We have previously reported (MNL 70:29-30) on the effect of different redox conditions on the protein synthesising activity in isolated mitochondria. It was found that under oxidising conditions created by the addition of potassium ferricyanide to mitochondria the protein synthesis was activated in organello, while under reducing conditions created by the addition of sodium dithionite it was strongly suppressed. We made an assumption of the existence of redox regulation of mitochondrial gene expression at the translational level. According to this hypothesis, the presence of a set of special redox sensors transmitting information on the changes in redox conditions to the genetic apparatus of the organelles seems to be a necessary element in the mechanism of this regulation.
Particular components of the respiratory chain complexes can, in our opinion, serve as such redox sensors of the mitochondrial system of redox regulation. To test this assumption, the present work studied the effect of different states of the respiratory chain of mitochondria created by different respiratory inhibitors on the activity of the protein synthesising system of these organelles.
The mitochondria were isolated from 3-day-old etiolated maize seedlings of hybrid VIR42 MV by a standard method of differential centrifugation. Mitochondrial protein was determined by the Lowry method. Protein synthesis reactions were registered according to Bhat et al. (Biochemistry 21:2452-2460, 1982) with the use of [14C]-leucine (specific radioactivity was 1760 GBq/mol). In order to study the effect of oxidative phosphorylation uncoupler on mitochondrial translation carbonyl cyanide chlorophenylhydrazone (CCCP) at a final concentration of 1 uM was used. 1 ug/ml of antimycin A, 1 mM of potassium cyanide and 0.003 mM of rotenon were added to mitochondria in the inhibitory analysis of the respiratory chain. The kinetic data were obtained from at least 3 to 4 experiments.
Table. Activity of protein synthesis in isolated maize mitochondria
under different redox states of respiratory chain. Incorporation of [14C]-leucine,
% of control.
|Conditions||5 min||10 min||15 min||20 min|
The table shows that the addition of CCCP, an uncoupler of oxidative phosphorylation, caused an activation of protein synthesis as in the case of oxidising conditions following the addition of ferricyanide (MNL 70:29-30). Such an effect of CCCP presumably results from redox states of the carriers of the respiratory chain, which, as shown elsewhere (Muraoka and Slater, BBA 180:221-226, 1969), are converted to more oxidising states under uncoupling of mitochondria.
The addition of rotenon blocking the electron transport from NAD to cytochrome b failed to produce any effect on the translational activity of mitochondria, which is not surprising as sodium succinate was used as a respiratory substrate and oxidation is not inhibited by this agent. The protein synthesis in organello was shown to be drastically inhibited when the respiratory chain was blocked by potassium cyanide ceasing the final stage of the electron transport at the level of cytochrome a + a3, which can be accounted for by the conversion of the respiratory carriers into a reduced state. In contrast, the activity of the protein-synthesising system of mitochondria declined to a lesser degree under antimycin A inhibition blocking the electron transport from cytochrome b to cytochrome c than under cyanide inhibition. The different level of suppression of the protein synthesis can be accounted for by the location of the antimycin block in the respiratory chain when only carriers localized left of the "crossing point" proved to be in the reduced state. It should be emphasized that energy deficiency was not the reason for the protein synthesis suppression in our experiments with the inhibitors of respiratory chain, since otherwise cyanide and antimicin A could produce similar effects on the translational activity. In conclusion, the results obtained suggest the localization of the redox sensors in all multienzyme complexes of the respiratory chain of mitochondria.
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