38 STUDIES ON ALKALINE PHOSPHATASE 



nique (in 1944), active kidney sections were placed upon in- 

 active sections of kidney, spleen, and healing wounds. I found 

 that with 12 hours' incubation there was no evidence for dif- 

 fusion of calcium phosphate. It is therefore clear that, when 

 the experiment is carried out with the conditions and timing 

 set out here, calcium phosphate does not diffuse during the ex- 

 perimental procedure to a degree that can produce a precipita- 

 tion which might be interpreted as indicating the presence of 

 enzyme in a position in which it is not intrinsically present. It 

 is also clear, of course, that neither the cobalt phosphate nor 

 the cobalt sulfide can be diffusing to such an extent as to pro- 

 duce an observable artefact. We must therefore conclude that 

 the site of precipitation of calcium phosphate is identical with 

 the site of enzyme activity. It is important in studies of effects 

 of this type that judgment should be based upon diffusion from 

 an active section into an inactive section of the same material. 

 If the underlying section is of a different material from the 

 active section, and evidence for a diffusion artefact should be 

 found, this would not constitute evidence that similar artefacts 

 would occur in the active section, since affinities for various sub- 

 stances differ from one tissue to another. 



When, as may occur in some instances, the enzyme itself is 

 diffusible, the technique given above may not make it possible 

 to make an independent study of the extent to which calcium 

 phosphate is diffusing in a section. We may then resort to the 

 device of carrying out the experiment in superimposed sections 

 in the presence of different concentrations of calcium ion in the 

 incubation mixture. Since calcium ion is present in the incuba- 

 tion mixture in a great excess, the rate at which calcium phos- 

 phate can be transferred from one site on the section to another 

 site depends upon the solubility of phosphate ion. We have the 



equation: [Ca 2 +] 3 [P0 4 3 -] 2 = constant 



defining the solubility of phosphate ion, i.e., 



[P0 4 3 ~] = VConstant/[Ca 2+ ] 3 



Thus we see that the rate at which an artefact can be established 

 due to the diffusion of calcium phosphate will depend upon the 

 calcium ion concentration in the incubation medium. 



