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  Section: Plant Lab Protocols
 
 
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Methodology for Enzymes

 
     
 
Glutamine Synthetase
(L-Glutamate : Ammonia Ligase EC 6.3A.2)
 
Glutamine  synthetase  (GS)   appears  throughout  the  plant  and  animal kingdoms. It catalyses the following reaction.
L-glutamate + NH3 + ATP
Mn++
L-glutamine + ADP + Pi

 
This enzyme has high affinity for ammonia. High concentration of this enzyme in the nodules is probably related to a role in assimilating ammonia formed as a result of nitrogen fixation.
 
 



Principle
The activity of the enzyme is measured by estimating the production of inorganic phosphate, GS also catalyses the g-glutamyl transfer reaction.
 
ADP, Mn++
 
Glutamine + Hydroxylamine

Glutamyl hydroxamate
 
Arsenate
 
 
Hence, it can be assayed by measuring the production of g-glutamyl hydroxamate. The latter method is described below. The g-glutamyl hydroxamate is made to react with ferric chloride to produce brown color in acidic medium.
 
Activity in the transferase assay generally gives rates several times greater than those obtained in the synthetase assay (measuring Pi) and hence, this method is often used for measurement of GS in relatively crude preparations.
 
When the activity is measured in the presence of Mn**, it represents total glutamine synthetase activity (adenylated and unadenylated forms). The biologically active unadenylated form may be measured by inhibiting the adenylated form by the addition of60mM Mg++.
 
 



Materials
Prepare the following reagents in 20mM Tris-HCl buffer (pH 8.0). The concentration of stock solution is indicated in parentheses.
L-Glutamine 0.2M (700mg/12mL)
Sodium Arsenate 20mM (500mg/10mL) (Disodium Hydrogen Arsenate)
MnCl2  3mM(83mg/10mL)
Hydroxylamine 50mM (278mg/10mL)
Adenosine diphosphate 1mM (40mg/10mL)
Ferric chloride reagent: Dissolve 10g trichloro-acetic acid and 8g ferric chloride in 250mL of 0.5N hydrochloric acid.
Enzyme Extract
   Extract 1g plant material in 5mL of 50mM imidazole-acetate buffer pH 7.8 containing 0.5mM EDTA, 1mM dithiothreitol, 2mM MnCl2 and 20% glycerol at 4°C. Centrifuge at 10,000g for 30 min. If purification is required precipitate the enzyme with (NH4)2SO4 at 60 % saturation. Resuspend the precipitate in extraction buffer. Desalt over sephadex G 25.
 
 
Procedure
1.
Pipette out the reagents (mL) as mentioned in the order below:
Glutamine                           2.0
Sodium arsenate               0.5
MnCl2                                   0.3
Hydroxylamine                   0.5
ADP                                      0.5
Enzyme extract                  0.2
2.
To set a blank, add 2mL 20mM Tris-HCl buffer instead of glutamine.
3.
Incubate the reaction mixture for 30min at 37°C.
4.
Stop the reaction by adding 1mL of ferric chloride reagent.
5.
Measure the brown color developed at 540nm.
6.
Prepare   a range of standards containing 100-500mg g-glutamyl hydroxamate in 4mL buffer solution and develop the color by adding 1mL of ferric chloride reagent.
 
 
Calculation
Find out the amount of g-glutamyl hydroxamate formed in the reaction using the standard graph. Express the enzyme activity as nanomole g-glutamyl hydroxamate formed per min per mg protein.
 
 


References
1.  Pateman,J A (1969) Biochem J 115 769.
 
     
 
 
     




     
 
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