46 STUDIES ON ALKALINE PHOSPHATASE 



In the various studies which I have made, artefacts due to the 

 diffusion of phosphatase have usually not occurred within the 

 time period of incubation which was necessary to obtain adequate 

 information about the distribution of enzyme within sections. 

 In the few cases where diffusion has occurred to a significant de- 

 gree, the methods indicated above have made it quite simple to 

 assess its importance, with one exception. The exceptional case 

 is that of tissue cultures, which will be discussed later. 



Martin and Jacoby, in a study of the distribution of phos- 

 phatase in the small intestine, appear to have encountered very 

 much greater difficulty with diffusion of phosphatase: this is no 

 doubt due to the fact that the small intestine usually contains 

 a considerable concentration of diffusible phosphatase which acts 

 as a digestive enzyme. They appear to have had difficulty also 

 in interpreting their results with other tissues : this seems in part 

 due to incubation for an excessive length of time. But there 

 are also two points in their experimental procedure which may 

 have caused them some difficulty. In the first place, many of 

 their experiments involved superimposing tissue sections on 

 sections of different material, usually guinea-pig liver. Now it 

 is of no interest to know to what extent enzymes will diffuse from 

 one tissue into another. What is important is to know to what 

 extent enzymes will diffuse within a tissue and thus give rise to 

 artefacts. It is therefore essential that experiments with super- 

 imposed sections should be carried out with an active section 

 superimposed on an inert section of the same material. The 

 second fault in their procedure lay in rinsing the sections in dis- 

 tilled water immediately after incubation. The section at this 

 stage contains precipitated calcium phosphate which has a very 

 significant solubility in water in the absence of calcium ions, or 

 if the pH is shifted much below pB. 9. The effect of rinsing in 

 distilled water is to shift the pH towards the acid side and at 

 the same time to diminish the calcium ion concentration. It is 

 thus possible that the precipitate of calcium phosphate formed 

 in their sections was partially dissolved by rinsing in distilled 

 water when, of course, it would be freely diffusible. Subse- 

 quently, when they transferred the sections from distilled water 

 to cobalt nitrate solution, reprecipitation would occur at many 

 cites, and simulate diffusion artefacts. 



