48 1. MALONATE 



modest stimulation of succinate oxidation by low concentrations of Ca^^ 

 is a permeability or structure-opening effect on the mitochondria, or due 

 to a more direct effect on the enzyme, is not known; certainly the oxidations 

 of other cycle substrates and pyruvate are strongly depressed by Ca++. 

 The effects of Mg++ concentratioti from 6.2 to 12.4 mM on trypanosomal 

 succinate dehydrogenase inhibition by malonate have been reported as 

 negligible (Agosin and von Brand, 1955). In connection with the relation- 

 ship between mitochondrial structure and malonate inhibition, it is interest- 

 ing to examine the effect of ATP, inasmuch as ATP protects or stabilizes 

 mitochondria after isolation from cells. ATP at 1 mM has very little effect 

 on the rate of succinate oxidation in rat heart mitochondria (in five exper- 

 iments a mean depression of 3%), but in the presence of succinate (5 

 mM) and malonate (5 mM) it stimulated the rate 67%, thus antagonizing 

 the inhibition by malonate (Montgomery and Webb, 1956 b). Similar re- 

 sults were seen with acetylene-dicarboxylate inhibition. If the effect of 

 ATP is to reduce the permeability to these inhibitors, it is surprising that 

 interference with succinate penetration does not also occur. Thus some 

 other explanation may have to be sought. 



Inhibition of Fumarate Reduction 



If succinate, fumarate, and malonate bind at the same site on succinate 

 dehydrogenase, the reverse reaction — the hydrogenation of fumarate to 

 succinate — should be inhibited by malonate; that is, malonate should 

 compete with fumarate as well as with succinate. The relative potencies 

 of the inhibitions on the two reactions would depend on the Michaelis 

 constants for succinate and fumarate, so that the inhibitions would not 

 necessarily be identical. Malonate was, indeed, found to inhibit the reduction 

 of fumarate by horse muscle succinate dehydrogenase, but less potently 

 than the oxidation of succinate (18 mM malonate required to inhibit 50% 

 in the former case and 3.6 mM in the latter) (Das, 1937 b). A more detailed 

 analysis was made by Forssman (1941) in Lund, using pig heart succinate 

 dehydrogenase and leucomethylene blue as a hydrogen donor, in this case 

 the inhibition by malonate being essentially equivalent for both forward 

 and backward reactions. More recent studies of beef heart succinate dehy- 

 drogenase, however, have given different values for K,: 0.025 mM for suc- 

 cinate oxidation and 0.12 mM for fumarate reduction (Singer et al., 1956 a). 

 This difference is unexpected and the suggestion was made that the binding 

 of malonate may be effected by the state of oxidation of the electron trans- 

 port components adjacent to the binding site. The affinities of fumarate 

 for the enzyme were also found to be different for each reaction. It may also 

 be of significance that iV-methylphenazine was the acceptor in the oxida- 

 tion of succinate, and FMNH, or leucodiethylsafranin the donor in the re- 

 duction of fumarate. 



