FIGURE 2.
Pretreatment of mitochondria with CAT and BKA do not prevent activation of MPTP opening by PAO. MPTP opening was assayed under de-energized conditions in KSCN buffer (see “Experimental Procedures”) by the swelling of mitochondria (1 mg protein per ml). In A the mitochondrial suspension was pretreated for 1 min with either 4 μm CAT or BKA prior to the start of recording and additions of PAO (20 μm) or Ca2+ (total concentration of 1.4 mm to give 180 μm free [Ca2+]) to the sample cuvette as indicated. In B, the mitochondrial suspension was pretreated with PAO (20 μm) for 1 min prior to the start of recording and additions of BKA, CAT, and Ca2+ made at the concentrations used in A. In the inset, rat liver mitochondria (5 mg/ml) were pretreated with 0.1 mm PAO, 20 μm CAT, or 20 μm BKA for 10 min at room prior to solubilization with Triton X-100 and application to the PAO affinity matrix as described under “Experimental Procedures.” After extensive washing, the bound proteins were eluted with dithiothreitol, separated by SDS/PAGE and bound ANT revealed by Western blotting with the ANT antibodies shown.