GLYCEROL KINASE [GKⅢ]
from Microorganism
(ATP:Glycerol 3-phosphotransferase, EC 2.7.1.30)
Glycerol + ATP → sn-Glycerol 3-phosphate + ADP
Preparation and Specification
- Appearance
- : White to brownish lyophilized powder
- Specific activity
- : More than 50 U/mg solid
- Contaminants
- : Catalase Less than 0.1%(U/U)
Properties
- Molecular weight
- : 50 kDa ( SDS-PAGE)
- Isoelectric point
- : 4.73 (estimated from amino acid sequence)
- Michaelis constants
- : Glycerol 5.3 × 10-5M
ATP 4.8 × 10-4M
- Optimum pH
- : 6.5-7.0Figure 1
- pH stability
- : 5.5–9.0 (37℃, 60 min) Figure 2
- Thermal stability
- : Stable at 50℃ and below (pH7.0, 15 min)Figure3
- Optimum temperature
- : 45℃Figure4
- ProClin stability
- : See Figure 5
Preparation and Specification
This enzyme is useful for enzymatic determination of triglyceride (TG) coupled with LP (Lipase; T-01, T-63, or T-116) and GPO (L-α-Glycerophosphate oxidase; T-60 or T-107).
| LP | ||
| TG + 3 H2O | → | Glycerol + 3 Fatty acid |
| GKⅢ | ||
| Glycerol + ATP | → | G-3-P + ADP |
| GPO | ||
| G-3-P + O2 | → | DHAP + H2O2 |
| POD | ||
| 2H2O2 + 4-AA + Phenol | → | Quinonimine dye + 4 H2O |
G-3-P: sn-Glycerol 3-phosphate
DHAP: Dihydroxyacetone phosphate
Fig.1 Optimum pH
〇: Acetate buffer
■: Phosphate buffer
△: PIPES-NaOH buffer
●: Tris-HCl buffer
□: Glycine-NaOH buffer
■: Phosphate buffer
△: PIPES-NaOH buffer
●: Tris-HCl buffer
□: Glycine-NaOH buffer
Fig.2 pH Stability
37°C, 60 min
〇: Acetate buffer
■: Phosphate buffer
△: PIPES-NaOH buffer
●: Tris-HCl buffer
□: Glycine-NaOH buffer
〇: Acetate buffer
■: Phosphate buffer
△: PIPES-NaOH buffer
●: Tris-HCl buffer
□: Glycine-NaOH buffer
Fig.3 Thermal stability
pH7.0, 15 min
50 mM Phosphate buffer
50 mM Phosphate buffer
Fig.4 Optimum temperature
pH7.0
100 mM PIPES-NaOH buffer
100 mM PIPES-NaOH buffer
Fig.5 ProClin stability
20°C, pH7.0
50 mM PIPES-NaOH + 0.5% ProClin
〇: GK III (T-223)
●: Competitor A
△: Competitor B
50 mM PIPES-NaOH + 0.5% ProClin
〇: GK III (T-223)
●: Competitor A
△: Competitor B
Assay
Principle
-
The assay is based on the increase in absorbance at 546 nm as the formation of quinoneimine dye in the following reactions:
| GKⅢ | ||
| Glycerol+ATP | → | Glycerol 3-Phosphate+ADP |
| GPOSP | ||
| Glycerol 3-Phosphate+O2 | → | Dihydroxyacetone phosphate+H2O2 |
| POD | ||
| 2H2O2+4-AA+TODB | → | Quinoneimine dye+4H2O |
GPOSP: L-α-Glycerophosphate oxidase
POD: Peroxidase
Unit definition
-
One unit is defined as the amount of enzyme which converts 1 μmole of glycerol to glycerol 3-phosphate per minute at 37°C under the conditions specified in the assay procedure.
Reagents
- Reaction mixture
0.5 M PIPES-NaOH buffer pH7.0 0.2 ml 5% Glycerol solution 0.05 ml 100 mM ATP solution pH7.0 0.1 ml 100 mM MgCl2 solution 0.1 ml 100 U/ml GPOSP solution 0.1 ml 100 U/ml POD solution 0.05 ml 0.2% TODB solution 0.1 ml 0.3% 4-AA solution 0.1 ml Distilled water 0.2 ml - Reaction stopper
0.5% SDS solution - Enzyme dilution buffer
50 mM PIPES-NaOH buffer pH 7.0 containing 0.1%(W/V) BSA. - Reagents
PIPES [Piparazine-1,4-bis(2-ethanesulphonic acid)]:
Dojindo Laboratories #345–02225Glycerol: FUJIFILM Wako Pure Chemical Corporation
Guaranteed Reagent #075-00616ATP (2Na･3H2O): Kyowa Hakko Co., Ltd.
