臨床検査において、α-アミラーゼおよび無機リン酸の測定に利用されます。
| 由来 | recombinant E. coli |
|---|---|
| 系統名 | Maltose : orthophosphate 1-β-D-glucosyltransferase |
| EC 番号 | 2.4.1.8 |
| 反応式 | Maltose + Orthophosphate →→→ D-Glucose + β-D-Glucose 1-phosphate |
Maltose Phosphorylase (MPL-EP) 60233 マルトースホスホリラーゼ(MPL-EP)
- 臨床診断用酵素
仕様
| Appearance | white lyophilizate | |
|---|---|---|
| Activity | ≧10 U/mg | |
| Stabilizer | lactose, ethylenediaminetetraacetic acid (EDTA) | |
| Storage condition | below -20℃ |
特性
| Molecular weight |
ca. 220 kDa (gel filtration) |
|---|---|
| Structure | 2 subunits of 90 kDa (SDS-PAGE) |
| Michaelis constant |
1.9×10-3M (phosphate) |
|
3.4×10-3 M (phosphate) |
|
|
8.3×10-3M (arsenate) |
|
| pH Optimum | 6.5–7.5 |
| pH Stability | 5.5–8.0 |
| Optimum temperature | 45–50°C |
| Thermal stability | below 55℃ |
| Stability (liquid form) | stable at 37℃ for at least one week |
| Stability (powder form) | stable at 30℃ for at least four weeks |
| Inhibitors |
Hg2+，Ag+，Zn2+，Cu2+ |
アプリケーション
The enzyme is useful for the determination of α-amylase and inorganic phosphate in clinical analysis.
分析手順
Principle
The appearance of d-glucose is measured spectrophotometrically at 505 nm.
Definition of unit
One unit (U) is defined as the amount of enzyme which produces 1 μmol of d-glucose per min at 30℃ and pH 7.0 under the conditions described below.
Reagents
- HEPES–NaOH buffer, 50 mM; pH 7.0: dissolve 2.38 g of HEPES in 160 ml of distilled water, adjust to pH 7.0 with 1 N NaOH and dilute with distilled water to 200 ml.
- Maltose solution, 0.2 M: 3.60 g of maltose monohydrate/50 ml of HEPES–NaOH buffer (Reagent A).
- Phosphate solution, 0.2 M: dissolve 1.36 g of KH2PO4 in 40 ml of HEPES–NaOH buffer (Reagent A), adjust to pH 7.0 with 4 N NaOH and dilute with Reagent A to 50 ml.
- HCl solution, 5 N: 43 ml of concentrated HCl/100 ml of distilled water.
- pH Adjusting solution (NaOH solution, 1 N): 4.0 g of NaOH/100 ml of distilled water.
- Glucose assay kit: “GLUCOSE C II-TESTWAKO” (Wako Pure Chemical), or a similar glucose assay kit.
Sample: dissolve the lyophilized enzyme to a volume activity of 0.15–0.55 U/ml with ice-cold HEPES–NaOH buffer (Reagent A) immediately before measurement.
Procedure
- Pipette the following reagents into a test tube.
0.2 ml HEPES–NaOH buffer (Reagent A) 0.1 ml Maltose solution (Reagent B) 0.1 ml Phosphate solution (Reagent C) - Equilibrate at 30℃ for about 5 min.
- Add 0.1 ml of sample and incubate for 10 min at 30℃ [test].
- The blank solution is prepared by adding HEPES–NaOH buffer (Reagent A) instead of sample [blank].
- Add 0.1 ml of HCl solution (Reagent D) to stop the reaction.
- Add 0.5 ml of pH adjusting solution (Reagent E).
- Pipette 0.3 ml of the test and blank mixture into respective test tubes.
- Add 3.0 ml of glucose assay kit (Reagent D) and incubate for about 5 min at 37℃.
- Read the absorbance at 505 nm in a cuvette (light path: 1 cm) [test: AS, blank: A0].
Calculation
Activity can be calculated by using the following formula:
6.24 : Millimolar extinction coefficient of quinoneimine dye under the assay conditions (cm2/μmol)
1/10 : A factor for correction of the reaction volume
df : Dilution factor
C : Content of maltose phosphorylase preparation in sample (mg/ml)
実験データ
FAQ
- MPL-EPの主な用途は何ですか？
- MPL-EPの分子量を教えてください。
- MPL-EPの至適pHと至適温度について教えてください。
- MPL-EPの熱安定性について教えてください。
- MPL-EPの安定化剤は何を使用していますか？
- MPL-EPの保存方法を教えてください。
- 開封後の取扱い方法に注意事項はありますか？
参考文献
- Hiruma, M. et al., Nippon Nogeikagaku Kaishi, 70, 773–780 (1996).
- Shirokane, Y. et al., Carbohydr. Res., 329, 699–702 (2000).