72 1. MALONATE 



in certain plants, but its role in animal tissues is as yet unknown (Krebs and 

 Lowenstein, 1960). As a result of these considerations, the fifth principle 

 of cycle block is that the degree of inhibition by malonate will depend on 

 the activity of various alternate pathways and shunts; in addition, it will 

 depend on what is measured, e.g., oxygen uptake, CO2 production, or 

 pyruvate disappearance. 



There is no doubt, therefore, tha the operation of the cycle and any 

 ancillary pathways will vary with the experimental or physiological condi- 

 tions, and that one must expect marked differences in the behavior of the 

 cycle in different species or tissues. In addition to the factors discussed a- 

 bove, there are several other reasons for variability in response to malonate; 

 the different susceptibilities of succinate dehydrogenase to inhibition (see 

 page 49), the failure of malonate to penetrate readily into cells, and the pos- 

 sibility that malonate can inhibit other enzymes. The reliability of malonate 

 as an indicator of cycle activity in a tissue must be evaluated in the light 

 of these considerations. Certainly the lack of an expected response to mal- 

 onate cannot be immediately interpreted as indicating the absence of the 

 cycle, and the production of a significant effect by malonate should be 

 substantiated by other more direct evidence before the operation of the 

 cycle is established. 



Inhibition of Cycle Substrate Oxidation by Malonate 



A summary of some of the effects of malonate on cycle oxidations is 

 given in Table 1-14. In many cases the concentration of malonate is too 

 high to act specifically on succinate dehydrogenase, and the results are to 

 some extent meaningless. It is very difficult to make any generalizations 

 but malonate concentrations above 10 mM in subcellular preparations must 

 be looked upon as probably not completely specific, whereas in cellular 

 systems it is impossible to evaluate the specificity because the intracellular 

 concentration is not known. In attempting to interpret the inhibitions 

 observed, it is often necessary to know the pathway of metabolism of the 

 substrate and how much oxygen is normally taken up per molecule utilized. 

 For example, when a-ketoglutarate is added to a mitochondrial suspension, 

 it may be oxidized to fumarate (or malate) taking up 2 atoms of oxygen, 

 or to oxalacetate taking up 3 atoms of oxygen, or completely taking up 8 

 atoms of oxygen. If succinate oxidation is completely blocked by malonate, 

 only 1 atom of oxygen will be taken up. Thus the maximal inhibitions in 

 these three cases are 50%, 67%, and 87.5%, respectively. Similar reasoning 

 applies to each substrate and in many cases the exact fate of the substrate 

 is not known so that it is difficult to estimate what effect might be expected 

 from malonate. It is also important in this connection, as pointed out in 

 the previous section, to distinguish between inhibition over an initial short 

 period of oxidation and inhibition of the total oxygen uptake. 



