Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state

Abstract

The membrane potential of mitochondria was estimated from the accumulation of tetraphenyl phosphonium (TPP+), which was determined with the TPP+-selective electrode developed in the present study. The preparation and some operational parameters of the electrode were described. The kinetics for uptake by mitochondria of TPP+ and DDA+ (dibenzyldimethyl ammonium) were analyzed, and it was found that TPP+ permeated the mitochondrial membrane about 15 times faster than DDA+. The final amounts of accumulation of TPP+ and DDA+ by mitochondria were approximately equal. For the state-4 mitochondria, the membrane potential was about 180 mV (interior negative). Simultaneous measurements of TPP+-uptake and oxygen consumption showed that the transition between states 3 and 4 was detectable by use of the TPP+-electrode. After the TPP+-electrode showed that state-4 was reached, the extra-mitochondrial phosphorylation potential was measured. The difference in pH across the membrane was measured from the distribution of permeant anion, acetate, so as to calculate the proton electrochemical potential. The ratio of extra-mitochondrial phosphorylation potential to proton electro-chemical potential, n was close to 3. This value of n was also found to be 3 when ATP was hydrolyzed under the condition that the respiratory chain was arrested. The implication that n = 3 was discussed.

Interactions of melatonin with mammalian mitochondria. Reducer of energy capacity and amplifier of permeability transition

“…across the mitochondrial membrane with a selective electrode, prepared according to published procedures (Kamo et al 1979). The obtained values were corrected as reported by Jensen et al (1986).…”

Role of cardiolipin on tBid and tBid/Bax synergistic effects on yeast mitochondria

“…The use of glycerol/ethanol as a carbon source ensured a full differentiation of mitochondria. Mitochondria were isolated from zymoliase-treated cells, as described in Law et al 41 For bioenergetic measurements, mitochondria (0.33 mg/ml) were suspended in respiration buffer (0.6 M mannitol, 5 mM tris/maleate, 2 mM EGTA, 3 mM potassium phosphate, 5 mM tetraphenylphosphonium bromide, pH 6.7) in a thermostatically controlled chamber (281C) connected to three electrodes, allowing the simultaneous measurement of oxygen concentration, tetraphenylphosphonium (TPP þ ) concentration 42 and pH, respectively. 43 Respiration noncoupled to ADP-phosphorylation (state 4) was initiated by the addition of 1 mM NADH (yeast mitochondria are able to oxidize directly cytosolic NADH via two intermembrane space-facing NADH : ubiquinone oxidoreductases).…”

Glutamate-mediated inhibition of oxidative phosphorylation in cultured retinal cells

“…TPP + (4 mM) uptake was followed by a decrease of TPP + concentration in Na + saline solution (0.8 ml, ®nal volume), at 378C, after incubation of the retinal cells (about 0.25 mg protein) with 10 mM±10 mM glutamate, under magnetic stirring. The mitochondrial DC was determined as relative changes in potential rather than absolute values, and estimated from the following equation (Kamo et al, 1979):…”

“…The membrane potential (DC) was monitored by following the accumulation of TPP + by retinal cells submitted to glutamate (10 mM±10 mM), by using a TPP + selective electrode. Insert shows the DC after addition of 100 mM glutamate, estimated according to Kamo et al (1979). Data, expressed as a percentage of control (C), in the absence of glutamate, are the means2SEM from six to seven determinations, by using three independent retinal cell suspensions.…”