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Aldolase

(D-Fructose-1, 6-diphosphate-D-glyceraldehyde-3-phosphate-lyase; EC 4.1.2.13)

Aldolase catalyzes the following key reaction in glycolysis:

                           ALDOLASE
D-Fructose-1,6 diphosphate <------->  Dihydroxyacetone phosphate + D-glyceraldehyde-3-phosphate 
Aldolase is found in all animal and plant tissues and most microorganisms. In animal tissues, three types of aldolases have been identified: Type A, the major form found in muscle; Type B found in kidney and liver and Type C found in brain. The enzyme may exist in five different isoenzyme forms which may be organ-specific.

Aldolase from rabbit muscle has been extensively studied. It has a molecular weight of 161,000 and consists of four catalytically active sub-units.

Measurement of serum aldolase activity is of clinical significance. Elevated serum aldolase activity has been observed in certain carcinomas, muscular dystrophy, hepatitis and myocardial infarction (Kaldor, J., Clin. Chim. Acta, 28, 145, 1970). Aldolase is also used for determination of metabolites, in coupled enzyme reactions. The is 9.38 and isoelectric point is 6.1. It is inhibited by heavy metal ions, like copper, silver and zinc.





The amount of enzyme which will convert one micromole of fructose-1,6-diphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate per minute at pH 7.6 and 25C.


 




  1. 0.1 M Triethanolamine HCl buffer, pH 7.6.
  2. 0.008 M NADH, (5 mg/ml), NADH disodium salt in buffer.
  3. 0.033 M Fructose-1,6-diphosphate, (11.22 mg/ml) in buffer.
  4. Glycerol-3-phosphate dehydrogenase (G3PDH) (76 U/ml) in buffer. Prepare fresh.
  5. Triose phosphate isomerase (TPI) (480 U/ml) in buffer. Prepare fresh.
  6. Aldolase (enzyme) solution - dissolve in buffer to a final concentration of 0.1 U/ml. Prepare fresh immediately prior to assay.



  1. Set the spectrophotometer (equipped with a strip chart recorder and temperature control) at 340 nm and 25C.
  2. Into a cuvette pipette the following:
    Triethanolamine buffer                  2.7 ml 
    NADH solution                           0.1 ml 
    Fructose 1,6-Diphosphate (substrate)    0.1 ml 
    
    Mix and incubate in the spectrophotometer at 25C for 5 min. to achieve temperature equilibration. Record blank at 340 nm, if any.
  3. Add the enzyme solutions to the cuvette as follows:
    Glycerol-3-phosphate dehydrogenase      0.01 ml
    Triose phosphate isomerase              0.01 ml
    Aldolase                                0.1  ml
    
  4. Record the change in absorbance at 340 nm for 5-10 min.
  5. Calculate (delta)E304nm/min



Activity (U/mg) = (ΔE304nm/min)(Total Vol.)(Enz. Diln.)
(6.22)(2)(Enz. Vol.)(mg Enz./ml)
 

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