Control of succinate oxidation by cucumber (Cucumis sativus L.) cotyledon mitochondria

Abstract

The aim of this work was to assess the extent to which mitochondria control the gluconeogenic flux in cucumber (Cucumis sativus L.) cotyledons, by quantifying the distribution of control of succinate oxidation by cotyledon mitochondria. The methods of metabolic control analysis were applied under state 3 and state 4 conditions and in the presence of cell-free extracts in order to simulate in-vivo conditions. Oxygen uptake by isolated cotyledon mitochondria oxidising succinate under state 3 conditions was examined using inhibitor titrations. During lipid mobilisation in light-grown cotyledons (3-4 d post-imbibition), control was shared between the adenine-nucleotide translocator (flux-control coefficient, C = 0.25–0.28) and the dicarboxylate-uptake system (C = 0.69–0.72). The dicarboxylate-uptake system was also important in dark-grown cotyledons at this stage (C = 0.55–0.57). In the photosynthetic phase of development (more than 5 d post-imbibition) control rested with the respiratory chain. Application of an external ATP demand provided either by cell-free extracts of cucumber cotyledons or a glucose/hexokinase ADP-regenerating system showed that the reactions outside the mitochondria exert control (C = 0.45–0.54 and C = 0.24–0.38, for cytosolic extract and glucose/hexokinase, respectively). The adenine-nucleotide translocator was a controlling step of both oxygen uptake (C = 0.11–0.32) and the flux between succinate and hexose phosphates (C = 0.28). Other mitochondrial steps made a significant contribution to control. Control of oxygen uptake was dependent on both the nature of the external load and on the rate of phosphorylation. A potential role for mitochondrial membrane-transport processes, including the adenine-nucleotide translocator, is proposed for the integration of lipid breakdown and gluconeogenesis in vivo.