1,5 – ANHYDROGLUCITOL – 6 – PHOSPHATE DEHYDROGENASE [AG6PDHⅡ]
from Escherichia coli
1,5 – Anhydroglucitol – 6 – phosphate + NADP+ C6H11O8P+NADPH + H+
Preparation and Specification
- Appearance
- : White amorphous powder, lyophilized
- Specific activity
- : More than 20 U/mg solid
Properties
- Substrate specificity
- : See Table 1
- Molecular weight
- : 78 kDa (TSK gel G 3000 SWXL gel filtration) 40 kDa (SDS–PAGE)
- Isoelectric point
- : pH 4.7
- Michaelis constants
- : 1,5–Anhydroglucitol–6–phosphate 25 mM (pH 10.0) NADP 0.09 mM (pH 10.0)
- Optimum pH
- : 9.0–10.0Figure 1
- pH stability
- : 7.0–9.0 (50℃, 30 min) Figure 2
- Optimum temperature
- : 37–50℃Figure 3
- Thermal stability
- : Stable at 42℃ and belowFigure 4
Applications for Diagnostic Test
This enzyme is useful for enzymatic determination of 1,5–AG.
| ADP-HKT Ⅱ | ||
| ADP + 1, 5-AG | → | AMP + 1, 5-AG-6-P |
| AG6PDH Ⅱ | ||
| 1, 5-AG-6-P + NADP+ | → | C6H11O8P + NADPH + H+ |
Table 1. Substrate specificity
| Substrate (5mM) | Relative activity (%) |
|---|---|
| 1,5–Anhydroglucitol–6–phosphate | 100 |
| Glucose–6–phosphate | 8 |
| Fructose–6–phosphate | 0 |
| Galactose–6–phosphate | 0 |
| Mannose–6–phosphate | 0 |
| Sorbitol–6–phosphate | 0 |
| Glucose–1–phosphate | 0 |
| Glucose–1, 6–diphosphate | 0 |
| 1,5–Anhydroglucitol | 0 |
| Glucose | 0 |
| Sorbitol | 0 |
| myo–Inositol | 0 |
Table 2. Effect of various chemicals on AG6PDH activity
| Additives | Concentration | Relative activity (%) |
|---|---|---|
| None | – | 100 |
| KCl | 100mM | 142 |
| NaCl | 250mM | 113 |
| CaCl2 | 1mM | 114 |
| MgCl2 | 1mM | 118 |
| MnCl2 | 1mM | 138 |
| NH4Cl | 1mM | 97 |
| MgSO4 | 1mM | 113 |
| EDTA | 1mM | 111 |
| Triton X–100 | 0.1% | 99 |
| Sodium Deoxycholate | 0.01% | 107 |
Fig.1 pH Optimum
50 mM buffer
□: Tris buffer
■: CAPS buffer
□: Tris buffer
■: CAPS buffer
Fig.2 pH Stability
50℃, 30 min.
50 mM buffer
〇: Citrate buffer
●: Bis Tris buffer
□: Tris buffer
■: CAPS buffer
50 mM buffer
〇: Citrate buffer
●: Bis Tris buffer
□: Tris buffer
■: CAPS buffer
Fig.3 Optimum Temperature
pH 9.5
50 mM CAPS buffer
50 mM CAPS buffer
Fig.4 Thermal Stability
pH 9.5, 30min.
50 mM CAPS buffer
50 mM CAPS buffer
Assay
Principle
-
The assay is based on the increase in absorbance at 340 nm as the formation of NADPH proceeds in the following reactions:
| ADP–HKT Ⅱ | |||
| 1,5–A+ADP | → | 1,5–AG–6–P+AMP | |
| Mg2+ | |||
| ＊ 1,5–AG: 1,5–Anhydro–D–Glucitol | |||
| AG6PDH Ⅱ | |||
| 1,5–AG–6–P+NADP + | → | C6H11O8P+NADPH+H + | |
| DIP | |||
| NADPH+H ++NTB | → | NADP+NTBH2 | |
Unit definition
-
One unit is defined as the amount of enzyme which converts 1 μmole of 1,5–AG–6–phosphate to C6H11O8P per minute at 37 ℃ under the conditions specified in the assay procedure.
Reagents
- Reaction mixture–Ⅰ
0.2M Tris–HCl buffer pH7.5 0.10 ml 0.4M ADP solution (pH7.5) 0.05 ml 0.4M MgCl2 solution 0.05 ml 200U/ml ADP–HKT Ⅱ solution 0.10 ml Distilled water 0.15 ml
- Substrate solution (400mM 1,5–AG Solution)
Dissolve 65.6mg of 1,5–AG with 1ml of distilled water. - Reaction mixture–Ⅱ
0.2M EDTA・2Na (pH10.0) solution 0.05 ml 1.0M Glycine–NaOH buffer pH10.0 0.10 ml 2.0% Triton X–100 solution 0.05 ml 0.5% NTB solution 0.05 ml 100U/ml DIP solution 0.05 ml 20mM NADP solution 0.10 ml Distilled water 0.10 ml - Enzyme dilution buffer
10mM Tris–HCl Buffer pH9.0 (25℃) - Reaction Stopper
0.1N HCl - Reagents
Tris (hydroxymethyl) aminomethane:Sigma Chemical Co. #T–1503ADP (Adenosine diphosphate・2Na) : Oriental Yeast Co.Ltd.
ADP–HKT Ⅱ: Asahi Kasei Pharma Corporation #T–93
1,5–Anhydro–D–Sorbitol ( 1,5–Anhydro–D–Glucitol)
FUJIFILM Wako Pure Chemical CorporationEDTA・2Na: KISHIDA CHEMICAL Co., Ltd.
