KOH: P5958 SIGMA (make a 5N stock) HCl: 339253 SIGMA (make a 5M stock)
Just before the experiment, add MgCl2. For isolated mitochondria, use 1 mM final concentration; for permeabilized cells, 1.5 mM (unless you calibrate the magnesium green signal, or determine Kds of ADP or ATP for Mg2+, see under 'Kds determination of ADP, ATP to Mg2+').
MgCl2: M1028 SIGMA (1 M stock solution).
Do NOT keep buffer containing MgCl2 for more than one day!
Magnesium Green, pentapotassiumsalt
Water (add 993 microl of water in 1 mg vial); make at least 10 aliquots; store in -20 oC
Water. pH between 6.2 and 6.9. Freeze to -20 oC in aliquots. Aliquots can be reused up to 3 times
A7699 SIGMA do NOT use other type of ATP
Water; pH to 7.0
Other chemicals required, depending on the experimental scheme:
Preparation of sodium orthovanadate (Na3VO4) and beryllium trifluoride (BeF3-): A 25 mM Na3VO4 solution is prepared in distilled water (>17 megaOhm resistance). The pH of the solution is adjusted to 8.7 with HCl, upon which it turns yellow. This solution is boiled until it turns colorless and cooled to room temperature. The pH is reassessed, and readjusted to pH 8.7 with HCl, upon which the solution turns yellow again. This cycle of boiling until colorless and readjusting the pH is repeated until the solution remains colorless at pH 8.7. Finally, the solution is brought up to the initial volume with distilled water and stored in aliquots at -20 °C. This treatment removes all decavanadate ions present in the Na3VO4 solution, which induces mitochondrial membrane depolarization and inhibition of oxygen consumption (Aureliano and Crans 2009). Orthovanadate inhibits the oxidation of only disrupted mammalian mitochondria (Byczkowski et al. 1979).
Likewise, fluoroberyllium nucleoside diphosphate complexes inhibit only the exposed F1F0-ATPase (Issartel et al. 1991). BeSO4 and NaF are prepared as aqueous stock solutions of 0.2 M and 0.5 M, respectively, and kept at +4 °C for several years. BeF3- is formed immediately in solution upon mixing of BeSO4 and NaF, provided that NaF is in excess in the working solution. BeF3- is formed immediately in solution upon mixing of 2 microliters of 0.2 M in a 2 ml cuvette volume containing the buffer of your choice (i.e. 200 micromolar BeSO4 final concentration) and 20 microliters of 0.5 M NaF, (i.e. 5 millimolar NaF final concentration). As such, final concentration of BeF3- will be 200 micromolar.
Vanadate, beryllium and fluoride salts are highly toxic to tissues and to the environment, and thus require proper handling and disposal. The combination of orthovanadate and BeF3- will inhibit kinases, mutases, phosphatases, and ATPases (Ray et al. 1990; Climent et al. 1981). However, some kinases, such as pyruvate kinase, will remain uninhibited (Lord and Reed 1990). In this respect, upon permeabilization of the cells one has to totally separate pyruvate kinase from its substrate, phosphoenol pyruvate, i.e. there must be no glucose present in the medium prior to permeabilization, and a few minutes lag time must be allowed prior to ADP-ATP exchange rate measurements in order for the remaining reactions by kinases to ‘die-out’.
*Concentration of ADP and ATP stock solutions is corrected by measuring absorbance at 260 nm using an extinction coefficient eM= 15,400 M-1*cm-1.