MUTUAL DEPLETION SYSTEMS 77 



since both enzyme and inhibitor are reduced in concentration. Care should 

 be taken to distinguish between these two situations when comparing in- 

 hibitions in tissues and in extracts. 



Mutual Depletion Systems in the Cell 



The behavior of systems showing mutual depletion has been discussed in 

 some detail because it is believed that they may be more common than 

 previously supposed, particularly within cells, and that they will become 

 more common as new inhibitors of high potency are applied to enzyme 

 study. In a cell, or within subcellular structures, or in localized cellular re- 

 gions, the concentration of enzyme may be very high, far greater than could 

 be practically attained with the extracted enzyme, and the possibility sug- 

 gests itself that zone B and C behavior would be observed. However, we must 

 again distinguish between two situations: (a) the substrate or inhibitor is 

 added to the system and has a diffuse distribution, and (6) the substrate 

 or inhibitor is also localized in the same region as the enzyme, being 

 formed in that region. Most experimental systems are of the former type 

 and in this case the enzyme will bind no more substrate or inhibitor 

 than if it were homogeneously distributed; mere concentration of enzyme 

 in localized regions will not reduce the concentration of free substrate or 

 inhibitor, which is what determines zone B and C behavior. A suspension 

 of mitochondria, each containing a particular enzyme in high concentra- 

 tion, will not yield kinetic data different from a system in which the same 

 amount of enzyme is dissolved uniformly in the same total volume. Only 

 the effects of mutual depletion are being considered at this time; there are 

 other possible factors which may alter the kinetic behavior when an enzyme 

 is released from its localized condition. In the second type of situation zone 

 behavior may be exhibited commonly, even with respect to substrate as 

 well as inhibitor, since the total amount of substrate or inhibitor present 

 is very small, even though the actual concentration may be comparable to 

 that used in isolated preparations. It is easy to visualize that, within a mi- 

 tochondrion for example, essentially all the substrate is bound to enzyme. 

 If an inhibitor is formed from a precursor and this occurs in the localized 

 region, it also may be mainly combined; examples of such inhibitors might 

 be fluorocitrate or the various substrate and coenzyme analogs that must 

 be phosphorylated or synthesized within the cell from simpler added sub- 

 stances. A simple example will illustrate this. Let us assume a mitochondrial 

 suspension of 10 ml containing an enzyme whose over-all concentration is 

 10"^ M; an inhibitor is added at 10"^ M — no reduction of free inhibitor 

 concentration occurs because there is a total of 10^^" moles of enzyme and 

 10"' moles of inhibitor present. However, if the inhibitor is produced within 

 the mitochondria and is localized there at the same concentration of 10"^ M, 

 the total amount in the system may be 1/1000 of that previously or 10"^*^ 



