INHIBITION OF SUCCINATE DEHYDROGENASE 19 



a decrease in absorption between 400 and 470 m// and an increase between 

 480 and 540 m//, with a maximum at 510 m// in the difference spectrum. 

 It is not known what this implies relative to the site of malonate binding. 

 The dyes may accept electrons from various sites in the succinate oxi- 

 dase system. The fragments obtained from the oxidase particles differ in 

 their abilities to reduce these dyes, and as one approaches the purest succi- 

 nate dehydrogenase the number of possible electron acceptors is reduced. 

 Indeed, soluble succinate dehydrogenase reduces only the A^-alkylphenazines 

 and ferricyanide at appreciable rates. Some dyes accept electrons chiefly 

 from the nonheme iron, some accept more efficiently from the flavin com- 

 ponent, and some, such as the indophenol dyes, involve ubiquinone. It may 

 well be that none of the commonly used dyes is completely selective. Fer- 

 ricyanide, for example, may react at other sites down the chain in addition 

 to the nonheme iron, since its reduction has a partially antimycin-sensitive 

 component, and even the A^-alkylphenazines, considered to be the most 

 reliable acceptors at the dehydrogenase level, may be able to react at other 

 sites. 



The question of importance with respect to malonate inhibition is whether 

 all of the methods of measuring succinate dehydrogenase activity are equiva- 

 lent for the purpose of obtaining accurate kinetic results. Unfortunately, 

 there have been very few reliable investigations wherein different methods 

 or acceptors have been compared. The inhibitions of succinate oxidase 

 (manometric) and succinate dehydrogenase (dye acceptors) at the same 

 concentrations of succinate and malonate have been reported sporadically. 

 It has generally been found that the oxidase is inhibited somewhat more 

 strongly: Arum spadix (Simon, 1957), Limidus gill cartilage (Person and 

 Fine, 1959), and potato tubers (Millerd, 1951). But in the enzymes from 

 Xanthomonas, this is reversed (Madsen, 1960). The results of Millerd are 

 particularly difficult to understand, inasmuch as she found a 40% inhibi- 

 tion of the oxidase at 0.1 raM malonate whereas the dehydrogenase (using 

 2,6-dichlorophenolindophenol as acceptor) was not inhibited at all. The 

 author is not aware of any quantitative studies comparing malonate inhi- 

 bition with different dye acceptors. It would appear that most workers 

 have assumed there would be no difference. 



Actually, from kinetic consideration, it is not at all necessary that the 

 inhibition produced by a chosen concentration of malonate be the same 

 when different acceptors are used. In fact, the inhibition may depend on the 

 acceptor concentration. Thorn (1953) showed that the inhibition of pig heart 

 succinate dehj^drogenase by 0.536 mM malonate increases with the con- 

 centration of methylene blue: at 0.15 n\M methylene blue the inhibition 

 is 15.9% and at 3 vaM methylene blue it is 28.8%. Succinate oxidase, is 

 inhibited 52.6% at the same malonate concentration. The transfer of hydro- 

 gen atoms from succinate to a dye acceptor always involves the interaction 



