Glycerol Kinase (GK)
(ATP:glycerol 3-phosphotransferase; EC 220.127.116.11)
Glycerol kinase (GK) catalyzes the following reaction:
Glycerol + ATP ------> Glycerol-3-P + ADP
Glycerol kinase has been isolated from microorganisms, who use
this enzyme for utilization of glycerol as a carbon
source. According to Kida, et al [Kida, K., Kobayashi, K,
Kimura, H., and Y. Yugari, J. Biochem (Tokyo), 73, 299, 1973],
the mammalian glycerol kinase represents an interfacing link
between carbohydrate and lipid metabolism. Glycerol kinase is
important in the determination of glycerol, triglycerides and
lipase activity. It can also be utilized in the assay of
glyceraldehyde and dihydroxyacetone.
The amount of enzyme which produces one micromole of glycerol-3-phosphate from glycerol, in the presence of ATP, per minute at pH 9.8 and 37°C.
The assay method is based on a coupled system through pyruvate kinase and lactate dehydrogenase. The rate of decrease in the absorbancy at 340 nm, resulting from the oxidation of NADH, is proportional to the GK activity.
- 0.2 M Glycine/0.002 M MgCl2 buffer, pH 9.8 (adjust pH with 1 M KOH).
- 0.006 M NADH-disodium salt (3.9 mg/ml) in cold 5 mM Potassium phosphate, pH 7.4.
- 0.10 M Glycerol (9.21 mg/ml).
- 0.045 M ATP-disodium salt (25 mg/ml) in cold 5 mM Potassium phosphate, pH 7.4.
- 0.085 M PEP-monocyclohexylammonium salt, (22.6 mg/ml) in cold 5 mM Potassium phosphate, pH 7.4.
- LDH (350 U/ml) and PK (400 U/ml) in Potassium phosphate buffer, pH 7.4; prepare fresh.
- Glycerol kinase (GK) solution: dilute to concentration of 0.24 U/ml with ice cold 0.01 M glycerol.
- Set spectrophotometer (equipped with strip chart recorder and temperature
control) at 340 nm and 37°C.
- Into a cuvette pipette the following reagents as indicated:
buffer, pH 9.8 2.40 ml
NADH 0.10 ml
Glycerol 0.10 ml
ATP 0.10 ml
PEP 0.10 ml
LDH/PK 0.10 ml
Mix and incubate in spectrophotometer at 37°C for 5 min. to achieve
- Determine blank rate, if any, at 340 nm.
- Initiate the reaction by adding 0.1 ml of enzyme (GK) solution to the
cuvette. Monitor the absorbance change at 340 nm for 5 min. and then calculate
|Activity (U/mg) =
||(ΔE340nm/min)(Total Vol.)(Enz. Diln.)
|(6.22)(Enz. Vol.)(mg Enz./ml)