Figure 5.

Isolation of rat heart nucleoids with increasing salt concentrations. Nucleoids were purified from rat heart. Top panel: MtDNA containing P1 fractions were quantified with SYBR Green. Nucleoids sedimented more slowly with increasing salt concentrations (20 and 200 mM NaCl), indicating that ionic forces are involved in the association of SOD2 with nucleoids. Middle panel: Under standard conditions (0 mM NaCl), the nucleoids migrated to the bottom of the gradient (fractions 1–3). At 20 mM NaCl, most of the SOD2 was still associated with mtDNA (fractions 4–5), but according to the mtDNA distribution the nucleoid complex started to dissociate at low salt (nucleoids were also identified by TFAM and Polγ; data not shown). At high salt (200 mM NaCl), attachment of the SOD2 was completely disrupted. Bottom panel: As markers for the nucleoid complexes in 0 mM fractions, we used antibodies against TFAM and Polγ. SOD2, TFAM, Polγ, and mtDNA were present in the same fractions. Data shown are representative of 3 independent experiments.