Pyruvate Kinase (PK)
(ATP:Pyruvate 2-0-phosphotransferase; EC 18.104.22.168)
Pyruvate kinase (PK) catalyzes the following reaction:
Phospho(enol) pyruvate + ADP ------> Pyruvate + ATP
Pyruvate kinase is an important enzyme in glucose
metabolism. Mammalian PK of various tissues have distinct
characteristics which are related to the specific metabolic
requirements of each tissue. PK, prepared from rabbit muscle,
is extensively used in the quantitative determination of ADP
and of enzymes that catalyze the formation of ADP. It can also
be used for the determination of phospho(enol) pyruvate and
2-phosphoglycerate. The assay of PK activity in red blood
cells has been described by Tanphaichitr and Van Eys
(Tanphaichitr, V.S. and Van Eys, J., Clin. Chim. Acta, 41, 41,
||Soluble in distilled water or dilute buffer
||-20° C; -4° F
||250 U/mg protein
||Myokinase <0.005% Glycerol-phosphate dehydrogenase <0.001% Creatine phosphokinase <0.001% Adenosine triphosphatase <0.001% NADH oxidase <0.001% Phospho-glycerate-mutase <0.001%
The amount of enzyme which produces one micromole of pyruvate
from phospho(enol)pyruvate, in the presence of ADP, per minute
at pH 7.5 and 25°C.
The method of assay has been described by Tietz and Ochoa
(Tietz and Ochoa, S., Arch. Biochim. Biophys. Acta., 78, 477,
1958). The rate of decrease in the absorbancy at 340 nm,
resulting from the oxidation of NADH, is proportional to the
- 0.06 M Tris-HCl buffer, pH 7.5, in distilled water.
- 0.12 M Magnesium chloride, in distilled water.
- 2.25 M Potassium chloride, in distilled water.
- 0.006 M Adenosine diphosphate (ADP), disodium, in buffer. Prepare fresh.
- 0.043 M Phospho(enol)pyruvate (PEP), trisodium in buffer. Prepare fresh.
- 0.006 M NADH, disodium salt, in buffer. Prepare fresh.
- Lactate dehydrogenase (LDH) solution, 40 U/ml in buffer. Prepare fresh.
- Pyruvate kinase (PK) solution. Using cold 1% bovine serum albumin
as solvent, dilute to a final concentration of 0.1-0.2 U/ml. Must be
prepared fresh immediately prior to assay.
- Set spectrophotometer (equipped with strip chart recorder and
temperature control) at 340 nm and 25°C.
- Into a cuvette, pipette the following reagents as indicated:
Tris-HCl buffer, pH 7.5 2.30 ml
Magnesium chloride 0.10 ml
Potassium chloride 0.10 ml
ADP 0.10 ml
PEP 0.10 ml
NADH 0.10 ml
LDH 0.10 ml
Mix and incubate in spectrophotometer at 25°C for 5 minutes to
achieve temperature equilibration.
- Determine blank rate, at 340 nm, if any.
- Initiate the reaction by adding 0.1 ml of enzyme (PK) solution
(0.1-0.2 U/ml) to the cuvette. Monitor the absorbance change at 340 nm
for 5 minutes and then calculate µE340 nm/min
|Activity (U/mg) =
||(ΔE340nm/min)(Total Vol.)(Enz. Diln.)
|(6.22)(Enz. Vol.)(mg Enz./ml)