Materials and Reagents

1.5 ml Eppendorf tubes (Eppendorf, catalog number: 022363204 )
Micropipette tips (200 μl) (Sigma-Aldrich, catalog number: CLS4866 )
Micropipette tips (1,000 μl) (Sigma-Aldrich, catalog number: CLS4868 )
Glass beads (unwashed) (212-300 μm) (Sigma-Aldrich, catalog number: G9143 )
1.5 ml polystyrene spectrophotometer cuvettes with 10 mm path length (Sigma-Aldrich, catalog number: C5416 )
Parafilm (Sigma-Aldrich, catalog number: P7793 )
Bacterial cells
Note: Amount of the cell depends on how many assays will be carried out. Supernatant yielded from a cell pellet of 50 ml well-grown cyanobacterial culture (OD₇₅₀ ≥ 1.0) and 10 ml overnight grown Escherichia coli will be sufficient for about 20 assays depending on what volume is used for each assay.
Trizma^® base (Sigma-Aldrich, catalog number: T1503 )
β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
Glucose-6-phosphate disodium salt (G6P) (Sigma-Aldrich, catalog number: G7250 )
Potassium phosphate dibasic trihydrate (K₂HPO₄·3H₂O) (Sigma-Aldrich, catalog number: P5504 )
Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P0662 )
Magnesium sulfate (MgSO₄) (Sigma-Aldrich, catalog number: M7506 )
Hydrochloric acid (HCl) (36.5-38.0%) (Sigma-Aldrich, catalog number: H1758 )
Maleic acid (Sigma-Aldrich, catalog number: M0375 )
β-nicotinamide adenine dinucleotide phosphate hydrate (NADP⁺) (Sigma-Aldrich, catalog number: N5755 )
Paraffin
50 mM Tris-maleate solution (see Recipes)
Extraction buffers
1. Tris-maleate buffer, pH 6.8 for Synechocystis sp. PCC6803 (see Recipes)
2. Potassium phosphate buffer, pH 6.8 for E.coli (see Recipes)
Assay buffers
a.Tris-maleate buffer, pH 7.4 for Synechocystis sp. PCC6803 (see Recipes)
b.Tris-HCl buffer, pH 8.0 for E.coli (see Recipes)
500 mM Tris-HCl stock solution (pH 8.0) (see Recipes)
500 mM G6P solution (see Recipes)
100 mM NADP⁺ solution (see Recipes)

Equipment

Micropipettes (20-200 μl capacity) (Nichiryo, catalog number: 00-NPX2-200 )
Micropipettes (100-1,000 μl capacity) (Nichiryo, catalog number: 00-NPX2-1000 )
Microcentrifuge, 1.5 ml Eppendorf tube rotor and at least 10,000 x g force (Sigma-Aldrich, Hettich^®, model: MIKRO120 )
FastPrep FP120 (BioSurplus, Thermo-Savant, model: FP120 )
Light microscope
Vortex mixer (Bibby Scientific, Stuart, model: SA8 )
Balance (Precision Weighing Balances, Salter Brecknell, model: ESA-150 )
pH meter (Hanna Instruments, model: HI5221 )
Vis-Spectrophotometer with 1.5 ml cuvette holder (Shimadzu Scientific Instruments, model: UV-1800 )

