Native Glycerol Kinase from Microorganism

Products

Native Glycerol Kinase from Microorganism

Product Specifications

Cat#	NATE-1006
Descriptions	High-purity Glycerol Kinase specifically phosphorylates free glycerol released from triglyceride hydrolysis in diagnostic reaction systems. The enzyme shows strict substrate specificity with no cross-reaction to other serum polyols, providing accurate intermediate reaction signals that guarantee linear and reliable triglyceride measurement results in clinical biochemical diagnostic reagents.
Applications	This product can be integrated with glycerophosphate dehydrogenase (G-3-PDH) and L-α-glycerophosphate oxidase (GPO), or pyruvate kinase and lactate dehydrogenase to detect serum triglycerides enzymatically in clinical diagnosis.
Synonyms	ATP: Glycerol 3-phosphotransferase
EC Number	EC 2.7.1.30
Catalysis	Glycerol +ATP → Glycerol-3-phosphate + ADP
Activity	≥ 10 U/mg (Glycerol kinase activity)
Contaminant	≤ 1.0 × 10⁻²% (ATPase activity); ≤ 1.0 × 10⁻¹% (Catalase activity); ≤ 5.0 × 10⁻²% (Hexokinase activity); ≤ 1.0 × 10⁻²% (NADH oxidase activity)
Appearance	White to light yellowish white powder, lyophilized
Additive	Sodium glutamate
Molecular Weight	82,000 (Gel filtration)
Isoelectric Point	4.3
Km	3.5 × 10⁻⁵ M (Glycerol); 3.7 × 10⁻⁵ M (ATP)
Optimum pH	10.0
pH Stability	5.0-9.5 (37°C, 1 h)
Optimum Temperature	50°C
Thermal Stability	up to 50°C (pH 7.0, 15 min)
Inhibitors	SH reagents
Stabilizers	ATP
Unit Definition	1 unit is defined as the enzyme quantity which produces 1 μ mole of NADH per minute under the conditions described below.
Storage	24 months from the date of analysis when stored at -20°C or below in a dry place under sealed conditions.

General Properties

Click image to enlarge

Assay Principle

Click image to enlarge

Activity Assay

Unit Definition

1 unit is defined as the enzyme quantity which produces 1 μ mole of NADH per minute under the conditions described below.

Reagents

A. 0.2 M Glycine-hydrazine buffer (containing 2 mM MgCl2, pH 9.8): Weigh 15 g of glycine, 51.1 g of hydrazine hydrate and 406 mg of MgCl2･6H2O then dissolve in 800 mL of deionized water. Adjust the pH to 9.8 at 25℃ with 1N KOH and fill up to 1000 mL with deionized water. (Can be used for 3 months if kept refrigerated)
B. 0.02 M Phosphate buffer (KH2PO4-Na2HPO4, pH 7.0)
C. NAD/ATP solution: Weigh 10 mg of β-NAD and 30 mg of ATP･2Na, then dissolve in 20 mL of 0.2 M Glycine-hydrazine buffer (A). (Can be used for 5 days if kept refrigerated)
D. Substrate solution: Weigh 92 mg of glycerol and dissolve in 0.2 M Glycine-hydrazine buffer (A). Fill up to 100 mL with 0.2 M Glycine-hydrazine buffer (A). (Can be used for 1 month if kept refrigerated)
E. G3PDH solution (Produced by Roche Diagnostics GmbH)
F. Diluent: Weigh 303 mg of ATP･2Na and dissolve in 0.02 M Phosphate buffer (B). Fill up to 500 mL with 0.02 M Phosphate buffer (B). (Can be used for 10 days if kept refrigerated)
G. Enzyme solution: Weigh out Glycerol Kinase and dissolve in chilled Diluent (F). Enzyme solution should be prepared so that the value of ΔOD/minute becomes in the range of 0.030±0.005.

Procedure

Pipette 2.0 mL of NAD/ATP solution (C), 1.0 mL of Substrate solution (D) and 0.02 mL of G3PDH solution (E) respectively into a quartz cell (d = 10 mm) and keep at 25±0.5℃ for 5 minutes. Then, pipette 0.1 mL of Enzyme solution (G) into the quartz cell and mix well immediately. Keep the reaction mixture at 25±0.5℃. Exactly at 2 minutes and 5 minutes after the addition of Enzyme solution (G), measure the absorbances of the reaction mixture at 340 nm (A2 an A5). As a blank, pipette Diluent (F) into another quartz cell (d = 10 mm) instead of Enzyme solution (G) and take the same procedure described above (Ab2 and Ab5).

Calculation

Click image to enlarge

Safe Handling Avoid inhalation; in the event of direct skin or eye contact, immediately flush the affected area with abundant water, and consult the SDS for further information.