Control of citrate and 2-oxoglutarate formation in Candida lipolytica mitochondria by adenine nucleotides

Abstract

Candida lipolytica accumulates citrate from glucose mainly in the post- logarithmic growth phase. In yeast mitochondria prepared from cells in the logarithmic and stationary phases, citrate formation from pyruvate and malate was investigated. In the absence of added adenine nucleotides, the mitochondria from the stationary phase produced citrate, 2-oxoglutarate and a small amount of isocitrate with a 90% yield from consumed pyruvate on a molar base, while the yield was 58% with log-phase mitochondria. The effects of added adenine nucleotides on the formation of citrate and 2-oxoglutarate by stationary-phase mitochondria were studied and the following results were obtained: 1. 1. On addition of 0.1 mM ADP, a decreased citrate level and a 4-fold increase of 2-oxoglutarate formation were observed; 2. 2. Addition of 0.1 mM 2,4-dinitrophenol encouraged the production of both citrate and 2-oxoglutarate; 3. 3. AMP, similar to ADP, stimulated mitochondrial oxidation of citrate to 2-oxoglutarate, but AMP was effective at much lower concentrations than ADP; 4. 4. On addition of 0.3 mM ATP, citrate formation increased by about 50%. To explain the above results, isocitrate dehydrogenase (NAD +) (EC 1.1.1.41) in the mitochondrial extract was studied and it was found that it requires allosteric activation by AMP or ADP and that this stimulation is partly reversed by ATP. Concentrations of the adenine nucleotides in the cells of both growth phases were estimated. AMP content of the stationary-phase ccells was about 10% of that of log-phase cells. These results indicate that citrate formation by C. lipolytica is susceptibly controlled by adenine nucleotides, especially by AMP via the allosteric regulation of the activity of NAD +-isocitrate dehydrogenase.