FIGURE 3.

The effect of Az-LPAF on isolated mitochondria is reversible. A, sequestration of Az-LPAF from energized mitochondria allows rapid repolarization. Isolated mitochondria were loaded with TMRM and energized by the addition of glutamate and malate, and 2.5 μm Az-LPAF was added at the time shown by the arrow. Subsequently, either PBS or 100 μg/ml BSA in PBS was added (third arrow) and fluorescence intensity was recorded as described in the legend to Fig. 2. B, Az-LPAF pre-treatment reversibly suppressed subsequent polarization by substrate oxidation. Mitochondria were loaded with TMRM and fluorescence in the absence of oxidizable substrate was recorded before the addition of Az-LPAF, PBS, or CCCP (first arrow). At the second arrow, PBS or 100 μg/ml BSA was added, and 2.5 min after that the oxidizable substrates glutamate and malate were added as shown by the third arrow (n = 3).