PRPS activity assay

Abstract

The PRPS (EC 2.7.6.1) catalyzes the reaction: ATP +R5P (D-ribose 5-phosphate)→ PRPP (5-phospho-alpha-D-ribose 1-diphosphate) +AMP . Here, we describe an assay to examine the activity of PRPS.  The forward reaction: OA (orotate) + PRPP  →OMP (orotidine 5-phosphate)  +PPi (diphosphate) catalyzed by  E. coli  orotate phosphoribosyltransferase (OPRT, EC 2.4.2.10) is coupled with PRPS reaction. The amount of PRPP generated in the reaction is indicated by the reduction of orotate (OA) in the mixture. The concentration of OA is measured by absorbance at 295 nm.

Keywords: PRPS, enzyme activity, OPRT, orotate, PRPP.

Materials and Reagents

1. Purified proteins: OPRT (EC 2.4.2.10), PRPS (EC 2.7.6.1)

2. Reagents: 10 mM OA (Orotic acid, Adamas, Catalog number: 01102798 (74736A))

   100/50/10 mM ATP (Takara, Catalog number: 4,041)

   10 mM ADP (Sigma, Catalog number: A2754-100MG)

   10 mM AMP (solarbio, Catalog number: A9860-1)

   50/10 mM R5P (Sigma, Catalog number: P8296-25 mg)

   1M MgCl₂, 5 M NaCl, 1M/10 mM Na₂HPO₄, ddH₂O.

3. 96-well clear flat bottom UV-transparent microplate (Corning, Catalog number: 3635)

4. BCA protein Concentration Determination Kit (Enhanced) (Beyotime, Catalog number: P0010)

    

Equipment and software

• The SpectraMax i3 platform comes with microplate detection system for absorbance measuring at 295 nm.

• Graphpad Prism for data analysis.

   

Procedure

1. Drawing the standard curve of OA concentration.

   OA concentration (mM)	0	0.125	0.25	0.5	0.75	1
   10 mM OA (μl)	0	2.5	5	10	15	20
   1 M Na2HPO4 (μl)	10	10	10	10	10	10
   5 M NaCl (μl)	10	10	10	10	10	10
   1 M MgCl2 (μl)	2	2	2	2	2	2
   ddH2O (μl)	178	175.5	173	168	163	158
   Total (μl)	200	200	200	200	200	200

   Add the reagents as listed in the table above to the 96-well UV-transparent microplate. Repeat each column for 3 times. Transfer the 96-well plate to the SpectraMax i3. Set the program in SpectraMax i3: Shake the 96-well plate gently for 5s (mix up the mixture)  and read the absorbance at 295 nm at 37 °C. Drawing the standard curve of OA concentration.

2. Measuring the concentrations of purified proteins

   Use the protocol of BCA Kit to determinate the concentrations of purified proteins.

3. PRPS activity measurement with different concentrations of ATP

   ATP concentration (mM)	Blank	0.2	0.3	0.4	0.5	0.6	0.7	0.8
   10 mM OA (μl)	20	20	20	20	20	20	20	20
   1 M Na2HPO4 (μl)	2	2	2	2	2	2	2	2
   5 M NaCl (μl)	10	10	10	10	10	10	10	10
   1 M MgCl2 (μl)	2	2	2	2	2	2	2	2
   1 mM OPRT (μl)	4	4	4	4	4	4	4	4
   15.4 μM PRPSWT (μl)	0	1.3	1.3	1.3	1.3	1.3	1.3	1.3
   10 mM ATP (μl)	0	4	6	8	10	12	14	16
   ddH2O (μl)	159.6	154.3	152.3	150.3	148.3	146.3	144.3	142.3
   50 mM R5P (μl)	2.4	2.4	2.4	2.4	2.4	2.4	2.4	2.4
   Total (μl)	200	200	200	200	200	200	200	200

   Add the reagents and proteins as listed in the table above to the 96-well UV-transparent microplate. Repeat each column for 3 times. Add the R5P last with a multi-channel pipette to initiate the reaction and immediately transfer the 96-well plate to the SpectraMax i3. Preset the program in SpectraMax i3: Shake the 96-well plate gently for 5 s (mix up the mixture)  and read the absorbance at 295 nm for 300 s  with internal time of 17 s at 37 °C. Convert the Abs_295nm to OA concentration referred to standard curve. Use the consumption of OA to indicate PRPP generation. Calculate the Vmax of PRPP generation with different ATP concentrations and draw the graph. The reaction mixture (200 μl) contains 0.1μM PRPS, 20 μM OPRT, 1 mM OA, 10 mM MgCl₂, 10 mM Na₂HPO₄, 250 mM NaCl, 0.6 mM R5P.

4. Measuring PRPS activity with different amount of other ligand (eg. R5P, ADP, AMP, Na₂HPO₄). 

   For activity measurement with different amount of R5P, add ATP last to initiate the reaction. 

   For Na₂HPO₄, add R5P last to initiate the reaction and the reaction mixture (200 μl) contains 0.1μM PRPS, 20 μM OPRT, 1 mM OA, 10 mM MgCl₂, 250 mM NaCl, 0.6 mM R5P, 0.6 mM ATP. 

   For ADP/AMP, add R5P last to initiate the reaction and the reaction mixture (200 μl) contains 0.1μM PRPS, 20 μM OPRT, 1 mM OA, 10 mM MgCl₂, 250 mM NaCl, 0.6 mM R5P, 0.6 mM ATP and 10 mM Na₂HPO₄.

    

General notes

#Note: the 96-well plate must be UV-transparent for absorbance measuring at 295 nm.

# For drawing the standard curve of OA concentration, set a proper concentration gradient to make sure the absorption value within the best reading range of the instrument.  

 # Conduct preliminary experiments to determinate the concentrations of PRPS and OPRT in the reaction mixture.  Detect the activity of purified OPRT and compare it with reported data in other literature.

# Use multi-channel pipette to add R5P/ATP to initiate the reaction and preset the program for measuring Abs_295nm. Make the reactions in different wells started and quickly detected at the same time.