Difference between revisions of "ATP"

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ATP

Description

Adenosine triphosphate is a nucleotid and functions as the major carrier of chemical energy in the cells. As it transfers its energy to other molecules, it looses its terminal phosphate group and becomes adenosine diphosphate (ADP).

Abbreviation: T

Reference: MiPNet03.02 Chemicals-Media

MitoPedia topics: Substrate and metabolite

Application in HRR

Rationale

ATP may be titrated to mt-preparations in ETS-competent substrate states to quantify ATPase activity before any addition of ADP. As ATP is hydrolysed to ADP, OXPHOS is activated by ADP and respiration increases accordingly. High-quality isolated mitochondria contain zero or very low ATPase activity, such that resipration in the absence and presence of ATP is identical.
Intracellular ATP levels are in the mM range, but many respiratory studies of mt-preparations are carried out at zero or µM concentrations of ATP. ADP kinetics of isolated rat liver mitochondria reveals a K´_m,D of 56 µM ADP in the presence of 2 mM ADP ( Gnaiger 2001), which is higher than the frequently cited value of 15 to 20 µM ADP.
ATP stabilizes mitochondrial function and is therefore used in preservation solutions BIOPS and MiP03 (MiPNet03.02 Chemicals-Media). This may provide a rational to add mM concentrations of ATP after stimulation of mt-preparations by ADP in various SUIT protocols.

Sources of ATP

T: ATP (Adenosine 5'-triphosphate disodium salt hydrate, C₁₀H₁₄N₅O₁₃P₃Na₂* (H₂O)_x); Sigma A 2383, 5 g, store at -20 °C; FW = 614.1 containing 3.5 mol H₂O/mol (FW_{anhydrous basis} = 551.1). Alternative source: Merck 1191-1GM (Calbiochem, Adenosine 5′-Triphosphate Disodium Salt; FW = 551.2; store at -20 °C).

Caution: Chemicals stored in the fridge or freezer should be allowed to reach room temperature before opening.

Preparation of 500 mM ATP stock solution

1) Weigh 614.1 mg of ATP (FW = 614.1 containing 3.5 mol H₂O/mol).

2) Add 1.2 ml H₂O.

3) Neutralize with 5 N KOH (approx. 400 µl) - ATP dissolves after addition of KOH (this is Solution A).

4) Check pH and adjust to pH 7 if necessary.

5) Adjust final volume to 2 ml and divide into 0.2 ml portions.

6) Store frozen at -80 °C.

Preparation of 500 mM ATP stock solution with 3 mM free Mg²⁺

To keep free [MgMg²⁺] constant during respiration measurement in MiR05 or MiR06, mix ATP with MgCl₂ (0.8 mol MgCl₂ /mol ATP).

MgCl₂ (Scharlau MA0036: MgCl₂.6H₂O, FW = 203.3)

1) Add 162.64 mg MgCl₂ to Solution A. A white precipitate forms, which dissolves after 1-2 min of stirring on magnetic stirrer at room temperature.

2) Follow steps 4-6 of the above instructions.

Comment: In some cases it was observed, that after defreezing of the 'ATP-Mg' a white precipitate forms again. If this is the case we recommend to prepare the 500 mM ATP stock solution and a MgCl₂ solution (0.8 mol MgCl₂ /mol ATP) seperately and perform a titration of both solutions in series.

Oxygraph-2k manual titrations

>> MiPNet09.12 O2k-Titrations

Titration volume: 4-20 µl using a 25 µl syringe.
Final conc. in 2 ml O2k-chamber: 1-5 mM.

@@ Line 2: / Line 2: @@
 |abbr=T
 |description='''Adenosine triphosphate''' is a nucleotid and functions as the major carrier of chemical energy in the cells. As it transfers its energy to other molecules, it looses its terminal phosphate group and becomes adenosine diphosphate ([[ADP]]).
-|info=[[MiPNet03.02]]; [[MiPNet09.12]]
+|info=[[MiPNet03.02 Chemicals-Media]]
 |type=Substrate PS
 }}
@@ Line 14: / Line 14: @@
 == Application in [[HRR]] ==
+== Rationale ==
+* ATP may be titrated to mt-preparations in ETS-competent substrate states to quantify ATPase activity before any addition of ADP. As ATP is hydrolysed to ADP, OXPHOS is activated by ADP and respiration increases accordingly. High-quality isolated mitochondria contain zero or very low ATPase activity, such that resipration in the absence and presence of ATP is identical.
+* Intracellular ATP levels are in the mM range, but many respiratory studies of mt-preparations are carried out at zero or µM concentrations of ATP. ADP kinetics of isolated rat liver mitochondria reveals a ''K''´<sub>m,D</sub> of 56 µM ADP in the presence of 2 mM ADP ([[Gnaiger 2001 Respir Physiol | Gnaiger 2001]]), which is higher than the frequently cited value of 15 to 20 µM ADP.
+* ATP stabilizes mitochondrial function and is therefore used in preservation solutions BIOPS and MiP03 ([[MiPNet03.02 Chemicals-Media]]). This may provide a rational to add mM concentrations of ATP after stimulation of mt-preparations by ADP in various [[SUIT]] protocols.
+== Sources of ATP ==
 '''T: ATP''' (Adenosine 5'-triphosphate disodium salt hydrate, C<sub>10</sub>H<sub>14</sub>N<sub>5</sub>O<sub>13</sub>P<sub>3</sub>Na<sub>2</sub>* (H<sub>2</sub>O)<sub>x</sub>); Sigma A 2383, 5 g, store at -20 °C; FW = 614.1 containing 3.5 mol H<sub>2</sub>O/mol (FW<sub>anhydrous basis</sub> = 551.1).  '''Alternative source:'''  Merck  1191-1GM (Calbiochem, Adenosine 5′-Triphosphate Disodium Salt; FW = 551.2; store at -20 °C).
@@ Line 24: / Line 32: @@
 ::1) Weigh 614.1 mg of ATP (FW = 614.1 containing 3.5 mol H<sub>2</sub>O/mol).
-::2) Add 1.2 ml H<sub>2</sub>O
+::2) Add 1.2 ml H<sub>2</sub>O.
 ::3) Neutralize with 5 N KOH (approx. 400 µl) - ATP dissolves after addition of KOH (this is '''Solution A''').
 ::4) Check pH and adjust to pH 7 if necessary.