Mitochondria were extracted from winter wheat shoots by differential centrifugation as described previously (Pobezhimova et al., J. Therm. Biol. 21:283-288, 1996). The activity of mitochondria was recorded polarographically at 27 C using a platinum electrode of a closed type in a 1.4 ml volume cell. The study of an influence of cold shock (0 C, 1 h) on the energetic activity of maize mitochondria showed that cold shock caused slight uncoupling in mitochondria. If the rate of non-phosphorylative (state 4) respiration was 27.8+1.5 nMol O2/mg of mitochondrial protein and respiratory control coefficient (RC) was 3.76+0.12 in non-stressed mitochondria, then in mitochondria isolated from stressed shoots these values were 33.5+1.6 nMol O2/mg of mitochondrial protein and 3.35+0.02, accordingly.
The study of an influence of CSP 310 on the energetic activity of isolated maize mitochondria showed that an addition of 0.5 mg CSP 310 per 1 mg of mitochondrial protein after 60 min incubation caused a significant increase of state 4 respiration (from 27.8+1.5 to 45.1+1.1 nMol O2/mg of mitochondrial protein) and a decrease of RC value from 3.76+0.12 to 2.32+0.11. So, we can suppose that uncoupling observed during cold stress can be caused by CSP 310 association with mitochondria in vivo.
To verify this presumption we studied an influence of anti-CSP 310 serum on energetic activity of maize mitochondria. The results obtained showed that if non-immune serum failed to result in any changes in mitochondrial energetic activity, anti-CSP 310 serum caused significant coupling of oxidation and phosphorylation in maize mitochondria. RC coefficient after this treatment increased up to 7.0+1.5.
Based on the data obtained we can conclude
that the maize defense mechanism against cold stress is associated with
uncoupling in mitochondria caused by cold stress protein CSP 310.
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