INTEGRATED OXIDATIONS IN ISOLATED MITOCHONDRIA 



75 



which in this case is directly oxidized by the cytochrome system, since, 

 even in mitochondria depleted of their endogenous substrates, glutamate 

 was oxidized immediately and rapidly. This was also the case when 

 ^-hydroxybutyrate, proline and malate served as substrates. 



However, even when glutamate is oxidized by the dehydrogenase 

 pathway, the rate of this reaction is intimately dependent upon the rate at 

 which a-ketoglutarate is removed. This can be shown clearly by a study of 

 the effects of /S-chlorovinylarsenious oxide on glutamate oxidation (Fig. 5). 



100 

 90 

 80 



B 70 



I 60 



: 50 

 > 



° 40 

 <v 



30|- 

 20I-' 

 10 



-»- 



OS 1-0 1-5 20 25 

 ^/S-chlorovinylarsenious oxide 

 (/^g/4ml,) 



Fig. 5. The eflFect of ^-chlorovinylarsenious oxide on ADP-stimulated oxida- 

 tion of glutamate (x — x ), a-ketoglutarate (D — D), succinate (H h) and 



proline(c — c). 



This arsenical inhibited oxidation of glutamate and a-ketoglutarate to the 

 same extent at the same concentrations. Under identical conditions 

 j8-hydroxybutyrate, malate, proline, and succinate oxidation and the 

 associated phosphorylation were unaffected. The inhibitory effect was 

 readily reversed by 2 :3-dimercaptopropanol (Fig. 6). The arsenical, at 

 these concentrations, does not inhibit the glutamate dehydrogenase itself, 

 since in disrupted mitochondria, when DPN and cytochrome c were added, 

 /S-chlorovinylarsenious oxide did not inhibit glutamate oxidation. In intact 

 mitochondria it appears that glutamate oxidation cannot occur when 

 a-ketoglutarate accumulates. Alternatively it may be concluded that the 

 glutamate dehydrogenase is not functional in intact liver mitochondria and 

 only serves to "spark" the oxidation of glutamate by the transaminase 



