180 1. MALONATE 



correct for this phenomenon is to determine the C^^02 formed from labeled 

 malonate. 



(f) Nature of the cycle operation and the presence of alternate pathways. 

 The inhibition of the oxygen uptake associated with the cycle will depend 

 on a number of factors in addition to the inhibition of succinate dehydro- 

 genase. The availability of a large pool of organic acids to form oxalacetate, 

 or the presence of pathways from which oxalacetate may arise (e.g., by 

 carboxylation of pyruvate, or from aspartate by transamination), will re- 

 duce the inhibition of oxygen uptake from that which would be observed 

 if all the oxalacetate had to be derived from the cycle. Other pathways 

 for the metabolism of succinate may circumvent the block to some extent. 

 These matters have been discussed in some detail (see pages 72-88). 



(g) Adaptive changes in the presence of malonate. Inhibition of the cycle 

 may accelerate other pathways. The increased uptake and metabolism of 

 glucose brought about by malonate have been noted in several types of 

 cells, and such a phenomenon will tend to counteract the malonate inhi- 

 bition on the oxygen uptake. Adaptive changes in enzyme concentrations 

 probably are seldom important in short-term experiments but cannot be 

 completely ignored in work with certain microorganisms. Inhibitions by 

 malonate have occasionally been noted to decrease with time, and adaptive 

 changes are the most obvious explanation. 



These and other more subtle factors determine the effect of malonate on 

 the total oxygen uptake of a preparation, and it should be apparent that 

 deductions based exclusively on the inhibitions of endogenous respiration 

 are frequently untenable. A definite inhibition with a reasonable malonate 

 concentration is more significant than a negative result, because there are 

 many factors which can reduce or abolish the action of malonate even 

 though the cycle is present and active. 



The Time Course of Malonate Inhibition 



The inhibition of respiration by malonate may occur fairly rapidly and 

 remain constant, or it may increase slowly to a level at which it is main- 

 tained, or it may gradually disappear, or it may vary in quite complex 

 fashion with time. A slowly developing inhibition would not be unexpected 

 with a substance which does not penetrate readily. The inhibition of suc- 

 cinate dehydrogenase is essentially instantaneous so that an approximately 

 linear increase in the inhibition to a constant level would imply that the 

 rate of inhibition is determined by the penetration. On the other hand, 

 secondary effects, such as would result from the depletion of ATP, may 

 also contribute to a progressive inhibition. Greville (1936) noted that mal- 

 onate does not immediately inhibit the respiration of rat diaphragm but 



