Kinetic analysis of the mitochondrial quinol-oxidizing enzymes during development of thermogenesis in Arum maculatum L

Abstract

The dependence of the rate of oxygen uptake upon the ubiquinone (Q)-pool reduction level in mitochondria isolated during the development of thermogenesis of Arum maculatum spadices has been investigated. At the alpha-stage of development, the respiratory rate was linearly dependent upon the reduction level of the Q-pool (Qr) both under state-3 and -4 conditions. Progression through the beta/gamma to the delta-stage resulted in a non-linear dependence of the state-4 rate on Qr. In the delta-stage of development, both state-3 and -4 respiratory rates were linearly dependent upon Qr due to a shift in the engagement of the alternative oxidase to lower levels of Qr. Western blot analysis revealed that increased alternative oxidase activity could be correlated with expression of a 35 kDa protein. Respiratory control was only observed with mitochondria in the alpha-stage of development. At the beta/gamma-stage of development, the addition of ADP resulted in a significant oxidation of the Q-pool which was accompanied by a decrease in the respiratory rate. This was due either to decreased contribution of the alternative pathway to the overall respiratory rate under state 3 or by deactivation of succinate dehydrogenase activity by ADP. Cold-storage of the spadices at the beta-stage of development led to increased activity of both the cytochrome pathway and succinate dehydrogenase, without any change in alternative oxidase activity. Results are discussed in terms of how changes in the activation level of the alternative oxidase and succinate dehydrogenase influence the activity and engagement of the quinol-oxidizing pathways during the development of thermogenesis in A. maculatum.

Targeting the Plant Alternative Oxidase Protein to Mitochondria Confers Cyanide-insensitive Respiration

“…4 indicate that the alternative oxidase is expressed in a functional form, indicating that following import and processing the protein is correctly folded and importantly, the necessary enzymes for the incorporation of iron are present in S. pombe. Furthermore such observations indicate the expression of a single polypeptide is sufficient for enzyme activity, which is in contrast to that observed in thermogenic tissues (39,40) and soybean (4,41).…”

Identification of a Gene for Pyruvate-Insensitive Mitochondrial Alternative Oxidase Expressed in the Thermogenic Appendices in Arum maculatum

“…Regarding region 4, which is located at the C terminus of the AOX protein, it has been shown that mutation of a Gly residue close to this region results in an AOX protein with an apparent decreased sensitivity to the salicylhydroxamic acid (Berthold, 1998). AOX has previously been successfully purified from A. maculatum thermogenic appendices (Affourtit and Moore, 2004), and such preparations show both pyruvate-sensitive and -insensitive activities (Leach et al, 1996).…”

Maesaquinone: A Novel Inhibitor of Plant Mitochondrial Respiratory Enzymes That React with Ubiquinone

“…Mitochondria isolated from this tissue provide a suitable example of a branched plant electron transfer chain in that external NADH is oxidised at a high rate (8) and AOX is engaged in the overall respiration to a considerable degree (9). We report that in this this system, MAQ does not act as an uncoupler but rather as an universal inhibitor of those respiratory enzymes that react with ubiquinone (Q).…”

“…maculatum spadices were collected from plants that were approximately at the b /c stage of thermogenesis (10) and growing in local Sussex woods. After collection, the mitochondria were immediately isolated and puri ed as previously described (9). Mitochondria were either used in experiments on the day of isolation or frozen at ¡ 18 ± C with subsequent storage at ¡ 70 ± C for use at a later date.…”