PHYSIOLOGICAL SITE OF ACTIVITY 45 



The first method is to use superimposed sections, as was done 

 in the study of the diffusion of calcium phosphate recorded above. 

 This method of using superimposed sections will, in fact, in one 

 operation record the diffusion, if any, of phosphatase, calcium 

 phosphate, cobalt phosphate, and cobalt sulfide. A detailed ex- 

 ample of the use of this method will be given later when the 

 occurrence of phosphatase in cell nuclei is discussed. 



The method of using superimposed sections is quite convenient 

 when the tissue components which are to be studied constitute a 

 considerable fraction of the area of the section. But when the 

 tissue component constitutes only a small fraction of the sec- 

 tion, it is difficult to superimpose a section containing active 

 enzyme upon a section containing no enzyme in such a manner 

 as to obtain an active tissue component immediately above the 

 corresponding inactive component in the underlying section. 

 Under these circumstances, both loss of time and waste of ma- 

 terial may be avoided by using series of sections which have been 

 incubated for the usual logarithmic series of times in a number 

 of dilutions of the same substrate. As the substrate concentra- 

 tion is diminished the rate of enzyme action is also diminished, 

 and, consequently, the rate at which a given component attains 

 a given degree of staining is diminished. On the other hand, the 

 rate of development of diffusion artefacts due to diffusion of 

 phosphatase will not be seriously affected by changing the 

 substrate concentration. Consequently, by comparing the series 

 of slides which have been incubated in the different substrate 

 concentrations, it is possible to determine the extent to which 

 phosphatase is diffusing within the section. 



As an alternative to various concentrations of the same sub- 

 strate, a given concentration of different substrates may be stud- 

 ied, the same logarithmic series of times of incubation being 

 used. If the substrates are split by the enzyme at different rates, 

 comparison of the series incubated in the various substrates 

 should again demonstrate the extent of diffusion of phosphatase. 



So far I have not made use of this last method. We are at 

 present studying the use of phenol phosphate and paranitrophenyl 

 phosphate to see whether these compounds together with glycero- 

 phosphate constitute a satisfactory series. The rate at which 

 phosphate is split from these esters by ordinary alkaline hydroly- 

 sis varies about a hundredfold or more. 



