EFFECTS ON TRICARBOXYLIC ACID CYCLE 85 



ever the explanation, it is obvious that in such mitochondria malonate 

 could not be used to isolate a-ketoglutarate oxidation. 



Another quantitative study on the effects of malonate on a-ketoglutarate 

 oxidation was made by Grafflin et al. (1952), who were attempting to find 

 a good assay system for a-ketoglutarate oxidase in rabbit kidney homogen- 

 ates. They concluded that the use of malonate is unsatisfactory and aban- 

 doned this procedure. The difficulty lies particularly in the inability, 

 except at high malonate concentrations (around 30 mM), to inhibit com- 

 pletely the oxidation of succinate, as determined from the total oxygen 

 uptake compared with the theoretical value for a one-step oxidation of 

 a-ketoglutarate. Although no evidence on the effect of malonate on the 

 a-ketoglutarate oxidase was presented, it would be surprising if concentra- 

 tions of malonate above 20 mM had no effect. Lewis and -Slater (1954) 

 also remarked that even in the presence of 10 milf malonate, the oxygen 

 uptake from the oxidation of a-ketoglutarate greatly exceeds the disap- 

 pearance of a-ketoglutarate, .lO/.Ja-KG ratios generally being above 2, 

 in blowfly sarcosomes. Also, the oxygen uptake over 35-45 min is depressed 

 only 10% at this concentration of malonate. 



It is difficult to understand why the oxidation of succinate is so resistant 

 to malonate under these circumstances. Taking the values of Kj that 

 have been found in mammalian tissue studies, malonate at 10 mM should 

 inhibit well over 95% even at succinate concentrations that might occur 

 experimentally. For example, in beef heart preparations, where K^ is about 

 0.04 WlM, 10 vnM malonate would inhibit over 99% at a succinate concen- 

 tration of 2 mM. It may be that in the intact system the oxidation of endo- 

 genously formed succinate from a-ketoglutarate via succinyl-CoA is ki- 

 neticaUy different than the oxidation of exogenous succinate, or that local 

 concentrations of succinate can reach much higher levels than predicted 

 on the basis of over all analyses. One must conclude at least at the present 

 time that the specific inhibition of succinate oxidation, even in these rela- 

 tively simple systems, is generally impossible. 



This problem has been approached recently by Jones and Gutfreund 

 (1964), who experimentally determined the steady-state concentrations of 

 succinate in guinea pig liver mitochondria during the oxidation of a-keto- 

 glutarate. The O2 uptake of uninhibited mitochondria is 40-45% due to 

 a-ketoglutarate oxidation, 40-45% due to succinate oxidation, and 10-20% 

 due to other oxidations. The effects of malonate on the oxidation of exoge- 

 nous succinate were compared with the effects on the oxidation of succin- 

 ate-C^* formed from a-ketoglutarate-C^^. The rate of utilization of a-keto- 

 glutarate is not altered up to 8 mM malonate. The steady-state succinate 

 concentration by total analysis is 0.04 mM, and this is not affected by 

 malonate until its concentration is higher than 0.04 mM; half the succinate 

 formed from a-ketoglutarate accumulates with 0.7 mM malonate, and 



