TISSUE SLICES AND ISOLATED TISSUE PREPARATIONS 479 



INHIBITION IN TISSUE SLICES AND ISOLATED TISSUE 



PREPARATIONS 



Tissue slices are used so commonly in metabolic studies that attention 

 will now be turned to some of the special problems encountered when using 

 inhibitors. The abnormality of tissue slices has been frequently emphasized 

 and experimentally investigated, both histologically and biochemically. 

 The outside layers of damaged cells; the possible anoxia of the innermost 

 cells; the leakage of ions, proteins, and enzymes from the slices during in- 

 cubation; these are problems in any work on such preparations, but here 

 only the points relevant to inhibition will be mentioned. 



Penetration of Inhibitors into Slices 



A slice of tissue 0.4 mm (400 /^) in thickness may be composed of 10 to 20 

 layers of cells and an inhibitor, if it cannot penetrate between the cells, 

 must penetrate successively many cell membranes to reach the interior 

 of the slice. Thus it may require a fairly long interval for the inhibitor to 

 be uniformly distributed in all the cells. Another factor may also slow 

 down penetration. This is the binding of certain inhibitors to cell material 

 as they come into contact with it. An inhibitor which reacts readily with 

 protein, such as a heavy metal ion, will be concentrated in the external 

 layers of the slice during the early period of incubation inasmuch as a major 

 fraction that enters the slice will be immediately bound. The inhibition 

 during these early phases can be very inhomogeneous and the measured 

 degree of inhibition only a kinetically meaningless average value. A pro- 

 gressive depression of metabolism might be interpreted in terms of the rate 

 of inhibition of an enzyme system but it could just as well be explained 

 on the basis of a delayed entrance of the inhibitor into the slice. A kinetic 

 analysis of an inhibition in slices, in the manner of the preceding section, 

 presupposes a uniform distribution of the inhibitor. 



Binding of the Inhibitor to Nonenzymic Material 



Using isolated enzymes one can often neglect any reduction in inhibitor 

 concentration arising from binding to nonenzymic substances but with 

 tissue slices or strips the amount of this nonenzymic material is much greater 

 relative to the enzymes attacked. The specific inhibitor concentration, 

 (F) = (I) IK I, is not sufficient to predict if depletion will occur, because 

 this will now depend on the various dissociation constants and concentra- 

 tions of all the substances to which the inhibitor may be bound. Let us 

 take an organic arsenical as an example. If its molecular weight is 175 

 and it is used at 0.01 mM concentration in a reaction medium of 3 ml, 

 the total amount of inhibitor present is 0.00525 mg. If the ability of the 



