FIGURE 7.

Phosphorylation of tyrosine 304 correlates with changes in CcO activity. To assess a time-dependent change in CcO phosphorylation (A) and respiration (B), bovine liver tissue was treated with a lower concentration of 10 ng/ml TNFα for the indicated time periods, followed by immediate mitochondria isolation and measurement of respiration. Specific activity is defined as consumed O₂ (μmol)/(min·total mitochondrial protein (mg)) (B). Western blot analysis of subsequently isolated CcO (6 μg/lane) using the phosphotyrosine 304-specific antibody showed a time-dependent increase of CcO subunit I phosphorylation, which was maximal after 12 min (A). C, correlation between tyrosine 304 phosphorylation (A) and CcO specific activity at maximal turnover (B, at 30 μm cytochrome c) are presented time-dependently. Films from Western analysis were scanned and intensities of individual bands analyzed with ImageQuant software (version 5, Molecular Dynamics) correcting for background signal. The signal obtained after 12 min was arbitrarily set to 100%. TN, turnover number.