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  Section: Plant Lab Protocols
 
 
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Methodology for Enzymes

 
     
 
Glutamate Synthase
(L-glutamate:NADP+ oxidoreductase (transaminating) EC 1.4.1.13)
 
The original name given to this enzyme was glutamine (amide) 2-Oxoglutarate amino transferase (oxidoreductase NADP+) from which the acronym GOGAT is derived. The enzyme has however been referred to as L-glutamate : NADP+ oxido reductase EC 1.4.1.13. The trivial name glutamate synthase is also in use. The bacterial enzyme is highly specific for the pyridine nucleotide electron donor (NADH or NADPH). In angiosperms ferredoxin glutamate synthase has been reported. Angiosperm pyridine nucleotide glutamate synthase appears to exhibit activity with both NADH and NADPH. The only exception to this is the NADH-specific enzyme from legume root nodules. The root enzyme has low affinity for 2-Oxoglutarate (Km 0.4-1.0mM). The reaction catalyzed by this enzyme is as follows.
Glutamine + 2-Oxoglutarate + NADPH + H+

2 Glutamate + NADP+
 
 



Principle
Glutamate synthase is assayed spectrophotometrically by recording the rate of oxidation of NADPH or NADH, as indicated by a change in absorbance at 340nm following the addition of enzyme extract.
 
 



Materials
Tris HC1 buffer 50mM pH 7.6.
Prepare the following reagents in Tris HCl buffer 50mM pH 7.6
   Glutamine, 5mM (36.5mg/10mL)
   2-Oxoglutarate 5mM (36.5mg/10mL)
   NADPH 0.25mM (10mg/10mL)
Enzyme Extract
   Extract 1g of the plant material with 5mL of 100mM phosphate buffer pH 7.5 containing 1mM disodium EDTA, 1mM  dithioerythritol and 1% Polyvinyl Pyrrolidone (PVP) and centrifuge at 10,000g for 30 min at 4°C. Collect the supernatant and use it for enzyme assay.
 
 
Procedure
1. Prepare reaction mixture as per the table.
Sr. No.
Reagent
mL to be added
1
Glutamine
1.0
2
2-Oxoglutarate
1.0
3
NADPH
1.0
4
Enzyme extract
0.2
5
Buffer
1.8
2. Omit 2-Oxoglutarate in the blank, instead add 1mL buffer.
3. Incubate for 15-30 min at 37°C.
4. Record the change in absorbance at 340nm.
 
 
Calculation
Activity is expressed as n mole of NAD(P)H oxidized per min per mg protein (refer GDH).
 
 


References
1. Tempest, D W, Meers, J C and Brown, C M (1970) Biochem J 117 405.
2. Van de Casteele, J P, Lemal, J and Coudest, M (1975) J Gen Microbiology 90 178.
 
     
 
 
     




     
 
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