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GIOVANNI FELICE AZZONE 



(Expt. (a)) to liver mitochondria in the presence of succinate ehcited a 

 respiration which was less than half of that obtained in the presence of 

 either dicoumarol (Expt. (a)), or phosphate and a phosphate acceptor. 

 The increased respiration elicited by arsenate was due to a partial release 

 of respiratory control. Subsequent addition of dicoumarol released the 

 respiratory control completely. When the mitochondria were preincubated 

 (Expt, (b) ) with arsenate for 3 min. prior to the addition of succinate the 

 respiration was about half maximal, but even this level was reached only 

 after a lag phase. If on the other hand dicoumarol was added during the 

 preincubation (Expt. (c)) together with arsenate, the rate of succinate 

 oxidation remained low upon prolonged incubation (about 10% of the 

 maximum). 



[02]= - 



Fig. I. Inhibition of succinate oxidation in rat liver mitochondria incubated 

 with arsenate and dicoumarol [6]. Concentrations of the reagents in a final 

 volume of 1-5 ml. were as follows: 0-05 M KCl, 0-033 ^ tris buffer pH 7-5, 

 o-oo8 M MgClo, 0-05 M sucrose, 0-013 m succinate (Succ), 0-002 m arsenate 

 (Arsen.), o- 00006 M dicoumarol (Die.)- Mitochondria from 400 mg. rat liver wet 

 weight (about 8 mg. protein). The substances were added at the points indicated. 

 "1-3 Medium" stands for KCl, tris, MgCl and sucrose added in a volume of i -3 

 ml. Oxidation rate of succinate is given in m/iatoms oxygen per min. 



A possible interpretation of these findings was suggested by experi- 

 ments in which arsenate was found to deplete the mitochondria of their 

 endogenous phosphate. When mitochondria labelled with ^'-P were 

 incubated in the presence of arsenate (Table I) an almost complete 

 depletion of mitochondrial phosphate took place within a few minutes. 

 Addition of succinate largely prevented this effect of arsenate. The pre- 

 vention was ascribed to a reincorporation of inorganic phosphate into 

 ATP by way of aerobic phosphorylation, since the oxidation of succinate 

 is only partly uncoupled by arsenate. Addition of respiratory inhibitors 

 such as antimycin A or KCN abolished the succinate effect on the arsenate- 

 induced depletion. 



Thus the initial low rate of succinate oxidation after preincubation with 

 arsenate was considered to be a consequence of the loss of high energy 

 phosphate from the mitochondria, and the gradual increase in the rate of 