#012–13533
#060–29133Glycine: FUJIFILM Wako Pure Chemical Corporation
#077–00735Triton X–100: The Dow Chemical Company
NTB (Nitrotetrazorium blue) : Dohjindo Laboratories
#344–02033
- DIP (Diaphorase) : Asahi Kasei Pharma Corporation
#T–10NADP (Nicotinamide adenine dinucleotide phosphate, oxidized form) :
FUJIFILM Wako Pure Chemical Corporation#308–50463
Enzyme solution
-
Accurately weigh about 10 mg of the sample and add enzyme dilution buffer to make a total of 10 ml.
Dilute it with enzyme dilution buffer to adjust the concentration as required.
Procedure
- Pipette accurately 0.45 ml of reaction mixture–Ⅰ into a small test tube.
- Add 0.05 ml of substrate solution.
※ In the case of a test blank, add 0.05 ml of distilled water in place of substrate solution. ※ above the mixed solution keep on ice until use. - Above the mixed solution incubate at 37 ℃ and after 15 min. add acculately 0.50 ml of reaction mixture–Ⅱ at 37 ℃.
- After 5 min. add accurately 0.05 ml of enzyme solution and mix to start the reaction at 37 ℃.
- At 10 min. after starting the reaction, add 2.0 ml of the reaction stopper to stop the reaction.
- Measure the absorbance at 550 nm.
△ A = (As−Ab) = 0.2–0.5 Abs.Absorbance sample : As blank : Ab
Calculation
-
Activity (U/mg of powder) = △ A/17.0 × 3.05/0.05 × 1/x
17.0 : millimolar extinction coefficient of NTB at 550nm ( cm2 /μmol)3.05 : final volume (ml) 0.05 : volume of enzyme solution (ml) 10 : reaction time (min) X : concentration of the sample in enzyme solution (mg/ml)
Storage
-
Storage at −80 ℃ in the presence of a desiccant is recommended.
AG6PDH Ⅱ活性測定法 (Japanese)
試薬液
- 反応試薬混合液−Ⅰ
0.2M トリス−HCl 緩衝液pH7.5
0.10 ml 0.4M ADP 溶液 (pH7.5) 0.05 ml 0.4M 塩化マグネシウム溶液 0.05 ml 200U/ml ADP–HKT Ⅱ溶液 0.10 ml 精製水 0.15 ml - 基質溶液 (400mM 1,5–AG 溶液)
1,5–AG 65.6mg を精製水1.0ml で溶解。 - 反応試薬混合液−Ⅱ
0.2M EDTA・2Na (pH10.0) 溶液
0.05 ml 1.0M Glycine–NaOH 緩衝液pH10.0 0.10 ml 2.0% トリトンX–100 溶液 0.05 ml 0.5% NTB 溶液 0.05 ml 100U/ml DIP 溶液 0.05 ml 20mM NADP 溶液 0.10 ml 精製水 0.10 ml - 酵素溶解希釈用液
10mM トリス−HCl 緩衝液pH9.0 (25℃) - 反応停止液
0.1N HCl - 試薬
トリス (ヒドロキシメチル) アミノメタン：シグマ製 #T–1503ADP (アデノシンニリン酸・2Na) ：オリエンタル酵母製
- ADP–HKT Ⅱ：旭化成ファーマ製 #T–93
1,5–Anhydro–D–Sorbitol
(1,5–Anhydro–D–Glucitol と同) ：富士フイルム和光純薬製 #012–13533EDTA・2Na (エチレンジアミン四酢酸ニナトリウム)：キシダ化学製 #060–29133Glycine (グリシン) ：富士フイルム和光純薬製 特級 #077–00735トリトンX–100：Dow Chemical 製
NTB (ニトロテトラゾリウムブルー) ：同仁化学工業製 #344–02033DIP (ジアフォラーゼ) ：旭化成ファーマ製 #T–10
NADP (ニコチンアミドアデニンジヌクレオチド・
リン酸酸化型) ：富士フイルム和光純薬製 #308–50463
酵素試料液
- 検品約10mg を精密に量り、酵素溶解希釈用液で溶解して全容10.0ml とする。
その液を酵素溶解希釈用液で適宜希釈する。
測定操作法
- 小試験管に反応試薬混合液–Ⅰを0.45ml を正確に分注する。
- 1,5–AG 基質液を0.05ml を正確に加えて混和する。
※ 盲検は1.5–AG 基質液の代わりに精製水0.05ml を加える。 ※ 上記混合液は使用するまで氷中に保存する。
- 次に37℃で第一反応を開始し、15 分後反応試薬混合液−Ⅱを0.5ml ずつ正確に加えて混和し、更に37℃で5 分間放置する。
- 5 分経過後、酵素試料液0.05ml を正確に加えて混和し、37℃で反応を開始する。
- 10 分間酵素反応を行った後、反応停止液 (0.1N HCl)
2.0ml を加えて混和し、反応を停止する。
反応を停止後、550nm における吸光度を測定する。
求められた吸光度変化を
試料液についてはAs
盲検液についてはAb とする。
※吸光度範囲：△ A = (As−Ab) ; 0.2～0.5Absの範囲とする。
計算
以下の計算式に従い、活性 (U/mg) を計算する。活性 (U/mg) = △ A/17.0 × 3.05/0.05 × 1/10 × 1/x
| 17 : | NTB の550nm におけるミリモル分子吸光係数
(cm2 / μmol) |
| 3.05 : | 反応総液量 (ml) |
| 0.05 : | 反応に供した酵素試料液量 (ml) |
| 10 : | 酵素反応時間 (min) |
| X : | 酵素試料液中の検品濃度 (mg/ml) |
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