Fig. 8. Decrease in the activity and amounts of the mitochondrial respiratory chain complexes. (A) Semiconfluent cells were trypsinized and harvested. The harvested cells were suspended at (0.7–1.5) × 10⁷ cells/ml in the culture medium and placed into a chamber (50 µl) of an oxygen electrode. The rate of oxygen consumption by cells was measured at 37°C using a Clark-type electrode (Strathkelvin Instruments, Glasgow). Left panel: traces of oxygen consumption by cells at 1 × 10⁷ cells/ml. Right panel: average values with SD normalized to cell number from three independent experiments. (B) The activities of NADH–cytochrome c oxidoreductase (I + III), succinate–cytochrome c oxidoreductase (II + III), ubiquinol–cytochrome c oxidoreductase (III) and cytochrome c oxidase (IV) were measured as described in Materials and methods. Results are shown with the relative activities. The level of activity in mitochondria isolated from SH-SY5Y cells was set to 100. Note that no complex IV activity in the mitochondria isolated from Z5 and Z21 clones was detected. Average values with the SD from three independent experiments are shown. (C) Proteins (10 µg) from the mitochondrial fraction prepared from each clone were subjected to western blotting using each antibody against the subunits of the respiratory complexes and ATP synthase. CO I and CO II are mitochondrial-encoded proteins, while the others are encoded by nuclear genes. F₁β, F1-ATPase β subunit. (D) Mitochondrial translation products labeled with [³⁵S]methionine in vivo were analyzed as described (Hayashi et al., 1993). Briefly, semiconfluent cells were labeled with [³⁵S]methionine in the presence of emetine (0.2 mg/ml) for 60 min at 37°C. Proteins of the mitochondrial fraction were separated by SDS–PAGE (15–25% gradient gel). The translation products were analyzed with a bioimaging analyzer FLA2000 (Fuji Film, Tokyo). ND 1–5, subunits of complex I (NADH–ubiquinone oxidoreductase); CO I–III, subunits of cytochrome c oxidase (complex IV); cyt b, the cytochrome b subunit of ubiquinol–cytochrome c oxidoreductase (complex III); ATP 6 and ATP 8, subunits of ATP synthase (complex V). (E) Poly(A)⁺ RNA from each clone was converted into cDNA and CO IV mRNA was quantified by real-time RT–PCR as described in Materials and methods. Average values with the SD from three experiments are shown after normalization to GAPDH cDNA.