Abstract
The enzyme γ-glutamyltranspeptidase (GGT, EC 2.3.2.2) is highly conserved among eukaryotic and prokaryotic organisms (Heisterkamp et al., 2008) and has a key function in glutathione metabolism. Although the enzyme is highly conserved and found throughout organisms ranging from bacteria to plants and animals several major difference between eukaryotic and prokaryotic GGT can be noticed. They mainly concern the enzyme localization and posttranslational modification. Eukaryotic GGT is cell membrane anchored and highly glycosylated whereas prokaryotic GGT does not undergo this posttranslational modification and is a soluble periplasmic protein. GGT amino acids sequences of diverse origin exhibit high amino acid similarity (Ong et al., 2008). The prokaryotic GGT enzymes are produced as proenzyme, equipped with a typical prokaryotic signal sequence and transported through the inner membrane into the periplasm where the enzyme undergoes autocatalytic cleavage. This proteolysis yields a mature dimer which transfers the γ-glutamyl moieties from extracellular glutathione and related compounds to amino acids or peptides (Hanigan et al., 1998). The GGT enzyme activity can be easily measured as it catalyzes the transfer of a γ-glutamyl group from a colorless substrate, L-γ-glutamyl-3-carboxy-4-nitroanilide, to the acceptor, glycylglycine with leads to the production of yellow colored product, p-nitroaniline (Figure 1) which can be measured by a spectrophotometer (Figure 2). Here we describe a protocol to measure the GGT activity in the Gram-negative bacterium Campylobacter jejuni, with some minor modifications; this protocol also works for other Gram-negative bacterial species.Figure 1. Yellow colored product, p-nitroaniline formed during the GGT enzyme assayFigure 2. Spectral curve of pNA in Tris/HCl buffer, recorded on a Biodrop µLite (Isogen)
Keywords: Campylobacter, Bacterial GGT assay, Glutamyltranspetidase, Periplasmic enzym assay, Glutathione
Materials and Reagents
Equipment
Procedure
Figure 3. A typical pNA standard curve Figure 4. Graph showing in time the pNA increase in the bacterial lysates made of C.jejuni wt (red) and racRS mutant (blue) To calculate the GGT activity first plot the pNA standard curve. Calculate the Δabsorbance (A time x - A time y) in a linear range. Next, compare the calculated Δabsorbance of each sample to the standard pNA curve to determine the amount of pNA generated between time x and time y (nmol pNA). The GGT activity is calculated by the formula: (nmol pNA) T x M T = the time incubated (in min) M = mg protein determined by BCA protein kit GGT activity is reported as nmole/min-1/mg protein-1 Example: Δabsorbance(wt) = 0.58 (15 min) - 0.43 (5 min)=0.15 Δabsorbance 0.15 = 40 nmole pNA Time x - time y = 10 min M = 0.02 mg protein GGT activity is: 40/(10 x 0.02) = 200 nmole/min-1/mg protein-1
Recipes
Acknowledgments
The γ-glutamyltranspeptidase activity protocol described here and in, van der Stel et al. (2015), is modified from a protocol described in Chevalier et al. (1999). This work was supported by the NWO-ECHO grant 711.012.007 to MW.
References
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