Recombinant Glycerol Kinase (GK), CAS No.9030-66-4

CAS: 9030-66-4 Cat. No.: G1493000 EC Number: 232-862-0 PubChem CID: 135333386

GRADE & PURITY Bioactive, Recombinant, ActiBioPure™, High Performance, EnzymoPure™, ≥30 U/mg enzyme powder

Accession #

P0A6F3

Expression system

E. coli

Bioactivity

≥30 U/mg enzyme powder

Overview

  Glycerol Kinase (GK, EC 2.7.1.30) is a key member of the kinase family, catalyzing the initial reaction in glycerol metabolism. In the presence of ATP, this enzyme can specifically catalyze the phosphorylation of glycerol, generating glycerol-3-phosphate (G-3-P) and ADP. 

Application

  In clinical analysis, this enzyme can be used for the enzymatic determination of glycerol and triglycerides by coupling with glycerol-3-phosphate oxidase, or with pyruvate kinase and lactate dehydrogenase, or with lipoprotein lipase.

Assay procedure

Principle

  The appearance of quinoneimine dye is measured at 500nm by spectrophotometry.

Unit definition

  One unit causes the formation of one micromole of hydrogen peroxide(half a micromole of quinoneimine dye)per minute under the conditions described below.

Reagents

A. Glycerol solution:0.3M(Should be prepared fresh)

B. 4-AA solution:0.1%(100mg of 4-aminoantipyrine/100ml of H₂O₂)

C. Phenol solution:0.1% (100mg of phenol/100ml of H₂O₂)

D. Peroxidase solution:20mg Peroxidase(110purpurogallin unit/mg)/100ml of H₂O₂

E. G-3POD Solution:20U/ml(dissolve in 200Mm HEPES buffer,pH7.9)

F. Buffer solution: 200Mm HEPES,pH7.9 contg.20Mm MgCl2 and 40mM ATP (Should be prepared fresh)

G. Enzyme diluent: 20mM K-phosphate buffer,pH7.5

Procedure

1. Prepare the following working solution in a brownish bottle and store on ice.

10ml	4-AA solution	(B)
20ml	Phenol solution	(C)
20ml	Preoxidase solution	(D)
40ml	G-3POD solution	(E)
10ml	Buffer solution	(F)

Concentration in assay mixture

HEPES buffer	95.2mM
Glycerol	4.76mM
ATP	3.81mM
MgCl 2	1.90mM
4-AA	0.469mM
Phenol	2.02mM
G-3-POD	ca.7.6 U /ml
POD	ca.5.2 U /ml

2. Pipette 3.0ml of working solution into a cuvette (d=1.0cm). (d=1.0 cm)

3. Add 0.1ml of the enzyme solution* mix by gentle inversion and equilibrate at 37℃ for about 5 minutes.

4. Add 0.05ml of glycerol solution(A)and mix by gentle inversion.

5. Record the optical density at 500nm against water for 3 to 4 minutes in a spectrophotometer thermostated at 37℃,and calculate the ∆OD per minute from the initial portion of the curve(∆OD test).

At the same time,measure the blank rate(∆OD blank)by using the same method as the test except that enzyme diluent (G) is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent(G)and dilute to 0.2~0.4U/ml with the same buffer,immediately before assay.

Calculation

  Activity can be calculated by using the following formula:

Vt: Total volume (3.15ml)

Vs: Sample volume (0.1ml)

13.3: Millimolar extinction coefficient of quinoneimine dye under the assay conditions (cm²/μmol)

1.0: Light path length (cm)

1/2: Factor based on the fact that one mole of H₂O₂​ produces a half of quinoneimine dye.

df: Dilution factor

C: Enzyme concentration in dissolution (C mg/ml)

Specifications

Product Name

Recombinant Glycerol Kinase (GK), CAS No.9030-66-4

Synonyms

Glycerokinase | GyK | glpK

Grade

ActiBioPure™, Bioactive, High Performance, Recombinant, EnzymoPure™

Specifications & Purity

Bioactive, Recombinant, ActiBioPure™, High Performance, EnzymoPure™, ≥30 U/mg enzyme powder

Biochemical and Physiological Mechanisms

Key enzyme in the regulation of glycerol uptake and metabolism. Catalyzes the phosphorylation of glycerol to yield sn-glycerol 3-phosphate. It also catalyzes the phosphorylation of dihydroxyacetone, L-glyceraldehyde and D-glyceraldehyde. It uses only ATP.

Bioactivity

≥30 U/mg enzyme powder

Accession #

P0A6F3

CAS

9030-66-4

Enzyme Commission Number

EC 2.7.1.30

Molecule Type

Enzyme

Storage and Shipping

Concentration

≥30 U/mg enzyme powder

Reconstitution

Reconstitute in 20mM K-phosphate buffer,pH7.5.

Storage

Store at -20°C,Avoid repeated freezing and thawing

Shipped In

Ice chest + Ice pads

Stability And Storage

Store at -20℃ long term (24 months). Upon reconstitution, it is recommended to aliquot. Avoid freeze/thaw cycle.

Unit definition

One enzyme activity unit is defined as the amount of enzyme that produces 1 micromole of hydrogen peroxide (1/2 millimole of quinone dye) per minute at pH 7.9 and 37°C.

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