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Glycerol Kinase (GK)

(ATP:glycerol 3-phosphotransferase; EC
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 37C.

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.

  1. 0.2 M Glycine/0.002 M MgCl2 buffer, pH 9.8 (adjust pH with 1 M KOH).
  2. 0.006 M NADH-disodium salt (3.9 mg/ml) in cold 5 mM Potassium phosphate, pH 7.4.
  3. 0.10 M Glycerol (9.21 mg/ml).
  4. 0.045 M ATP-disodium salt (25 mg/ml) in cold 5 mM Potassium phosphate, pH 7.4.
  5. 0.085 M PEP-monocyclohexylammonium salt, (22.6 mg/ml) in cold 5 mM Potassium phosphate, pH 7.4.
  6. LDH (350 U/ml) and PK (400 U/ml) in Potassium phosphate buffer, pH 7.4; prepare fresh.
  7. Glycerol kinase (GK) solution: dilute to concentration of 0.24 U/ml with ice cold 0.01 M glycerol.

  1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 37C.
  2. Into a cuvette pipette the following reagents as indicated:
    Glycine 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 37C for 5 min. to achieve temperature equilibration.

  3. Determine blank rate, if any, at 340 nm.
  4. 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 (delta)E340 nm/min

Activity (U/mg) = (ΔE340nm/min)(Total Vol.)(Enz. Diln.)
(6.22)(Enz. Vol.)(mg Enz./ml)

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