ENZYME INHIBITION AND CHANGES IN CELL FUNCTION 473 



VI. Use of several inhibitors or analogs. If the enzyme suspected of con- 

 nection with the function can be depressed to varying degrees by several 

 inhibitors, a very useful approach is to examine the evidence for parallel 

 effects on enzyme and function. Substances chemically similar to the basic 

 inhibitor, but without inhibitory action on the enzyme, should not alter 

 the function if there is indeed a relationship between the two processes. 

 This technique is particularly valuable when an homologous series of sub- 

 stances can be tested and the degree of inhibition correlated with functional 

 disturbance. 



The functional side of the picture may also be complex and the final 

 cellular activity measured may be dependent on both metabolic and non- 

 metabolic processes. One example may be cited. The dependence of cardiac 

 muscle contraction on high-energy phosphate is being studied by using 

 the uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, and it is 

 found that the contractile force is depressed readily by this substance. 

 The conclusion might be drawn that the cyclic changes in the state of the 

 actomyosin complex are directly controlled by the availability of ATP 

 and that 2,4-dinitrophenol is producing its effect by depressing the for- 

 mation of ATP to be used by the contractile elements. On the other hand, 

 when the actions of 2,4-dinitrophenol on the cell membrane potentials are 

 recorded simultaneously with the contractile response, it is seen that at 

 least the initial contractile depression is the result of changes brought about 

 in the ionic fluxes across the membrane (Webb and Hollander, 1956). 

 In this case, the membrane processes are more sensitive to 2,4-dinitrophenol 

 than are the contractile events, but the latter, being controlled by the na- 

 ture of the action potential, are secondarily affected. A method for deter- 

 mining the relative effects of inhibitors on membrane and contractile pro- 

 cesses has been suggested (AVebb and Hollander, 1959); if this method is 

 valid, it would appear that most inhibitors affect both processes simultan- 

 eously but to different degrees. Similar relations between membrane events 

 and the measured function may occur in other types of contractile cells 

 and in cells carrying out various activities, such as secretion or cleavage. 



An example of the difiiculty in establishing a correlation between the 

 inhibition and the effect produced in a relatively simple situation will be 

 cited for illustration. Many of the modern and most potent insecticides 

 are organophosphorus compounds that are called cholinesterase inhibitors, 

 and it has been generally assumed that the lethal action is the result of 

 the inhibition of this enzyme. However, these substances are known to 

 inhibit other enzymes and recently the question as to the mechanism of 

 their insecticidal action has been argued. In houseflies there are at least 

 two enzymes that are very sensitive to the organophosphorus compounds, 

 a cholinesterase and an aliesterase (hydrolyzing low molecular weight 

 aliphatic esters). Flies were exposed to several insecticides and the inhi- 