MgCl2･6H2O:
FUJIFILM Wako Pure Chemical Corporation Guaranteed Reagent #131-00162GPOSP: Asahi Kasei Pharma Corporation #T-60 TODB (N,N-Bis(4-sulfobutyl)-3-methylaniline,disodium salt):Dojindo Laboratories #OC224-AA: NACALAI TESQUE, INC. Guaranteed Reagent #01907–52
POD: Sigma Chemical Co. Type II #P-8250
BSA: Millipore Fraction V pH 5.2 #81–053
SDS (Sodium Dodecyl Sulfate): NACALAI TESQUE, INC. #31606
Enzyme solution
Accurately weigh about 20 mg of the sample and add enzyme dilution buffer to make a total of 20 ml. Dilute it with enzyme dilution buffer to adjust the concentration as required.
Procedure
- Pipette accurately 1.0 ml of reaction mixture into a small test tube and preincubate at 37°C.
- After 5 min, add 40 μl of enzyme solution and mix to start the reaction at 37°C.
※ In the case of a test blank, add 40 μl of enzyme dilution buffer in place of enzyme solution. - At 5 min after starting the reaction, add 2.0 ml of the reaction stopper to stop the reaction.
- Measure the absorbance at 546 nm.
Absorbance sample : As
blank : Ab
△ A = (As−Ab) ≦ 0.6 Abs
Calculation
Activity (U/mg of powder) = {(△A/5)/(39.2 × 1/2)} × 3.04/0.04 × 1/x-
39.2 : millimolar extinction coefficient of quinoneimine dye at 546 nm (cm2/μmol) 1/2 : a multiplier derived from the fact that 2 mole of H2O2 produce 1 mole of quinoneimine dye 5 : reaction time (min) 3.04 : final volume (ml) 0.04 : volume of enzyme solution (ml) X : concentration of the sample in enzyme solution (mg/ml)
Storage
-
Storage at -20°C in the presence of a desiccant is recommended. Enzyme activity will be retained for at least one year under this condition.
GKⅢ活性測定法 (Japanese)
試薬液
- 反応試薬混合液
0.5M PIPES-NaOH 緩衝液pH 7.0 0.2 ml 5% グリセロール液 0.05 ml 100mM ATP 溶液 pH7.0 0.1 ml 100mM 塩化マグネシウム溶液 0.1 ml 100U/ml GPOSP 0.1 ml 100U/ml POD 0.05 ml 0.2% TODB 0.1 ml 0.3% 4-AA 0.1 ml 精製水 0.2 ml - 反応停止液
0.5% SDS 溶液 - 酵素溶解希釈用液
0.1% (W/V) BSA を含む50 mM PIPES-NaOH 緩衝液pH7.0 - 試薬
PIPES (ピペラジン-N,N'- ビス (2- エタンスルフォン酸) ) : 同仁化学製 #345-02225
グリセロール:富士フイルム和光純薬製 特級 #075-00616
ATP ( アデノシン三リン酸・2Na・3H2O) : 協和発酵製
塩化マグネシウム: 富士フイルム和光純薬製 特級 #131-00162
GPOSP: 旭化成ファーマ製 #T-60
TODB(N,N-Bis(4-sulfobutyl)-3-methylaniline, disodium salt): 同仁化学製 #OC22
4-AA (4- アモノアンチピリン) ナカライテスク製 特級 #01907–52
POD: シグマ製 Type II #P-8250
BSA: ミリポア製 Fraction V pH 5.2 #81–053
SDS (ドデシル硫酸ナトリウム) ナカライテスク製 #31606
酵素試料液
- 検品約20mg を精密に量り、酵素溶解希釈用液で溶解して全容20ml 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1.0ml を正確に分注して37℃で予備加温する｡
- 5 分経過後、酵素試料液40 μl を正確に加えて混和し、37℃で反応を開始する｡
※盲検は酵素試料液の代わりに酵素溶解希釈用液40μl を加える｡ - 5 分経過後、反応停止液2.0ml を加えて混和し、反応を停止する。
- 546nm における吸光度を測定する。求められた吸光度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 ΔA = (As － Ab) ≦ 0.6 Abs
計算
活性 (U/mg) = {(ΔA/5)/(39.2 × 1/2)} × 3.04/0.04 × 1/x| 39.2 : | キノンイミン色素の546nm におけるミリモル分子吸光係数 (cm2/μmol) |
| 1/2 : | H2O2 2 モルからキノンイミン色素1 モルが生成することによる係数 |
| 5 : | 反応時間 (min) |
| 3.04 : | 反応総液量 (ml) |
| 0.04 : | 反応に供した酵素試料液量 (ml) |
| X : | 酵素試料液中の検品濃度 (mg/ml) |
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