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Glutamic Pyruvic Transaminase (ALT)

(L-alanine:2 oxoglutarate aminotransferase; EC Glutamic pyruvic transaminase (GPT) is also known as alanine aminotransferase. It catalyzes the following reaction: GPT L-Alanine + a-Ketoglutarate ------> Pyruvate + L-Glutamate Transamination reactions play a significant role in intermediary metabolism. Transaminases require pyridoxal phosphate as a coenzyme for their catalytic activity. GPT is found in many animal and plant tissues and it's activity is especially high in mammalian heart and liver. The enzyme exists in two distinct isoenzyme forms in mammalian tissues, the mitochondrial form and the cytoplasmic form. GPT from porcine heart has been extensively studied. It has a molecular weight of 100,000. Human GPT levels in serum are used in clinical diagnosis of liver and heart disease (Methods of Enzymatic Analysis, Bergmeyer, H.U. ed. Vol I, 14, 1974. Academic Press, New York).

1.Source: Porcine Heart
   Form: Freeze-dried powder 
   Solubility: Distilled water or dilute buffer 
   Stability: Store at -20 C (-4 F) 
   Activity (approx.): 100 U/mg 
   Protein (approx.): 40% (biuret) 
   Contaminants (approx.): Lactate dehydrogenase <0.005%; Glyceraldehyde-3-P-dehydrogenase <0.001%; Malate dehydrogenase <0.002%; Glutamate oxaloacetate transaminase <0.001% 
   Catalog No.: 081A0100 

That amount of enzyme which will catalyze the conversion of one micromole of a-ketoglutarate to L-glutamate, in potassium phosphate buffer, at pH 7.5 and 25C.

The decrease in the absorbance at 340 nm, caused by the oxidation of NADH, is proportional to the catalytic activity of GPT.

  1. 0.1 M Potassium phosphate buffer, pH 7.5.
  2. 1.15 M L-Alanine (103 mg/ml) in buffer.
  3. 0.31 M a-Ketoglutarate (45 mg/ml) in buffer.
  4. 0.008 M NADH disodium salt (5 mg/ml) in distilled water. Prepare fresh.
  5. Lactate dehydrogenase (LDH), 250 U/ml in buffer. Prepare fresh immediately prior to assay.
  6. Enzyme (GPT) solution. Prepare in buffer to yield a final concentration of 0.1-0.2 U/ml. Must be prepared fresh immediately prior to assay.

  1. Set spectrophotometer (equipped with strip chart recorder and temperature control) at 340 nm and 25C.
  2. Into a cuvette pipette the following reagents in the amounts indicated:
    L-alanine 2.60 ml
    a-ketoglutarate 0.10 ml
    NADH 0.10 ml
    LDH 0.10 ml
    Mix and incubate in spectrophotometer for 5 minutes to attain temperature equilibration, then record blank rate at 340 nm.
  3. Initiate the reaction by adding 0.1 ml of enzyme (GPT) solution to the cuvette.
  4. Record the rate of decrease in absorbance at 340 nm for 5-8 minutes.
  5. Calculate (delta)E340nm/min

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

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