86 1. MALONATE 



80% accumulates at 8 mM malonate. The Og uptake due to the oxidation 

 of succinate formed from a-ketoglutarate is reduced 50% by 0.2 mM mal- 

 onate. The data indicate that endogenously formed succinate is at a much 

 higher concentration than that determined by total analysis. The inhibition 

 studies indicate the steady-state succinate concentration in the mitochon- 

 dria to be 4.6-13 mM and the isotopic studies suggest concentrations ex- 

 ceeding 4 mM. Such high succinate concentrations would protect the suc- 

 cinate dehydrogenase and result in less malonate inhibition than expected. 

 Since there appear to be no permeability barriers in the mitochondria to 

 succinate, the authors suggested that there is a spatial relation between 

 the succinate dehydrogenase and the enzyme forming the succinate. If 

 this is true, it raises the interesting possibility that certain enzymes are 

 specific not only for their substrates but also for other enzymes with 

 which they interact to form functional metabolic complexes. 



(c) Effects on tricarboxylate utilization. Malonate has variable affects on 

 the oxidation of citrate and isocitrate, often inhibiting rather well, but 

 sometimes having little effect or actually stimulating (Table 1-14). The 

 relationship between malonate inhibition and isocitrate oxidation is prob- 

 ably complex in most instances. There seems to be minimal direct inhibi- 

 tion of isocitrate dehydrogenase, although data are lacking. Hiilsmann 

 (1961) showed that malonate at 11 mM reduces quite potently the utiliza- 

 tion of isocitrate by rabbit heart mitochondria when acetate or /?-hydroxy- 

 butyrate is the additional substrate (these accelerating the utilization of iso- 

 citrate). The stimulation of isocitrate utilization by these substrates was 

 claimed to be due to the formation of acetoacetyl-CoA, an intermediate in 

 fatty acid synthesis, and this alters the oxidation-reduction states of NAD- 

 NADH and NADP-NADPH through the transhydrogenase reaction, 

 isocitrate oxidation rate being dependent on the concentration of NADP. 

 Kasamaki et ol. (1963) showed that malonate inhibits citrate and isocitrate 

 oxidations in Proteus vulgaris much less when NADP is added. Chappell 

 (1964 a) suggested that the effects of malonate on tricarboxylate utilization 

 in rat liver mitochondria are due to the block in the formation of malate 

 from succinate, malate being necessary for the oxidation of isocitrate, since 

 it provides a means for reoxidation of NADPH mediated through the 

 transhydrogenase and malate dehydrogenase. We see here a possibly im- 

 portant control in the operation of the cycle and how malonate can exert 

 effects indirectly on steps distant from the succinate oxidation reaction, 



{d) Effect of cycle substrate concentration on the inhibition. Some claims 

 have been made that increases in the citrate concentration will tend to over- 

 come inhibition of the oxygen uptake by malonate, and sometimes this 

 has been attributed to a competitive effect, it being assumed that the higher 

 concentration of substrate will give rise to a higher succinate level. Thus 



