1-phosphate and 2-amino-6-mercapto-7-methyl-purine, which can
be measured at 360 nm, hence allowing for the quantification of
the PPi released during the enzymatic reaction.
In this article, the method is used to measure the activity of the
Arabidopsis thaliana GGPPS11 protein (At4g36810), a GGPP
synthase (EC 2.5.1.29) that supplies GGPP for the production of
carotenoids and other groups of plastidial isoprenoids [32]. This
enzyme has been well characterized in previous studies [21, 31,
33], which served as a reference to validate the results obtained with
the reported assay.
2 Materials
1. Reaction buffer (20Â): 1 M Tris–HCl, pH 7.5, 20 mM MgCl 2
(see Note 1).
2. Solid Tris Base. Tris(hydroxymethyl)aminomethane.
3. Hydrogen chloride (HCl) 37%.
4. Solid magnesium dichloride (MgCl 2 ).
5. 2-Amino-6-mercapto-7-methylpurine ribonucleoside (MESG):
1 mM in milli-Q water (store at À20
C) (see Note 2).
6. Purine nucleoside phosphorylase (PNP): 100 U/mL in milli-Q
water (store at 4
C) (see Note 3).
7. Inorganic pyrophosphatase: 3 U/mL in 1Â Reaction buffer
(store at 4
C) (see Note 4).
8. PPi standard: 500 μM in milli-Q water (see Note 5).
9. IPP and DMAPP (Echelon Biosciences Inc.): 1 mM in milli-Q
water (store at À80
C) (see Note 6).
10. Eppendorf Safe-Lock Tubes™ of 1.5 and 2 mL capacity.
11. Sterile polystyrene 96 well-plates (costar
® ) with low evaporation lid and flat bottom.
12. SpectraMax M3 multi-mode microplate reader (Molecular
Devices).
13. GraphPad Prism Software.
3 Methods
3.1 Standard GGPPS
Activity Assay
All 200 μL enzyme reactions should be carried out in 96-well plates
(Fig. 2). The empty wells can be reused for subsequent experiments. Always perform three technical replicates per condition
and at least two independent experiments.
1. Prepare the reaction mixture (Table 1) by adding 10 μL of 20Â
reaction buffer (see Note 7), 40 μL of 1 mM MESG substrate,
30
M. Victoria Barja and Manuel Rodrı ´guez-Concepcio ´ n
be measured at 360 nm, hence allowing for the quantification of
the PPi released during the enzymatic reaction.
In this article, the method is used to measure the activity of the
Arabidopsis thaliana GGPPS11 protein (At4g36810), a GGPP
synthase (EC 2.5.1.29) that supplies GGPP for the production of
carotenoids and other groups of plastidial isoprenoids [32]. This
enzyme has been well characterized in previous studies [21, 31,
33], which served as a reference to validate the results obtained with
the reported assay.
2 Materials
1. Reaction buffer (20Â): 1 M Tris–HCl, pH 7.5, 20 mM MgCl 2
(see Note 1).
2. Solid Tris Base. Tris(hydroxymethyl)aminomethane.
3. Hydrogen chloride (HCl) 37%.
4. Solid magnesium dichloride (MgCl 2 ).
5. 2-Amino-6-mercapto-7-methylpurine ribonucleoside (MESG):
1 mM in milli-Q water (store at À20
C) (see Note 2).
6. Purine nucleoside phosphorylase (PNP): 100 U/mL in milli-Q
water (store at 4
C) (see Note 3).
7. Inorganic pyrophosphatase: 3 U/mL in 1Â Reaction buffer
(store at 4
C) (see Note 4).
8. PPi standard: 500 μM in milli-Q water (see Note 5).
9. IPP and DMAPP (Echelon Biosciences Inc.): 1 mM in milli-Q
water (store at À80
C) (see Note 6).
10. Eppendorf Safe-Lock Tubes™ of 1.5 and 2 mL capacity.
11. Sterile polystyrene 96 well-plates (costar
® ) with low evaporation lid and flat bottom.
12. SpectraMax M3 multi-mode microplate reader (Molecular
Devices).
13. GraphPad Prism Software.
3 Methods
3.1 Standard GGPPS
Activity Assay
All 200 μL enzyme reactions should be carried out in 96-well plates
(Fig. 2). The empty wells can be reused for subsequent experiments. Always perform three technical replicates per condition
and at least two independent experiments.
1. Prepare the reaction mixture (Table 1) by adding 10 μL of 20Â
reaction buffer (see Note 7), 40 μL of 1 mM MESG substrate,
30
M. Victoria Barja and Manuel Rodrı ´guez-Concepcio ´ n