Procedure

Extraction of G6PDH from bacterial cells
Note: Bacterial cells and cell-free extracts must be kept on ice or at 4 °C throughout all steps of extraction.
1. Resuspend the cell pellets in 500 μl extraction buffer (Tris-maleate buffer pH 6.8 for cyanobacterial cells or potassium phosphate buffer pH 6.8 for E.coli cells). The extraction buffers keep G6PDH enzyme in an active state.
  Note: G6PDH of the photosynthetic prokaryotes like cyanobacteria is a redox modulated oligomeric enzyme. In case, redox modulation properties are planned to be tested, β-mercaptoethanol must be omitted from the buffers.
2. To wash glass beads, add 500 μg glass beads and 1 ml extraction buffer to two separate Eppendorf tubes, mix with a whirly mixer well, centrifuge at 10,000 x g for 1 min, and discard the supernatant.
3. Add 500 μl cell suspension to each glass beads containing tubes and mix the tube to resuspend the glass beads.
4. Put the tubes in fast prep FP120 and run the device twice at 5.5 m/sec for 40 sec.
  Note: This step may be done by mixing the tubes vigorously on a whirly mixer for 1-2 min until most of the cells are disrupted. This may be confirmed by examining a small amount of the crude extract for unbroken cells under a light microscope.
5. Centrifuge the extract at 10,000 x g for 10 min.
6. Transfer the supernatant to a new Eppendorf tube and keep at 4 °C. Use this cell-free extract as enzyme solution.
  Note: It is better to use the extract immediately. However, the extract may be stored at 4 °C for a week albeit some degree of activity loss.
Assay of G6PDH activity in cell-free extract
1. Set the wavelength of spectrophotometer to 340 nm. Select absorbance function.
2. Prepare a blank and a test mixture as follows (Table1).
  
  Table 1. Regent contents of test and blank mixtures
3. Add assay buffer, NADP⁺ and enzyme solutions first to a 1 ml spectrophotometer cuvette, then, start the enzyme reaction by adding G6P and mix the content by flipping the paraffin sealed cuvette.
  Note: Assay buffer is Tris-maleate (pH 7.4) for cyanobacteria and Tris-HCl (pH 8.0) for E.coli. A blank reaction mixture is needed to check any endogenous NADP⁺ reduction in enzyme solution. A minimum amount of 200 mg protein per assay is necessary albeit it may vary depending on factors such as growth phase of the culture used.
4. Place the cuvette in the cell holder of the spectrophotometer, close the lid and follow the absorbance for 3-5 min at room temperature by recording absorbance value in 15-30 sec intervals. Temperature of the reaction mixture should be kept at a certain point. Some enzymes need specific temperature like 37 °C. In this case reaction cuvette must be treated with a thermostatic apparatus integrated to the spectrophotometer. However, some enzymes work well at room temperature.
5. Estimate ∆A₃₄₀ (rate) dividing the total absorbance change to the total assay time in min. For example, if the absorbance change in a reaction mixture at 340 nm is 0.450 after 3 min, the rate is 0.150.
  Note: Some spectrophotometers have rate measurement function. Using such a device, the rate may be determined directly.
6. Repeat steps B4 to B6 for the blank tube to test whether any detectable endogenous NADP⁺ is present in the enzyme solution. If so, subtract this value from the test value to find net ∆A₃₄₀ value.
Estimation of unit of activity and specific activity of G6PDH
One of the standard expression ways of enzyme activity is the unit of activity. Unit of G6PDH activity is defined here as the formation of 1 μmol NADPH in one min. NADP⁺ reduction (G6PDH activity) determined as rate (∆A₃₄₀) is needed to be converted to concentration in μmol. Once the unit of activity is determined, it is often converted to specific activity. Specific activity is expressed as units per mg protein.
1. Estimate G6PDH activity in units by using the formula as follows:
  
  In this protocol the dilution factor is 20 (50 μl extract in 1,000 μl test or blank mixture). For example, if a rate of 0.85 is yielded in the test assay and 0.05 in the blank assay, the net rate will be 0.8. Then the units of activity will be estimated as 0.8 x 20/6.22 = 2.572 μmol min^-1 ml^-1.
2. To estimate specific activity, the protein amount in the extract must be determined by a standard method such as Bradford assay. Specific activity is then estimated as the amount of units per mg protein by using the formula below:
  
  For example, if the protein content of the extract is 0.5 mg ml^-1, the specific activity will be 2.572/0.5 = 5.44 units/mg protein.

