42 STUDIES ON ALKALINE PHOSPHATASE 



The third substrate, p-nitrophenylazo-a-naphthol phosphate, 

 is itself the phosphate ester of an insoluble dye. The solution 

 of the phosphate ester is probably colloidal. When kidney sec- 

 tions are immersed in a solution of this dye phosphate at pH 

 9.3, the dye appears both in the brush borders and in the nuclei. 

 However, not all the nuclei are in fact stained in this manner. 

 Of two adjacent nuclei, one may stain and one may not. As 

 was the case when naphthol phosphate was used as a sub- 

 strate, this is probably due to the necessity for the rate of liber- 

 ation of dye in a particular site to reach a critical level before 

 precipitation can occur. In those nuclei which have a concen- 

 tration of enzyme high enough for this critical rate of libera- 

 tion to be reached, precipitation occurs. In those nuclei in 

 which the critical rate is not reached, no precipitation occurs. 

 The same phenomena can also be observed with this substrate 

 in the brush borders themselves. Some regions of the brush 

 borders contain a rather low level of phosphatase activity and 

 do not stain at all with this substrate, whereas most of the 

 brush-border region has a high concentration of phosphatase 

 and is deeply stained by the substrate. 



When we consider the results of studying the precipitation of 

 both the phosphate moiety and the alcoholic moiety of a phos- 

 phate ester after liberation by enzyme action, it seems impos- 

 sible to escape the conclusion that in all cases, within the limits 

 denned by the study of diffusion artefacts, the precipitate pro- 

 duced is in fact at the site of enzyme activity. If this were 

 not so, the same region of tissues would need to have an affinity 

 for calcium phosphate, phenolphthalein, and various azo dyes. 

 Kidney tissue has no affinity for any of the azo dyes or for 

 phenolphthalein. Calcium phosphate has an affinity for one 

 of the two main sites of activity in kidney sections but, on the 

 other hand, has been shown to be unable to diffuse within a sec- 

 tion to a significant degree. It is thus certain that the site of 

 alkaline phosphatase in tissue sections is truly demonstrated 

 by these cytochemical methods. 



The Effect of Thickness of the Section 



All the various procedures which have been outlined above in 

 connection with different techniques were worked out for sec- 

 tions between 5 and 8 p in thickness. It is sometimes necessary 



