54 STUDIES ON ALKALINE PHOSPHATASE 



able amount of an apoenzyme, which is not normally active 

 when the phosphatase reaction is carried out but which can be 

 activated by an activator which can diffuse from kidney sec- 

 tions, the diffusing substance must be alkaline phosphatase 

 itself. 



Investigators who are not familiar with diffusion phenomena 

 may perhaps not be entirely convinced by the arguments given 

 above that the phosphatase of the nuclei of kidney cells is intrin- 

 sic phosphatase. We were fortunate in obtaining quite inde- 

 pendent evidence that there is intrinsic phosphatase in cell nuclei 

 by the use of another substrate. This is 



N0 2 < >N=N< >OP0 3 H; 



which was prepared in an investigation with Dr. A. Loveless. 

 This substance is the phosphate of a dye, which gave a reddish- 

 yellow solution. Our first preparations of this material were puri- 

 fied rather carefully and, to our surprise, were not readily hy- 

 drolyzed by alkaline phosphatase. A preparation by a different 

 route, which was not highly purified, on the other hand, was 

 readily hydrolyzed by alkaline phosphatase. When kidney sec- 

 tions were incubated in the solution of this impure ester, there 

 was a heavy deposit of red dye in the brush borders and the nuclei 

 of the sections, Plate VI, Fig. A. The dye is precipitated because 

 the free dye is much less soluble in water than is the ester with 

 phosphoric acid. We then set out to investigate why it was that 

 the more highly purified material did not give a phosphatase re- 

 action. It was quickly found that, when a little of the products 

 of hydrolysis of the dye phosphate were added to purified dye 

 phosphate, the purified material was readily split by the enzyme. 

 The split products which were able to facilitate the display of 

 enzyme activity were, of course, phosphate ion and free phenol. 

 By chance, we discovered that the enzyme present in the nuclei 

 was more readily activated by free phenol, whereas the enzyme 

 present in the brush borders was more readily activated by phos- 

 phate. When a kidney section was equilibrated with purified 

 dye phosphatase to which a trace of free phenol had been added, 

 we were sometimes able to obtain a reaction in the nuclei only. 



