[J.Inst.Enol.Vltic.Yamanas
hiUniv.2321‑271988]

資料

Lipomyce
sstarkeyiの酵素 活性測定 の ため の反応液組成 (その 2)‑

トリカルボン酸 回路 とその周辺の酵素‑

長 沼 孝 文 ･伊 藤

功 ･横 山等 ･辻 井 栄 作

竹 下 昌 利 ･武 井 雅 也 ･兎 束 保 之PracticalAssay Procedure
sofEnzyme
son TricarboxylicAcid Cycleand Related Pat
hwaysin Liz)
omycesstarkeyi
TAKAFUMINAGANUMA,IsAO ITO,H汀OSHIYoKOYAMA,EISAKU TsUzlI,M ASATOSHITAKESHI
TA,M ASAYA TAKEIa｡
d YASUYUKI UzUKA

DepartクWntOfFer
ment
ation Technology,Facultyo
f Enginem'ng,YanwnashiUniue
7
Sity.Kofu400

生化学的見地か ら微 生物 の代謝 活性 を調べ る場合,酵素 活性 を 測定す る方法が 用 い られ る こ とが 多い.
しか し,一 つの代謝経路上 の幾つかの酵素 の活性 測
定 に必要 な反応液組成 を知 るだけで も, 多 くの文献調査 が要求 され る. これ らの 煩 わ し さ を解 決 す るには, 各種 酵素 の反応液組成 をま とめ た資料が あ る
こ とが望 ま しい. この よ うな観点か ら,我 々は前報(1)において Glycolyt
ic pathway, Pentose
phos‑e,Glyce
ro13‑
phosphatepathway
上のphatecycl
13
種類 の酵素 の反応液組成 を示 した.
本報 では, 引 き続 いて Trl
Carboxyl
i
c aci
dc
y‑
cle,Am onl
um assi
mil
at
i
onpat
hway,Anapl
e‑
rot
i
cre
acti
on pathway(2), Fatty‑acid blOSyn‑t
hesi
spat
hway上 の 17
種 類の酵素 について記載 した.
ただ し,前報 (1)において も述べ たが,ここに記述 した反応液組成は, 各種 文献 に記載 されてい る組成 を
基 に油脂酵母 LIPomycesstarkeyiI
AM4753の粗酵素溶液用に改変 した もの であ る.従 って,研 究対 象 とな る微 生物 の種類 が異 なる場合 には,試薬や粗 酵素液の濃度 ,pH, 活性 化 剤 等 に つ い て再検討 を行 う
必要が あ る と考 え られ る.本報 で使用 した細胞か ら
酵素 を取 り出す方法(3),活性 測定 の条件,試薬 (アル ドリッチ フ ァ イ ン ケ ミアルの製品 を追加 )等
は前報 (1)に準 じた.なお,前報 (1)には記述 しなか ったが, 反応液に用 いる試薬 溶液の 多 くは, p
Hを緩衝 液の p
H付 近 に調 牡 す る とか,使用直前 まで冷却あるいは調製後短時間内に使 用す るな どの注意が心要 であ る｡

22
Table 1. Composition of assay mixtures forenzymes on the tricarboxylic acidcycle.
Common abbreviations used in' alltables are as follows : Triehanolamine, triethanolamine hydrochlorideNaOH buffer ; Tris, Tris(hydroxymethyl) aminomethane hydrochloride-HCI buffer; R., reference; B.,
blank ; S., sample.

1-1. Citrate synthase (EC 4.1. 3. 7)(4)
Pipette into cuvette (ml)

0.1 M Triethanolamine(pH8.2)
5 mM Oxalacetic acid·1 ruM Acetyl coenzyme A
2 mM DTNBb
Distilled water
1/4 Crude extract
Final volume

R.

B.

S.

2.1

2.1

0.3
0.6
0.01
3.01

0.3
0.3
0.3
0.01
3.01

2.1
0.3
0.3
0.3
0.01
3.01

Wavelength, 412nm ; <:412=13.60 [cm 2/pmole]·Solution prepared should be used soon.
b5,5'- Dithiobis- (2-nitrobenzoic acid)

1-2. Aconitate hydratase (EC 4.2.1. 3)(5)
Pipette into cuvette (ml)B.

S.

0.1 M Tris(pH8.2)
2.7
50 mM DL-isocitric acid, trisodium salt
Distilled water
0.3
Crude extract
0.05
Final volume
3.05

2.7
0.3

R.

1-3. Isocitrate dehydrogenase(NAD) (EC 1. 1. 1.
41)(6)

Pipette into cuvette (ml)R.

B.

