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  Section: Plant Lab Protocols
 
 
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Methodology for Enzymes

 
     
 
Glutamate Dehydrogenase
(L-glutamate: NAD oxidoreductase (deaminating) EC 1.4.1.2)
(L-glutamate: NADP oxidoreductase (deaminating) EC 1.4.1.4)
 
Glutamate dehydrogenase (GDH) occurs in almost all living organisms. In higher plants, GDH activity has been found in most species tested. The existence of two distinct GDH enzymes in higher plants is now well documented.
a.  mitochondrial enzyme which specifically requires coenzyme NAD.
b.  a chloroplast enzyme which specifically requires coenzyme NADP.
Both enzymes have dual coenzymes specificity to a certain extent and have different pH optima.
The fungal and bacterial enzyme has single coenzyme specificity. GDH activity has been found in both bacteriod and cytosol fractions of root nodules of a number of legumes. The enzyme has high Km for ammonia (10-80mM).
The reaction catalyzed by this enzyme is given below:
 
L-glutamate + H2O+NAD+(P)

2-Oxoglutarate + NH4+ + NAD(P)H + H
 
 



Principle
GDH is like other dehydrogenases assayed by following the oxidation of the reduced coenzyme, NADH or NADPH These reduced coenzymes absorb light at 340nm, which in most biological systems is uniquely uncluttered with interfering absorption by other compounds. Thus even in crude extracts the absorption of NADH at 340nm is easily detected.
The molar extinction coefficient of NADH at 340nm is 6.22 x 103. This indicates one micromole NADH per mL will have an absorbance 6.22.
 
 



Materials
Potassium Phosphate Buffers 1.0M (pH 7.8 and pH 7.0)
2-Oxo-glutarate 0.1M. Dissolve 14.6g in one liter distilled water
NH4Cl (1.0M). Dissolve 53.5g in one liter of distilled water
NADH 10mg/mL
NADPH 10mg/mL
Enzyme Extract: Follow the method given under glutamate synthase.
 
 
Procedure
1. Proceed for the assay of NADH- and NADPH-dependent GDH as per the composition of assay given in table.
Reagent
NADH assay
NADPH assay
 
mL
mL
Potassium phosphate buffer pH 7.0
1

Potassium phosphate buffer pH 7.8

1
2-Oxoglutarate
0.3
0.3
NH4CI
0.5
0.5
NADH
0.12

NADPH

0.12
Enzyme extract
0.2
0.2
Water
8
8
 
2.
Add 0.3mL of water in the blank instead of 2-Oxoglutarate.
3.
Incubate the reaction mixture at 37°C for 15-30 min.
4.
Record the change in absorbance at 340nm.
 
Calculation
The amount of NADH or NADPH oxidized is calculated from the molar extinction coefficient. Activities are expressed as n mole NAD(P)H oxidized per min per mg protein.
 
Nanomole of NAD(P)H oxidized/min/mg protein
=
A 340 X vol of assay solution X 1000
6.22 X Time of incubation (min) X mg protein in enzyme extract used
 
 


References
1.  Delma Dohertry (1970) In: Methods in Enzymol Vol 17 Part A (Eds Tabor, H and Tabor, CW) p 850.
 
     
 
 
     




     
 
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