52 STUDIES ON ALKALINE PHOSPHATASE 



sion artefacts. But the amount of phosphatase which may have 

 diffused into the nuclei by this time is less than 5 percent of 

 the phosphatase which was intrinsically present in the nuclei. 

 After longer periods of incubation the amount of phosphatase 

 which may be present in the nuclei as a result of diffusion may, 

 of course, be much greater, so that the magnitude of the diffu- 

 sion artefact becomes increasingly serious. However, all the 

 detail of the distribution of phosphatase in mammalian kidney 

 is readily observed after only 10 or 20 minutes' incubation, so 

 that in this material there is no difficulty in eliminating diffu- 

 sion artefacts, so far as the distribution of alkaline phosphatase 

 is concerned. 



The experiment mentioned above, however, shows quite clearly 

 that, where protracted incubations are carried out with mate- 

 rial which contains a rich source of phosphatase, the diffusion 

 artefact may become quite serious. Plate V, Fig. A, is a low- 

 power view of a liver section which was devoid of enzyme 

 activity, superimposed upon which was a guinea-pig kidney sec- 

 tion containing active enzyme. These sections were incubated 

 for 1280 minutes. It will be seen that the enzyme has spread 

 from the active section to a distance equal to more than seven 

 cell diameters of the kidney section. Some tissues, such as the 

 small intestine, when under normal physiological conditions con- 

 tain active diffusible phosphatase. It seems very probable that 

 with such tissues diffusion artefacts may be much more serious. 

 The work of Martin and Jacoby has, indeed, provided some evi- 

 dence that this is the case. 



I have made a few experiments to try to ascertain the nature 

 of the substance diffusing from an active kidney section which 

 leads to the apparent appearance of phosphatase activity in the 

 underlying inactive section. The substances most likely to be 

 diffusing were calcium phosphate, alkaline phosphatase itself, 

 or an activator for phosphatase. As was indicated on p. 38, 

 if the diffusing substance is calcium phosphate, the rate of ap- 

 pearance of the artefact should be a function of the calcium 

 concentration. However, when the Gomori-Takamatsu method 

 for alkaline phosphatase was carried out in the presence of cal- 

 cium concentrations 10 times greater than, or only y 10 , that of 

 the normal, no significant difference was observed in the rate of 

 appearance of the diffusion artefact. Had the diffusing sub- 



