The mitochondria were isolated from 3-day-old etiolated seedlings of W64AxSg25 by a conventional method of differential centrifugation. The activity of mtRNA synthesis was estimated by the incorporation of (3H-UTP) into acid insoluble mitochondrial material. To inhibit DNA transcription in the mitochondria, ethidium bromide (2µg/ml) and actinomycin D (40µg/ml) were used. The mitochondrial respiration was analysed polarographically with a closed Clark electrode. The rate of phosphorylative oxidation was measured in terms of oligomycin-sensitive component of respiration. The protein was determined by the Lowry method with bovine serum albumin as a standard.
The kinetics showed that both inhibitors caused a significant decrease in the rate of mtDNA transcription, ethidium bromide exhibiting greater effect (Fig.). Furthermore, in the presence of ethidium bromide mtRNA synthesis quickly reached its stationary level.
Figure. Kinetics of mtRNA synthesis inhibition in isolated mitochondria of maize hybrid W64AxSg25. 1, control; 2, actinomycin D; 3, ethidium bromide.
Mitochondrial parameters of oxidation and phosphorylation revealed that mitochondria exposed to the inhibitors of transcription yielded a significant increase of the respiration rate in the 4th metabolic state and concomitant decrease in the value of respiration control after Lardy-Wellman (Table). No significant changes in the 3d metabolic state were observed in this case, which may indicate maintenance of the activity of enzymes of oxidative phosphorylation. It can be seen that of the two inhibitors, ethidium bromide had greater effect in the 4th metabolic state. The reduction in the phosphorylation activity of mitochondria in the presence of ethidium bromide and actinomycin D is likely to be due to a failure of the inner mitochondrial membrane to retain electrochemical potential of H+-ions causing uncoupling of oxidation and phosphorylation. The synthesis of mtRNA coding protein components of the inner mitochondrial membrane in isolated maize mitochondria appears to be necessary to maintain the energy transformation capacity of the organelles in such conditions. Dewey et al. (Plant Physiol. 79:914, 1985, and PNAS 82:1015, 1985) showed that the three genes encoding ATPase subunits in maize mitochondria were most actively transcribed. However, it is quite possible that the changes in membrane permeability may be associated with certain disturbances in the genetic system at the translation level resulting from the inhibition of ribosomal and transport RNA gene transcription. The data obtained suggest that the disturbances of mtDNA transcription in maize mitochondria in vivo may result in variations of the H+-permeability of the inner mitochondrial membrane. Thus it appears to be the main reason of the mitochondria failing to synthesize ATP.
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