36 STUDIES ON ALKALINE PHOSPHATASE 



If the glycerophosphate technique is to have biological sig- 

 nificance, one needs to establish that the cobalt sulfide precipi- 

 tate produced by this technique demonstrates with precision the 

 site of enzyme activity, and also that the enzyme is in its 

 physiologically normal position. This can be best demonstrated 

 by separate consideration of the three groups of diffusion arte- 

 facts just mentioned. It should, however, be noted that as yet 

 no studies have been made of the concentrations of activators, 

 inhibitors, substrates, etc., for phosphatase which are found 

 in the immediate vicinity of the enzyme in the living cell. Until 

 such studies are made, even the demonstration of the presence 

 of enzyme in its physiologically normal site does not enable 

 us to foretell with certainty the extent to which the enzyme is in 

 fact active at its site. 



Sites of Affinity for Calcium Phosphate 



In the glycerophosphate technique the initial precipitate 

 which is formed is calcium phosphate. It is therefore of interest 

 to determine to what extent fixed material has local sites of 

 special affinity for such substances. The best way to test this 

 is to cause the liberation of phosphate in the incubation mix- 

 ture by the spontaneous decomposition of a labile ester, and to 

 observe the extent to which the liberated phosphate is precipi- 

 tated in sections which are devoid of enzyme activity. The 

 phosphatase of tissue sections may readily be inactivated by 

 exposure to wet steam for 10 minutes, or by being placed in 

 water at 90° C. for 2 minutes. 



A solution containing a labile ester may be obtained simply 

 by adding hydrogen peroxide to the normal glycerophosphate 

 incubation mixture. The glycerophosphate undergoes oxidation, 

 and phosphate ion is slowly liberated and precipitated by the 

 calcium ions present in the medium. After addition of hydrogen 

 peroxide it is necessary to readjust the pH to 9.3, after which 

 inactive sections may be inserted and incubation carried out 

 at 37° C. Sections of kidney and of healing wounds in rat 

 skin were studied in this manner. The nuclei of the kidney 

 section were found to take up calcium phosphate with much 

 avidity, but the brush borders did not display any significant 

 affinity for calcium phosphate. In sections of healing wounds 

 the nuclei and the newly formed collagen fibres displayed a 