0.1 M Tris(pH7.6)
2.5 2.5
50 mM DL-isocitric acid, trisodium salt
0.2 M MgCIz ·6H20
0.1 0.1
0.2 M Citric acid, monohydrate 0.03 0.03
10 mM AMp·
0.3 0.3
10 mM NADb
0.05
Distilled water
0.11 0.06
Crude extract
0.05 0.05
Final volume
3.09 3.09

S.
2.5
0.06
0.1
0.03
0.3
0.05
0.05
3.09

Wavelength, 340nm ; <:340=6.22 [cm 2/pmole]•Adenosine-5' -monophosphate. disodium salt
bl3-nicotinamide -adenine dinucleotide, oxidized form
1-4. Isocitr ate dehydrogenase (NADP) (EC 1.
1.1.42)(7)
Pipette into cuvette (ml)
R.

B.

S.

0.1 M Tris(pH8.0)
2.7 2.7 2.7
0.02
50 mM DL-isocitric acid, trisodium salt
0.1
0.2 M MgCIz ·6H 20
0.1 0.1
10 mM NADp·
0.05 0.05
Distilled water
0.07 0.02
Crude extract
0.02 0.02 0.02
Final volume
2.89 2.89 2.89
Wavelength, 340nm ; <:340=6.22 [cm 2 /pmole]·Nicotinamide -adenine dinucleotide phosphate, oxidized form

0.05
3.05
1-5. 2-0xoglutarate dehydrogenase comlex(8.9)

Wavelength, 240nm ; <:240=4.88 [cm2/pmole]
Pipette into cuvette (m!)

0.5 M Tris(pH7.6)
2 mM Coenzyme A
40 mM 2-Ketoglutaric acid

R.

B.

S.

1.2

1.2

1.2
0.3
0.3

0.3

-2332 mM MgS04 ·7H2078 mM L-cysteine
2 mM Tppb
30 mM NADc
Distilled water
Crude extract
Final volume0.15 0.15
0.1
0.1
0.15 0.15
0.3
1.35 0.75
0.05 0.05
3.0 3.0

0.150.130.15
0.3
0.45
0.05
3.0

Wavelength, 340nm ; 10340 =6.22 [cm 2/ ,umole]'L-cysteine monohydrochloride, momohydrateb5, 10, 15, 20- Tetraphenyl-21l!, 23H-porphine
cp-nicotinamide -adenine dinucleotide, oxidized form
1-6, Succinyl-CoA hydrolase (EC 3.1. 2. 3)00)

1-8. Fumarate hydratase (EC 4.2. 1. 2)02)
Pipette into cuvette (m!)
R.

B.

0.5 M Tris(pH8.2)
1.2
0.5 M L-malic acid, sodium salt
Distilled water
1.75
Crude extract
0.05
Final volume
3.0

S.
1.2
0.3
1.450.05
3.0

Wavelength, 240nm ; 10240 = 2.44 [cm 2/ ,umole]

1-9. Malate dehydrogenase(NAD) (EC 1.1.1.37)
03.14)

Pipette into cuvette (ml)
R.

B.

S.

1.2
1.0
0.3
0.5

1.2
1.0
0.3
0.48
0.02
3.0

Pipette into cuvette (mI)R.

0.2 M Bicine·KOH buffer(pH8.0)1.2
3 mM Succinyl coenzyme A
2 mM DTNB'
0.3
Distilled water
1.48
Crude extract
0.02
Final volume
3.0

3.0

Wavelength, 412nm ; 10412 = 13.60 [cm 2/ ,umole]'5,5'·Dithiobis -(2-nitrobenzoic acid)1-7. Succinate dehydrogenase (EC 1. 3. 99.1)01)

0.1 M triethanolamine(pH7.6)
5 mM Oxalacetic acid'
10 mM NADHb
Distilled water
Crude extract
Final volume

2.8
0.05
0.33
0.01
3.16

B.

S.
2.8
0.3
0.05
0.01
3.16

Wavelength, 340nm ; 10340 = 6.22 [cm2 / ,umole]'Solution prepared should be used soon. bp-nicotinamide -adenine dinucleotide,
reduced form

Pipette into cuvette (ml)R.

0.25 M Tris(pH8.0)
1.2
20 mM Succinic acid, sodium salt
2.5 mM MTT'
0.3
Distilled water
1.45
Crude extract
0.05
40 mM Phenazine methosulfate bFinal volume
3.0

B.

S.

1.2

1.2
0.3
0.3 0.3
1.15 0.85
0.05 0.05
0.3 0.3
3.0 3.0

Wavelength, 578nm;
10·578 =approx. 13.0 [cm 2/ ,umole]'3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H-tetrazolium bi-omidebReaction start by addition of the reagent.

Table 2.

