MITOCHONDRIA. CELLS. AND TISSUES 573 



rate at which an inhibitor alters mj^ocardial contraction, for example, 

 even in isolated preparations to which the inhibitor can be directly ap- 

 plied, will bear little relationship to the primary inhibition rate of the 

 enzyme. Finally, these problems are particularly evident when only the 

 death of cells or organisms is used as an indication of the inhibition be- 

 cause it is likely that in all cases a number of reactions intervene between 

 the susceptible enzyme and the processes upon which the life of the cell 

 depends. The death rate of microorganisms has often been treated in a 

 quantitative fashion and from the data various conclusions as to the mech- 

 anism of inhibition have been derived. However, this approach is extremely 

 limited. Justification for such an approach by adducing the principle 

 of limiting reactions must be examined very critically. These problems 

 can best be presented by considering some of the possible factors that affect 

 inhibition rates in cells and by discussing some examples of cellular inhi- 

 bition studies. 



Factors Commonly Affecting Inhibition Rates in Cells 



The lag or latent period between the application of the inhibitor and 

 some metabolic or functional response is generally composed of three phas- 

 es: {a) the time reciuired for the inhibitor to reach the susceptible enzyme 

 from the site of its application (including the times to reach the cells and 

 to penetrate into the cells), (b) the time for the reaction of the inhibitor 

 with the enzyme, and (c) the time for the cells to respond to the loss of 

 this enzyme activity. These phases may be of any relative duration. In 

 many cases, one phase is much longer than the other two and thus pre- 

 dominantly determines the rate of the inhibition. Occasionally other fac- 

 tors may be of importance, such as the time required for the lethal synthesis 

 of the active inhibitor. The important thing is — unless we know what 

 phase, or phases, is controlling the rate, we cannot interpret kinetic data 

 mechanistically. 



(A) Rates of pe net ratio)} into cells. The rate of development of the inhibi- 

 tion may be determined by the rate at which the inhibitor penetrates into 

 the cells and may have nothing to do with the enzymic process. That the 

 cell membrane can slow down the reaction of a substance with an intracel- 

 lular component, even in situations where diffusion would be expected to 

 be rapid, is shown by the fact that the oxygenation of hemoglobin in ery- 

 throcytes is about eighteen times slower than when the hemoglobin is in 

 solution (Hartridge and Koughton. 19'27). If the diffusion of a small un- 

 charged molecule such as oxygen is reduced by the cell membrane, it is 

 evident that most inhil^itors will gain entrance to cells relatively slowly and 

 that the combination of the inhibitor with the enzyme would have to be 

 a reasonablv slow reaction in order to limit the rate at which the inhibition 



