EFFECTS ON OXIDATIVE PHOSPHORYLATION 119 



ent P : ratios, and (4) an effect on the hydrolysis of ATP or other high 

 energy substances. Only the first mechanism should be considered as true 

 uncoupling, although it is often difficult to determine the exact mechanism. 

 Included in the first mechanism would be the chelation of malonate with 

 Mg++ or Mn++, since these cations are beheved to be cofactors in phospho- 

 rylation. 



Claims for an uncoupling action will be discussed first. Lehninger (1951) 

 stated that malonate uncouples oxidative phosphorylation associated with 

 the oxidation of /5-hydroxybutyrate by rat liver mitochondria but no data 

 were given. In a previous report (Lehninger, 1949) 7.5 mM malonate was 

 shown to inhibit oxygen uptake 48%, the formation of acetoacetate from 

 /?-hydroxybutyrate 64.7%, and phosphorylation 37.3% (as determined by 

 the incorporation of P''- into the ester fraction). Since phosphorylation is 

 inhibited less than oxidation, no uncoupling is evident, and indeed the P:0 

 ratio should increase. Berger and Harman (1954) claimed that malonate 

 inhibits phosphorylation associated with the one-step oxidation of a- 

 ketoglutarate and completely suppresses phosphorylation during the oxida- 

 tion of L-glutamate by muscle mitochondria. However, the malonate con- 

 centration is not given and the absence of data prevents evaluation of the 

 results. An inhibition of phosphorylation does not necessarily mean an 

 uncoupling action. Malonate at 30 milf drops the P:0 ratio from 1.6 to 0.5 

 in the oxidation of choline by rat liver mitochondria (Rothschild et al., 

 1954). Although no control with malonate alone was reported, it would 

 appear that this is the most valid instance of uncoupling by malonate. 

 The malonate concentration was high and a reduction of Mg++ (total con- 

 centration was 5.7 mM) must be considered. Finally, the phosphorylation 

 associated with succinate oxidation in lupine mitochondria was shown to 

 be strongly depressed by 10 mM malonate (Conn and Young, 1957), but 

 it may be observed that the oxygen uptake was inhibited even more (Ta- 

 ble 1-23), so that no uncoupling occurred. 



All of the reports in which P:0 ratios were calculated are summarized 

 in Table 1-23 and in all cases, except for E. coli, Carcmvs maenas, and the 

 oxidation of choline, it is seen that the P:0 ratio is actually increased by 

 malonate. However, most of these only illustrate mechanism (2) above, 

 because in the oxidation of a-ketoglutarate a rise in the P:0 ratio would 

 be expected upon blocking succinate oxidase due to the fact that P:0 for 

 succinate oxidation is 2, whereas for the one-step oxidation of a-ketoglu- 

 tarate to succinate usually it is experimentally between 3 and 4. All one can 

 say from such data is that there is no evidence for an uncoupling action by 

 malonate. Copenhaver and Lardy (1952) used 3-20 mM malonate in all 

 their media in the study of the phosphorylation associated with a-keto- 

 glutarate oxidation and obtained high P:0 ratios, again providing evidence 

 against any uncoupling activity. It was shown by Slater and Holton in 