Composition of assay mixtures for
enzymes on the ammonium assimilation pathway.
Common abbreviations used in all
tables are as follows: Imidazole,imidazole- HCI buffer ; otherabbreviations are the same as table 1.

a24

2-1. Glutamate dehydrogenase (NAD, NADP)(EC 1. 4. 1. 2, 1. 4. 1. 4)(15· 16)
Pipette into cuvette (m!)
R.

B.

2.4

2.4

S.

2-3. Glutamine synthetase (EC 6.3. 1. 2)(ZO)(The enzyme is assayed with two-stepsreaction)I) 1st reaction
Pipette into cuvette (ml)

0.1 M Tris(pH8.0)
0.1 M 2-Ketoglutaric acid
3.2 M NH 4Cl
10 roM NADHa(or NADPHb)
Distilled water
Crude extract
Final volume

2.4
0.4
0.2 0.2 0.2
0.03 0.03
0.43 0.4
0.02 0.02 0.02
3.05 3.05 3.05

Wavelengt?, 340nm ; E340 = 6.22 [cm Z I tLmole]a{3-nicotinamide -adenine dinucleotide, reducedform
bNicotinamide -adenine dinucleotide, phosphate, reduced form

2-2. Glutamate synthase(NADP, NADP) (EC1.4.1.13, 1.4.1.14)(17,18,19)
Pipette into cuvette (mI)R.

0.1 M Tris(pH8.0)
0.1 M 2-Ketoglutaric acid
0.17 M L-glutamine
10 roM NADHa(or NADPHb)
Distilled water
Crude extract
Final volume

B.

S.

2.57
0.15
0.2 0.2 0.2
0.03 0.03
0.18 0.15
0.05 0.05 0.05
3.0 3.0 3.0R.

0.5 M Imidazole(pH7.2)
0.5 M L-glutamic acid
1 M MgS0 4 '7H zO
1 M L-cysteinea
50 mM ATpb
1 M Hydroxylamine hydrochloride<2.25
Distilled water
Crude extract
2.25
Final volume

B.

S.

0.5

0.5
0.5
0.1 0.1
0.1
0.1
0.5 0.5
0.1
0.1
0.9 0.4
0.05 0.05
2.25 2.25

aL-cysteine monohydrochloride, momohydratebAdenosine-5'-triphosphate,
disodium salt
<5 mI of a stock soluti on of 2 M ]1.ydroxylamine hydrochloride is neutralized to pH
volume7.2 with 2N NaOH and theadjusted to 10ml with distilled water.
. Neutral solution should be prepared freshly
before use.

2.57 2.57

Wavelength, 340nm ; E340 = 6.22 [cm Z I tLmole]
aj3-nicotinamide - adenine dinucleotide, reducedbNicotinamide - adenine dinucleotide phosphate, reduced formII) 2nd reaction
After 1st reaction for 20 min, 0.75 ml offollowing FeCb soluti on is added into 1streaction mixture.
FeCb solution:
10 % FeCb' 6H zO in 0.2 N HC1
0.25m!
24 % Trichloroacetic acid
0.25m!
50 % (v Iv) HCl0.25m!
After mixing 1st reaction mixture and FeCb
solution, it is refrigerated immediately andsubsequently is centrifuged at 2,500g for 5 min.
Enzyme activity is determined with adsorbanceat 540 nm. L-glutamic acid y-monohydroxamate (0-2 tLmole)is used for the calibration
curve. One unit of enzyme is defined as thatamount which produces 1.15 micromoles of Lglutamic acid y-monohydroxamate(ZO).

25Table 3.

Composition of assay mixtures for
enzymes on the anaplerotic reaction
pathway.
Common abbreviations are the sameas table 1 and 2.

b5, 5' -Dithiobis- (2-nitrobenzoic acid)3-3. Phosphoenolpyruvate carboxykinase (ATP)' (EC 4. 1. 1. 49)(23.24)
Pipette into cuvette (ml)
R.

B.

S.

1.2

1.2

1.2
0.12
0.1
0.1
0.2
0.03
0.3
0.3
0.62
0.03
3.0

3-1. Pyruvate carboxylase (EC 6.4.1. 1)(4.21)
Pipette into cuvette (ml)R.

B.

