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  Section: Plant Lab Protocols
 
 
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Methodology for Enzymes

 
     
 
Aminotransferases

Amino transferases are enzymes catalyzing the transfer of an amino group plus a proton and an electron pair, from an amino donor compound to the carbonyl position of an amino acceptor compound. Usually an amino acid acts as the amino donor and a 2-oxo acid as the amino acceptor. In some cases aldehydes may serve as amino acceptors, and amines may act as donors. The enzymatic transfer of amino groups plays an important role in many metabolic processes where the interconversion of nitrogen containing molecules is involved. Nitrogen, following its initial assimilation into glutamine and glutamate can be distributed to many other compounds by the action of aminotransferases. Glutamate is often the amino-donor substrate in biosynthetic transamination reactions. This reaction regenerates 2-oxoglutarate for necessary ammonia assimilation through GDH and the glutamine synthetase/glutamate synthase routes.
 
 


Content
i) Asparatate Amino Transferase
ii) Alanine Aminotransferase
 
 
i) Asparatate Amino Transferase (Glutamate: Oxaloacetate Aminotransferase EC 2.6.1.1)
 
Principle
Glutamate oxaloacetate aminotransferase (GOT) catalyses the reversible interconversions between glutamate and aspartate and their 2-oxo analogues.
 
Glutamate + Oxaloacetate
GOT
2-Oxoglutarate + Aspartate
 
The oxaloacetic acid is measured colorimetrically by a reaction with 2.4-dinitrophenylthydrazine giving a brown-colored hydrazone after the addition of 0.4N sodium hydroxide.
 
 



Materials
Phosphate buffer, pH 7.4. Add 11.3g dry anhydrous disodium hydrogen phosphate and 2.7g dry anhydrous potassium dihydrogen phosphate in one liter volumetric flask and make up to the mark with water. Check the pH
and store at 4°C.

Substrate Solution: Dissolve 13.3g DL-aspartic acid in minimum amount of 1N sodium hydroxide and prepare a solution with pH 7.4 (about 90mL is required).  Add 0.146g 2-oxoglutarate and dissolve it by adding a little
more sodium hydroxide solution. Adjust to pH 7.4 and then make to 500mL with phosphate buffer. Divide into 10mL portions and store frozen at-15°C.

Pyruvate Standard: Dissolve 22mg sodium pyruvate in 100mL water in a standard flask.
2,4-dinitrophenyl Hydrazine  (DNPH):  Dissolve  19.8mg dinitrophenyl hydrazine in 10mL concentrated hydrochloric acid and make to 100mL with water. Store it in an amber bottle at room temperature.
Sodium Hydroxide 0.4N: Dissolve 16g sodium hydroxide in one liter water.
Enzyme Extract: Prepare the crude extract by grinding the plant tissue in 0.2M potassium phosphate, pH 7.5 in a homogenizer for 2min. Pass the slurry through eight layers of cheesecloth and then centrifuge at 25,000g for 15min to get the enzyme fraction.
 
 
Procedure
>1.
Warm 0.5mL of substrate solution in a water bath at 37°C for 3min.
>2.
Add 0.2mL enzyme extract and mix gently.
>3.
Incubate for 60min at 37°C.
4.
Remove the tubes from the bath and immediately add 0.5mL dinitrophenyl hydrazine solution and mix well.
5.
Mix 0.5mL substrate with 0.5mL DNPH solution and then add 0.1mL enzyme extract for control.
6.
Allow the DNPH to react for 20min at room temperature.
7.
Add 5mL of 0.4N sodium hydroxide, mix well and leave for a further 10min.
8.
Record the absorbance at 510nm.
9.
Pipette out pyruvate standard 0.05 to 0.20mL and make up to 0.2mL. Add 0.5mL substrate and 0.5mL DNPH solution. For blank mix 0.5mL substrate, 0.2mL water and 0.5mL DNPH solution.
10.
Then proceed with steps 6-8.
 
 
Calculation
The pyruvate formed by enzyme is responsible for the absorbance difference between test and control. The pyruvate in standard produces the difference between standard and blank. Express the enzyme activity as micromole of pyruvate formed per min per mg protein.
 
 
ii) Alanine Aminotransferase (Glutamate: Pyruvate Aminotransferase EC 2.6.1.2)
 
Principle
Glutamate pyruvate aminotransferase catalyses the reversible interconversions between glutamate and alanine and their 2-oxo analogues.
2-oxoglutarate + Alanine
 
Glutamate + Pyruvate
 
The pyruvate formed after 30min incubation period is measured colorimetrically by reaction with DNPH as explained earlier.
 
 



Materials
Phosphate buffer, pyruvate standard, DNPH and sodium hydroxide, 0.4N - same as in Glutamate oxaloacetate aminotransferase.
Substrate Solution: Dissolve 9.0g alanine in 90mL water with addition of about 2.5mL sodium hydroxide, IN and adjust to pH 7.4.  Then add 0.146g 2-oxoglutarate and dissolve it by adding a little more sodium hydroxide solution and adjust to pH 7.4, make up to 500mL with phosphate buffer. Divide into 10mL portions and store frozen at -15°C.
 
 
Procedure
Similar to GOT procedure, use alanine as substrate and incubate for 30min.
 
 
Calculation
Express the enzyme activity as micromole of pyruvate formed per min per mg protein.
 
 


References
1.  Bergmeyer, H U and Bernt, E (1974) In: Methods of Enzymatic Analysis {Ed Bergmeyer) Vol2p735.
2.  Splittstoesser, W E, Chu, M C, Steward, S A and Splittstoesser, S A (1976) Plant Cell Physiol 17 83.


 
     
 
 
     




     
 
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