Figure 3.
Aconitase Activity Is Reduced in Fh1KO MEFs
(A) Aconitase activities of Fh1WT, Fh1KO, Fh1KO+FH, and Fh1KO+FHcyt MEF cell lysates normalized to cell number. The aconitase inhibitor oxalomalate was used as a negative control.
(B) CE-TOFMS analyses of fumarate concentration in the four MEF cell lines.
(C) Extent of succination of ACO2 at C385 and C451/448 in the four MEF cell lines determined by measuring abundance of the relevant peptides by LC-MS/MS.
(D) CE-TOFMS analyses confirmed significant differences between Fh1WT and KO MEFs in the levels of the key Krebs cycle metabolites fumarate, succinate, malate, 2OG, isocitrate, and citrate.
(E) Mass isotopomer analysis of key Krebs cycle metabolites in Fh1WT and Fh1KO MEFs cultured with [d5]glutamine for 24 hr.
(F) Schematic of glutamine metabolism by the Krebs cycle in Fh1KO MEFs. Abbreviations are as follows: Ac-CoA, acetyl coenzyme A; ACL, ATP citrate lyase; ACO1 and ACO2, Aconitases 1 and 2; FH, fumarate hydratase; FUM, fumarate; Gln, glutamine; Glu, glutamate; IDH, isocitrate dehydrogenase; Mal, malate; OAA, oxaloacetate; Succ, succinate; Succ-CoA, succinyl coenzyme A; SDH, succinate dehydrogenase; 2OG, 2-oxoglutarate; αKGDH, α-ketoglutarate dehydrogenase.
All error bars indicate SEM. See also Figure S3.