0.25 M Imidazole(pH7.2)
1.2 1.2
0.3 M Pyruvic acid, sodium salt
0.3 M NaHC03
0.16 0.16
0.1 M MgS0 4.7H20
0.15 0.15
0.06 0.06
50 mM ATP'2.2 IU Iml Citrate synthase
0.03 0.03
1 mM Acetyl coenzyme A
0.3
2 mM DTNBb
0.3
0.3
Distilled water
1.07 0.77
Crude extract
0.03 0.03
3.03.0
Final volume

S.
1.2
0.2
0.16
0.15
0.06
0.03
0.3
0.3
0.57
0.03
3.0

Wavelength, 412nm ; e4l2 = 13.60 [cm 2I j1mole]'Adenosine-5'-triphosphate, disodium salt
b5,5' -Dithiobis- (2-nitrobenzoic acid)
3-2. Phosphoenolpyruvate carboxylase(EC 4.1.
1.31)(22)0.25 M Tris(pH7.4)
0.1 M PEPb
0.3 M NaHC03
0.1 M MgS0 4 ·7H2060 mM ADpc
2.2 IU Iml Citrate synthase
1 mM Acetyl coenzyme A
2 mM DTNBb
Distilled water
Crude extract
Final volume

0.1
0.1
0.2
0.03

0.1
0.1
0.2
0.03
0.3
0.3 0.3
1.04 0.74
0.03 0.03
3.0 3.0

Wavelength, 412nm ; e4l2 =13.60 [cm2I j1mole]'True activity of this enzyme(PEPCK) is
calculated by following equation.
True value of PEPCK activity =(PEPCK activity) - activity ofphosphoenolpyruvate carboxylase)bphosphoenolpyruvic acid, monopotassiumsaltcAdenosine-5' -diphosphate, disodium salt
d5,5' -Dithiobis-(2-nitrobenzoic acid)

Pipette into cuvette (ml)0.25 M Tris(pH7.4)
0.1 M PEP'
0.3 M NaHC03
0.1 M MgS04 ·7HzO
2.2 IU/ml Citrate synthase
1 mM Acetyl coenzyme A
2 mM DTNBb
Distilled water
Crude extract
Final volume

R.

B.

S.

1.2

1.2

0.1
0.1
0.03

0.1
0.1
0.03
0.3
0.3
0.94
0.03
3.0

1.2
0.12
0.1
0.1
0.03
0.3
0.3
0.82
0.03
3.0

0.3
1.24
0.03
3.0

Wavelength, 412nm ; e4l2 = 13.60 [cm ZI j1mole]'Phosphoenolpyruvic acid, monopotassium
salt

3-4. Malic enzyme (EC 1. 1. 1. 40)(25)
Pipette into cuvette (m])

0.4 M Triethanolamine(pH7.6)
60 mM L-malic acid0.12 M MnC12 ·6HzO
50 mM NADP'Distilled water
Crude extract
Final volume

R.

B.

S.

1.2

1.2

0.1

0.1
0.06
2.06
0.03
3.0

1.2
0.25
0.1
0.06
1.82
0.03
3.0

2.12
0.03
3.0

Wavelength, 340nm ; .0340 =6.22 [cm ZI j1mole]'Nicotinamide -adenine dinucleotide phosphate, oxidized formE26

Table 4.

Composition of assay mixture forenzyme on the fatty-acid biosynthesis pathway.
Common abbreviations are the sameas table 1 and 2.

4-1. ATP citrate lyase (EC 4.1. 3. 8)(26)
Pipette into cuvette (ml)R.

0.5 M Triethanolamine(pH8.2) 1.7
0.2 M Citric acid, monohydrate 0.3
0.15
0.2 M MnCh ·6H2O
0.2 M 2·Mercaptoethanol
0.15
50 mM ATP'
10 mM NADHb
0.05- 20 IU Iml MDHc
0.02
Distilled water
0.6
Crude extract
0.05
2 mM Coenzyme b
3.02
Final volumeB.
1.7
0.30.150.15
Q.3
0.05
0_02
0.3
0.05

S.
1.7

0.3
0.15
0.15
0.3
0.05
0.02

0.05
0.3
3.02 3.02

Wavelength, 340nm ; E340 = 6.22 [cm 2 1tLmole]•Adenosine-5' -triphosphate, disodium saltb,B-nicotinamide-adenine dinucleleotide, redureduced form
cMalate dehydrogenase(NAD)dReaction start by addition of the regent.

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17) '£1t¥li¥~,¥[I'i](.£ft'ffi, Li.JJII~{I~) p.379,

A, ** (1984).

**1t18) Stewart, G. R, Mann, A. F., Fentem, P.
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22) H.I0, ~fJ: *!1~MU: f"O)iIJl]~, (T~, H.Iq"*#-{£i!!fl . *,jjji~), (~B'ffi[ ttl!\!Z M* l1.fi;aif:li!lflJ), Vol. 22, No.14, p.260, :!t:ll.I±\!t&,(1977).
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Chem., 235, 9 (1960).

**~) T~:.~mtf"O)~~,(T~,H.Iq,,*#-{£

M:*i!!fl . *,jjji~),~B'ffi[ ttl!l!Z
l1.fi;aif:li!lflJ),
Vol. 22, No.14, p.249, :;Jt:ll.I±\!t&,(1977).
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**