INTEGRATED OXIDATIONS IN ISOLATED MITOCHONDRIA 01 



and saline-phosphate treated Hver mitochondria. An alternative hypothesis 

 is that the ATP is required for removal of the oxaloacetate by the phos- 

 phoenolpyruvate carboxylase reaction. 



Malate oxidation 



EFFECT OF FLUOROMALATE 



DL-3-fluoromalate is a competitive inhibitor of purified mitochondrial 

 malate dehydrogenase; 2 mM fluoromalate causes a 99" u inhibition of 

 DPN reduction with i mM L-malate as substrate [5]. Malate oxidation 

 occurs at relatively low rates in liver mitochondria, especially when DNP 

 is used to stimulate respiration. However, when glutamate and /3-chloro- 

 vinyl arsenious oxide (0-5 /xg./ml.) were used the oxaloacetate produced 

 by the malate dehydrogenase was removed by transamination and a rapid 

 rate of oxygen uptake resulted. The arsenical inhibited the oxidation of 

 glutamate, as was described previously, and aspartate was shown to 

 accumulate in the medium. In this system 5 niM fluoromalate inhibited 

 the oxidation of 10 mM malate by more than 90'^* o ; at lower malate con- 

 centrations the inhibition was even more marked. 



However, fluoromalate, even at a concentration of 10 mM, had no 

 observable effect on two systems which are thought to involve the oxidation 

 of malate, namely the isocitrate system (Fig. 4) and the inhibition of 

 succinate oxidation which occurs when mitochondria are preincubated 

 with arsenate and DNP. Both these latter systems may be thought of as 

 " internal " and it may be that fluoromalate is unable to penetrate to them. 

 iVIitochondria behave as though they are partly impermeable to oxalo- 

 acetate and it is not inconceivable that they are also impermeable to fluro- 

 malate. 



Figure 11 is a summarv of some of the findings which have been dis- 

 cussed and an attempt to correlate the structural relationships of the 

 enzymes within the mitochondrion with their function. 



It is apparent that the level of oxaloacetate in mitochondria can under 

 certain conditions control the rates of glutamate, isocitrate, succinate and 

 malate oxidation. Under conditions in which the rate of oxaloacetate 

 production is inhibited (lewisite, malonate) glutamate and isocitrate oxida- 

 tion occur at markedly reduced rates. On the other hand succinate and 

 malate oxidation are inhibited by oxaloacetate accumulation. If these 

 factors are suitably controlled, e.g. by providing sufiicient oxaloacetate for 

 isocitrate oxidation or by preventing the accumulation of this keto-acid in 

 the case of malate and succinate oxidation, the rates of glutamate, isocitrate 

 and malate (in the presence of lewisite and glutamate) occur at the same 

 rate (360 400 /d. 02/mg. N/hr.) when ADP is used to stimulate respiration. 

 Succinate oxidation occurs at 50 60",, greater rates. However in the 



VOL. n. — (; 



