INTEGRATED OXIDATIONS IN ISOLATED MITOCHONDRIA 73 



observation the less obvious was the second slower rate. When io~^ to 

 10"* M-2 :4-dinitrophenol (DNP) was used to stimulate isocitrate oxidation 

 an initial rapid rate was observed followed by a very much slower rate, 

 which persisted for at least 15 min. 



When the phosphate concentration in the medium was reduced below 

 5 niM, linear rates of isocitrate oxidation occurred, both when ADP and 



DNIP 



12 3 4 5 

 Time (min) 



Fig. 3. The stimulation of isocitrate oxidation by malate. Three separate 

 experiments are shown. In each case 10^ M DNP was added followed 4 min. 

 later (*) either by i -o mM-L-malate or 10 niM -L,( + )— isocitrate, or isocitrate and 

 then malate. 



DXP wert used to stimulate respiration. However, even in a medium 

 containing a low concentration of phosphate, if the mitochondria were 

 depleted partly of their endogenous substrates by preincubating them 

 with DNP or ADP for 5 min., added isocitrate was not oxidized at ap- 

 preciable rates for many minutes. The addition of low concentrations of 

 malate, fumarate or higher concentrations of oxaloacetate led to a marked 

 increase in oxygen consumption. In the absence of isocitrate, and under 

 these conditions, malate, fumarate or oxaloacetate did not produce any 

 marked oxygen uptake (Fig. 3). 



