*2.6. Mitochondrial Transmembrane Potential (*ΔΨ*m) and Enzyme Activities*

Mitochondrial membrane energization (transmembrane potential; ΔΨm) was measured by spectrofluorimetry using safranin as fluorescent probe [44]. The wavelengths for excitation and emission were 520 and 580 nm, respectively. Energization was achieved by addition of 50 μM succinate plus 2 μM rotenone and the full de-energization was achieved by the addition of carbonyl cyanide-4-(trifluoro-methoxy) phenylhydrazone (FCCP; 10 μM).

The activity of NADH dehydrogenase in disrupted mitochondria was estimated by spectrophotometry at 420 nm using potassium ferricyanide as the electron acceptor and the results were calculated using the molar extinction coefficient (ε) of 1.04 <sup>×</sup> 103 <sup>M</sup>−<sup>1</sup> cm−<sup>1</sup> [45].

The ATPase activity was measured in intact (coupled and uncoupled) and disrupted mitochondria as previously described [46]. Briefly, intact mitochondria were incubated in a medium (0.5 mL) containing 50 mM KCl, 0.2 M sucrose, and 10 mM Tris–HCl (pH 7.4) plus 0.2 mM EGTA and 5.0 mM ATP for 20 min, at 37 ◦C. The reaction was initiated by the addition of ATP and stopped with 5% trichloroacetic acid. The ATPase activity was quantified by measuring inorganic phosphate release from ATP.

The activity of succinate dehydrogenase was measured by spectrophotometry (500 nm) in a medium containing 100 mM triethanolamine (pH 8.3), 0.5 mM EDTA, 2 mM KCN, 6.5 μM phenazine methosulfate, 0.6 mM iodonitrotetrazolium, and aliquots of disrupted mitochondria [40]. The reaction was initiated with the addition of 10 mM succinate. Values were calculated using the molar extinction coefficient (ε) of reduced iodonitrotetrazolium (1.93 <sup>×</sup> 104 <sup>M</sup>−<sup>1</sup> cm<sup>−</sup>1).

The activity of malate dehydrogenase was assayed by spectrophotometry at 340 nm in a medium (1.5 mL) containing 120 mM phosphate buffer (pH 7.8), 0.25 mM NADH, and aliquots of the supernatant obtained after centrifuging disrupted mitochondria at 10,000*g*. The reaction was started by the addition of 0.1 mM oxaloacetate [47]. Values were calculated using the molar extinction coefficient (ε) of NADH (6.22 <sup>×</sup> <sup>10</sup><sup>3</sup> <sup>M</sup>−<sup>1</sup> cm<sup>−</sup>1).

The activity of the NADP+-dependent isocitrate dehydrogenase was assayed in a reaction medium (1 mL) containing 0.1 M TRIS (pH 7.4), 2 mM MgCl2, 2 mM NADP<sup>+</sup>, and aliquots of the supernatant [48]. The reaction was started by the addition of 1.25 mM isocitrate and the increase in absorbance was monitored at 340 nm (<sup>ε</sup> <sup>=</sup> 6.22 <sup>×</sup> <sup>10</sup><sup>3</sup> <sup>M</sup>−<sup>1</sup> cm<sup>−</sup>1).

The activity of L-glutamate dehydrogenase was measured in a reaction medium (1 mL) containing 50 mM triethanolamine (pH 8.0), 0.1 M ammonium sulfate, 95 μM NADH, 2.5 mM EDTA, 1 mM ADP, and aliquots of the supernatant described above [49]. The reaction was started by addition of α-ketoglutarate (8.0 mM) and the decrease in absorbance was monitored at 340 nm.

The activity of α-ketoglutarate dehydrogenase was measured in a medium containing 100 mM phosphate buffer (pH 7.4), 2 mM NAD<sup>+</sup>, 0.2 mM thiamine pyrophosphate, 1 mM MgCl2, 0.1% Triton X-100, 0.3 mM dithiothreitol, 10 mM α-ketoglutarate and aliquots of disrupted mitochondria suspensions [40]. The reaction was initiated by the addition of coenzyme A (0.2 mM) and monitored spectrophotometrically as the reduction of NAD<sup>+</sup> at 340 nm (<sup>ε</sup> <sup>=</sup> 6.22 <sup>×</sup> 103 <sup>M</sup>−<sup>1</sup> cm<sup>−</sup>1).
