ࡃぢࡽࢀࡓࠋ㠃ⓑ࠸ࡇ࡜࡟㸪APIᾘ໬ࡢῧຍࡣ full length࡜㢮ఝࡋࡓ⺯ගᙉᗘࢆ♧ࡋࡓࠋ௨ୖࡢ⤖ᯝࡼࡾ㸪ᾘ໬ᚋࢧࣥࣉࣝࡣseedຠᯝࢆ

ᣢࡗ࡚࠸ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋ

ᅗ3-6㸬㓝⣲ᾘ໬ᚋRed-SOD1ࡢSeedຠᯝ☜ㄆࡢࡓࡵࡢThioflavin T assay (ە)ࡣNo seed㸪(ڦ)ࡣ1% full length seed㸪(ی)ࡣ5% full length seed㸪(ڸ)ࡣ1% API seed㸪

(ی)ࡣ5% API seed㸪(㸩)ࡣ1% PK seed㸪(™)ࡣ5% PK seedࢆ♧ࡍࠋ ᖹᆒ್ࢆࣉࣟࢵࢺࡋ㸪࢚࣮ࣛࣂ࣮ࡣ3ᅇ ᐃࡢᶆ‽೫ᕪࢆ♧ࡍࠋ

33-4 㸬⪃ᐹ

ᮏ❶࡛ࡣ㸪㔝⏕ᆺSOD1ࡢRed-SDO1㸪4SCAM-SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ࢆ⏝࠸࡚㸪᰾

㒊఩ࡢྠᐃࢆ⾜ࡗࡓࠋ

Red-SOD1㸪4SCAM-SOD1ࡢ࢔࣑ࣟ࢖ࢻ⥺⥔ࡣ3࠿ᡤࡢࣉࣟࢸ࢔࣮ࢮ⪏ᛶ㡿ᇦࡀᏑᅾࡋ ࡓࠋ3ࡘࡢ㡿ᇦࡣ SOD1ࡢ࢔࣑ࣀ㓟㓄ิ࡛ࡣ඲య࡟ᗈࡀࡗ࡚࠾ࡾ㸪Nᮎ➃㡿ᇦ㸪ࢭࣥࢺࣛ

ࣝ㡿ᇦ㸪Cᮎ➃㡿ᇦ࡟Ꮡᅾࡋ࡚࠸ࡓ㸦ᅗ3-7㸧ࠋ≉࡟㸪Cᮎ➃㡿ᇦ࡟ࡣCys57࡜ศᏊෆࢪࢫ

ࣝࣇ࢕ࢻ⤖ྜࢆᙧᡂࡋ࡚࠸ࡿCys146ࡀᏑᅾࡋ࡚࠸ࡓࡓࡵ㸪ศᏊෆࢪࢫࣝࣇ࢕ࢻ⤖ྜࡀ㑏ඖ ࡉࢀࡿࡇ࡜࡛Cᮎ➃㡿ᇦࡣᦂࡽࡂ㸪⥺⥔ᙧᡂ࡟స⏝ࡋ࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋࡲࡓ㸪Waltz

࡟ࡼࡾண ࡉࢀࡿ᰾㒊఩㡿ᇦࡣ¹⁴Val-Gln-Gly-Ile-Ile-Asn-Phe-Glu-Gln²²㸪⁹⁵Ala-Asp-Val- Ser-Ile-Glu-Asp-Ser-Val-Ile-Ser-Leu-Ser-Gly-Asp-His-Cys-Ile-Ile-Gly¹¹⁴㸪¹⁴⁵Ala-Cys-Gly- Val-Ile-Gly-Ile-Ala-Gln¹⁵³࡛࠶ࡗࡓࠋࡇࢀࡽࡢ㡿ᇦࡣࣉࣟࢸ࢔࣮ࢮ⪏ᛶ㡿ᇦ࡜㔜࡞ࡗࡓࠋࡲ

ࡓ㸪㠃ⓑ࠸ࡇ࡜࡟㸪4SCAM-SOD1 ࡢ⥺⥔᰾ࡣ Red-SOD1 ࡜᏶඲࡟୍⮴ࡋࡓ㸦ᅗ 3-7㸧ࠋ

4SCAM-SOD1ࡣ࢔ࣝ࢟ࣝ໬ࡢᙳ㡪ࢆཷࡅ㸪᏶඲࡟ኚᛶࡋ࡚࠾ࡾ࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂࡀ᪩

࠿ࡗࡓ㸦ᅗ2-2㸪ᅗ2-4㸧ࠋ୍᪉࡛㸪Red-SOD1ࡢᵓ㐀ࡣ୙Ᏻᐃ࡛࠶ࡿࡀ㸪஧ḟᵓ㐀ࢆ࡜ࡾ㸪

࢔࣑ࣟ࢖ࢻ⥺⥔ࡢᙧᡂࡣ 4SCAM-SOD1 ࡟ẚ࡭㐜࠿ࡗࡓࠋ௒ᅇࡢ⤖ᯝࡼࡾ㸪୧⪅ࡢ᰾㒊఩

㡿ᇦࡣ㔜࡞ࡗࡓࡓࡵ㸪ྠࡌ㡿ᇦࡀ┦஫స⏝ࡋ᰾ࢆᙧᡂࡍࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋRed-SOD1

࡜4SCAM-SOD1ࡢ᰾ᙧᡂ᫬㛫ࡢᕪࡣRed-SOD1ࡢᵓ㐀ࡀᦂࡽࡂ㸪unfoldࡋ㸪᰾㒊఩ࡀ㟢 ฟࡍࡿࡲ࡛࡟࠿࠿ࡗࡓ᫬㛫࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋࡘࡲࡾ㸪SOD1 ࡣ୍᪦㸪unfold ࡍࡿࡇ࡜

࡛᰾㒊఩ࡀ㟢ฟࡋ㸪᰾㒊఩ࡀ┦஫స⏝ࡋ㸪࢔࣑ࣟ࢖ࢻ⥺⥔ࢆᙧᡂࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗ ࡓࠋࡲࡓ㸪㓝⣲ᾘ໬ᚋࢧࣥࣉࣝࡣseedຠᯝࢆ♧ࡋࡓ㸦ᅗ3-6㸧ࠋ≉࡟㸪API seed࡟ࡘ࠸࡚

ࡣfull length seed࡜㢮ఝࡋࡓ⺯ගᙉᗘࡢቑຍࡀぢࡽࢀࡓࠋAPI seedࡣ20 hᾘ໬ᚋࢧࣥࣉ

ࣝࢆ౑⏝ࡋࡓࠋྵ᭷࣌ࣉࢳࢻࡣ¹⁰Gly-Asp-Gly-Pro-Val-Gln-Gly-Ile-Ile-Asn-Phe-Glu-Gln- Lys²³㸪⁹²Asp-Gly-Val-Ala-Asp-Val-Ser-Ile-Glu-Asp-Ser-Val-Ile-Ser-Leu-Ser-Gly-Asp-His-