Figure 3

Argininosuccinate is produced from arginine and fumarate. (A) Isotopologue distribution analysis of intracellular metabolites after incubation with U-¹³C-glutamine for 24 hours. (B) In light of the isotopologues distribution (¹³C₄) of glutamine-derived ¹³C-labeled argininosuccinate in FH-deficient cells (panel A), this scheme represents the most likely biochemical pathway that links TCA cycle metabolites in FH-deficient cells to the urea cycle and to argininosuccinate production. Arginine and fumarate were mixed in a physiological buffer in the presence or absence of protein lysates from (C) Fh1^−/− cells or (D) Fh1^−/− cells in which ASL expression was silenced. The formation of argininosuccinate is enzymatic and requires ASL. Where indicated, the cell extracts were heat inactivated (HI) prior to the in vitro reaction. Results were obtained from three independent experiments and expressed as average ± SEM. ASL, argininosuccinate lyase; FH, fumarate hydratase; HI, heat inactivated; SEM, standard error of the mean.