Figure 4: Channelling of TCA cycle intermediates in isolated plant mitochondria.

(a) Schematic representation of the isotope dilution experiment to assess the channelling of citrate and 2OG. ¹³C-labelled pyruvate was fed to isolated potato mitochondria and the label accumulation in succinate was monitored. The TCA cycle reaction was inhibited by malonate to avoid the complication of multiple turns of the cycle. Non-labelled citrate and 2OG were added into the medium once the fractional enrichment of ¹³C-label in succinate had reached steady state. In the case of channelling, the addition of non-labelled intermediates does not affect the subsequent labelling of succinate. (b) The result of isotope dilution experiments for citrate. The time course plot shows the changes in fractional ¹³C enrichment in succinate following the addition of unlabelled citrate at 0 min. Each point represents the difference in fractional enrichment of label (%) from that at 0 min in each of duplicated samples. The line is the smoothed conditional mean with the shadow showing confidence interval of 0.95. The metabolite is considered not to be channelled when the confidence interval line comes below 0. (c) The result of isotope dilution experiments for 2OG. (d) Schematic representation of the isotope dilution experiment to assess the channelling of succinate, fumarate and malate. The principle is the same as that for citrate and 2OG but ¹³C-glutamate was fed and the label accumulation in citrate was monitored in the presence of fluorocitrate that inhibits further metabolism of citrate. (e–g) The results of isotope dilution experiments for succinate (e), fumarate (f) and malate (g).