Notes

One important point is to keep the enzyme in the active state in the supernatant especially by protecting it from oxidation damage. To avoid oxidation damage and keep the enzyme in the active state, the reducing agent β-mercaptoethanol may be added to the supernatant. However, this agent also reduces disulphide bonds in the enzyme and should be omitted when studying the redox properties of the enzyme.
It is known that higher amounts of G6PDH, G6P and NADP positively affect the enzyme’s activity. If the effects of some agents on G6PDH activity are studied, minimal amounts of enzyme solution and substrates should be used. It is difficult to define standard minimal concentrations for enzyme and substrates since several factors may affect these values. Therefore, the researchers must determine minimal concentrations themselves.

Recipes

50 mM Tris-maleate solution
Dissolve 6.05 g Trizma-base in 800 ml water and adjust pH to 6.8 (for extraction buffer) or 7.4 (for assay buffer) by adding maleic acid.
Keep the solution at room temperature for a few months.
Tris-maleate extraction buffer
50 mM Tris-maleate (pH 6.8)
0.1% β-mercaptoethanol
10 mM G6P
Mix the reagents just before use.
Tris-maleate assay buffer
50 mM Tris-maleate (pH 7.4)
0.1% β-mercaptoethanol
10 mM MgSO₄
Potassium phosphate extraction buffer (pH 6.8) (Barnell et al., 1990)
50 mM K₂HPO₄
50 mM KH₂PO₄
Titrate against each other to adjust pH to 6.8.
500 mM Tris-HCl stock solution pH 8.0
Dissolve 60.57 g Trizma base in 800 ml water and adjust pH to 8.0 with 2 N HCl solution.
Keep the solution at room temperature for a few months.
Tris-HCl assay buffer for E.coli (Hylemon and Phibbs,1972)
50 mM Tris-HCl (pH 8.0)
0.1% β-mercaptoethanol
10 mM MgSO₄
500 mM G6P solution
Prepare 500 mM G6P solution (152.05 mg G6P in 1 ml ultrapure water), separate in aliquots and store at -20 °C for a few months.
100 mM NADP⁺ solution
Prepare 100 mM NADP⁺ solution (74.34 mg NADP⁺ in 1 ml ultrapure water), separate in aliquots and store at -20 °C for a few months.

Acknowledgments

This protocol was adapted and modified from previously published studies by Schaeffer and Stanier (1978), Hylemon and Phibbs (1972) and Barnell et al. (1990) and applied in the studies of Karakaya et al. (2008 and 2012) which were supported by TUBITAK (Project TBAG 1985 100T100) and Ondokuz Mayıs Üniversity (Projects F261 and PYO 1904 09 21).

References

Barnell, W. O., Yi, K. C. and Conway, T. (1990). Sequence and genetic organization of a Zymomonas mobilis gene cluster that encodes several enzymes of glucose metabolism. J Bacteriol 172(12): 7227-7240.
Hylemon, P. B. and Phibbs, P. V., Jr. (1972). Independent regulation of hexose catabolizing enzymes and glucose transport activity in Pseudomonas aeruginosa. Biochem Biophys Res Commun 48(5): 1041-1048.
Karakaya, H., Ay, M. T., Ozkul, K. and Mann, N. H. (2008). A Delta zwf (glucose-6-phosphate dehydrogenase) mutant of the cyanobacterium Synechocystis sp PCC 6803 exhibits unimpaired dark viability. Annals of Microbiology 58(2): 281-286.
Karakaya, H., Erdem, F., Özkul, K. and Yilmaz, A. (2012). Analysis of glucose-6-phosphate dehydrogenase of the cyanobacterium Synechococcus sp. PCC7942 in the zwf mutant Escherichia coli DF214 cells. Annals of Microbiology 63: 1319-1325.
Schaeffer, F. and Stanier, R. Y. (1978). Glucose-6-phosphate dehydrogenase of Anabaena sp. kinetic and molecular properties. Arch Microbiol 116: 9-19.