70 1. MALONATE 



cycle intermediate (sparker) is provided to furnish oxalacetate to condense 

 with the acetyl-CoA. A very small amount of such a sparker may suffice to 

 initiate the entry of the acetyl-CoA into the cycle, and the cycle will then 

 perpetuate itself through the continuous formation of oxalacetate, in which 

 case pyruvate will be completely oxidized to COg and water. Such a system 

 should be quite sensitive to malonate, because an inhibition of the oxidation 

 of succinate will reduce the amount of oxalacetate formed and consequently 

 the amount of acetyl-CoA entering the cycle. On the other hand, if an 

 approximately molar equivalent of fumarate, malate, or oxalacetate is 

 initially present with the pyruvate, there wiU be an adequate concentration 

 of oxalacetate to incorporate acetyl-CoA at a rapid rate, and the process will 

 not depend on a regeneration of oxalacetate. This system will not be very 

 sensitive to malonate, because a block of succinate oxidase will not apprec- 

 iably reduce the amount of oxalacetate present. The first important princi- 

 ple is, therefore, that the degree of cycle inhibition by malonate will depend 

 on the source of oxalacetate. 



When the cycle is operating in a steady state, the concentrations of 

 intermediates are low, and oxalacetate is formed just as rapidly as pyruvate 

 is incorporated, the cycle rate being limited by the entry of acetyl-CoA, 

 This may be the normal state of the cycle (Krebs and Lowenstain, 1960) but 

 probably in cells, and certainly in isolated preparations, there are times when 

 the cycle is not in a steady state. There is an initial rise in citrate concentra- 

 tion during the oxidation of pyruvate by heart mitochondria in the presence 

 of malate (Montgomery and Webb, 1956 a), indicating that the tricarboxy- 

 lates cannot be handled as rapidly as pyruvate can enter the cycle when the 

 supply of oxalacetate is not limiting. The rate of oxygen uptake is initially 

 very high, falls to a new level during the first 40 min, maintains this level 

 for 2-3 hr, and then suddenly fails when the pyruvate is completely utilized. 

 The first phase occurs when oxalacetate is readily available, and corresponds 

 to the accumulation of citrate; the rate of oxygen uptake during this period 

 is not an accurate measure of the rate of operation of the entire cycle — 

 block of succinate oxidation may have very little effect on the oxygen up- 

 take because relatively little of the respiration arises from this region of the 

 cycle. The second steady-state phase should be more sensitive to malonate 

 because the oxidations of succinate and malate now contribute 2 of the 

 total of 5 oxygen atoms taken up per molecule of pyruvate. The second 

 principle is thus that malonate inhibition will sometimes depend on the time 

 interval during which the oxygen uptake is measured, particularly whether 

 it is the initial rate or the total oxygen consumed. 



Succinate usually accumulates during malonate inhibition (see page 90) 

 and this will progressively reduce the degree of inhibition due to the com- 

 petitive nature of the inhibition. Eventually a new steady state may be 

 reached during which the succinate concentration remains constant. This 



