Materials and Reagents

1. p-aminobenzoic acid (Sigma-Aldrich, catalog number: P5669 )
   
2. d-Biotin (Sigma-Aldrich, catalog number: B4501 )
   
3. Calcium-D-pantothenate (Sigma-Aldrich, catalog number: 21210 )
   
4. Riboflavine (Sigma-Aldrich, catalog number: R4500 )
   
5. Pyridoxine hydrochloride (Sigma-Aldrich, catalog number: P9755 )
   
6. KCl
   
7. MgSO_4^.7H₂O
   
8. KH₂PO₄
   
9. Na₂B₄O₇^.10H₂O
   
10. CuSO₄^.5H₂O
    
11. FeO₄P^.4H₂O
    
12. MnSO_4^.H₂O
    
13. Na₂MoO₄^.2H₂O
    
14. ZnSO₄^.7H₂O
    
15. NaOH
    
16. Tween 80 (Sigma-Aldrich, catalog number: P1754 )
    
17. Radiolabelled substrate
    e.g. [8-³H]-xanthine, 22.8 Ci/mmol (Moravek Biochemicals, catalog number: MT537 )
    [2,8-³H]-hypoxanthine, 27.7 Ci/mmol (Moravek Biochemicals, catalog number: MT700 )
    [5-³H]-uracil, 23 Ci/mmol (Moravek Biochemicals, catalog number: MT610 )
    
18. Non radiolabelled substrate
    e.g. Xanthine (Sigma-Aldrich, catalog number: X7375 )
    Hypoxanthine (Sigma-Aldrich, catalog number: H9377 )
    Uracil (Sigma-Aldrich, catalog number: U0750 )
    
19. Toluol (AppliChem GmbH, catalog number: A3393 )
    
20. Triton X-100
    
21. 2,5-Diphenyloxazole (PPO) (Sigma-Aldrich, catalog number: D4630 )
    
22. 1,4-bis (5-phenyloxazol-2-yl) benzene (POPOP) (Sigma-Aldrich, catalog number: P3754 )
    
23. Complete Media (CM) (see Recipes)
    
24. Minimal Media (MM) (see Recipes)
    
25. Scintillation Fluid (see Recipes)
    

Equipment

1. Petri dishes
   
2. Neubauer counting-chamber slide
   
3. Spatula
   
4. Orbital shaking incubator
   
5. Incubator at 37 °C
   
6. Nylon net filter 60 μm (Merck KGaA, catalog number: NY60 )
   
7. 50 ml Falcon tubes
   
8. 1.5 ml centifuge tubes
   
9. Centrifuge
   
10. Vortex
    
11. Scintillation vials
    
12. Scintillation counter
    
13. Heat block
    
14. Magnetic stirrer
    
15. Magnetic strirr bar
    
16. pH meter
    
17. Pasteur pipette
    

Software

1. GraphPad Prism software (Amillis et al., 2004)

Procedure