PHOSPHAMIDASE 



207 



PHOSPHATE ION 



in place of eosin in the following method 

 because it gives (as Conn suggested) a 

 more brilliant color. Deparaffinize sec- 

 tions of Zenker fixed material in usual 

 way. Remove mercury with 0.5% 

 iodine in 95% alcohol 5-10 min. and the 

 iodine with 0.5% aq. sodium thiosulfate 

 (hypo) 5 min. Wash thoroughly in 

 water. 2.5% aq. phloxine in paraffin 

 over 1 hr. or more. Cool stain, drain 

 and rinse in water. Take 5 cc. 1% methy- 

 lene blue on 1% borax, 5 cc. 1% aq. 

 azure II, add 90 cc. aq. dest., filter onto 

 the sections. Pour on and off several 

 times. After required time differentiate 

 in 100 cc. 95% alcohol plus 2-5 cc. 10% 

 colophony (rosin) in absolute alcohol. 

 Control differentiation under micro- 

 scope. Dehydrate in several changes 

 abs. ale. Clear in xylol and mount in 

 balsam. Nuclei and bacteria, blue: 

 collagen, etc. bright rose. The method 

 yields beautiful preparations of intra- 

 nuclear inclusions in yellow fever and 

 is extensively used for many purposes. 



Phosphamidase an enzyme capable of 

 hydrolyzing para-chloranilido phos- 

 phonic acid, at acid pH (5.6). Gomori, 

 G., Proc. Soc. Exp. Biol, and Med., 

 1948, 69, 407-409, describes the method. 

 Phosphamidase is reported to be espe- 

 cially intense in the grey matter of the 

 central nervous system and in malig- 

 nant tumors. 



Phosphatases — Written by E. W. Dempsey, 

 Dept. of Anatomy, Washington Uni- 

 versity, St. Louis. February 26, 1951 — 

 Since the original papers of Gomori, G. 

 (Proc. Soc. Exp. Biol, and Med., 1939, 

 42, 23-26), and Takamatsu, H. (Trans. 

 Jap. Path. Soc, 1939, 29, 492-498), a 

 host of papers on the localization of 

 these enzymes has appeared. The 

 Gomori and Takamatsu methods in- 

 volve incubating sections in a solution 

 of organic phosphate, during which 

 free phosphate radicals are liberated. 

 These are instantly precipitated as the 

 calcium or the lead salt, for the alkaline 

 and acid phosphatase reactions, respec- 

 tively. The insoluble metal phosphate 

 is then visualized by staining with 

 alizarin or silver solutions (Kabat, 

 E. A. and J. Furth, Am. J. Path., 1941, 

 17, 303-318) or by transformation to 

 black cobaltous sulfide. For technical 

 procedures, see Gomori's Method. 



The question whether alkaline phos- 

 phatase is a single enzyme, or whether 

 several, substrate-specific enzymes ex- 

 ist, is an actively debated topic. 

 Dempsey, E. W. and H. W. Deane (J. 

 Cell, and Comp. Physiol., 1946, 27, 

 159-179) suggested that there are several 

 different alkaline phosphatases, and 

 Emmel, V. E. (Anat. Rec, 1946, 96, 



423-438) has demonstrated that the 

 enzyme of the intestine is easily poi- 

 soned by KCN whereas that of the kid- 

 ney is not. Contrariwise, Gomori, G. 

 (Proc. Soc. Exp. Biol, and Med., 1949, 

 70, 7-11) reports no difference in locali- 

 zation of phosphatase when 19 different 

 substrates were employed, although a 

 phosphonate substrate gave different 

 results in the acid range. Later, 

 Gomori, G. (Proc. Soc. Exp. Biol, and 

 Med., 1949, 72, 449-150) found that 

 5-nucleotidase was localized differently 

 than is glycerophosphatase. The pos- 

 sible multiplicity of the alkaline phos- 

 phatases is therefore unsettled, but the 

 weight of evidence suggests there is 

 more than one enzyme for dephospho- 

 rylating mechanisms. 



A new chemical approach to the 

 phosphatases appeared when Menten, 

 M. L., J. Junge and M. H. Green (J. 

 Biol. Chem., 1944, 153, 471-477) em- 

 ployed beta naphthyl phosphate as a 

 substrate. After enzymatic hydrolysis, 

 beta naphthol was visualized by cou- 

 pling with a diazonium salt. This pro- 

 cedure has been greatly improved by 

 Mannheimer, L. H. and A. M. Selig- 

 man, J. (Nat. Cancer Inst., 1948, 9, 

 181-200) and by Seligman and Mann- 

 heimer {ibid., 1949, 9, 427-434) for 

 alkaline and acid phosphatase respec- 

 tively. The present methods give 

 results fully comparable in precision 

 with the Gomori procedures. 



The possible occurrence of artifacts 

 of various sorts in the phosphatase 

 methods has been actively debated. 

 Most observers agree that histochemi- 

 cal preparations are of considerable 

 value, but many warn of the possible 

 migration of enzyme or of its products 

 during the technical procedures. The 

 problem therefore becomes one of re- 

 solving power — -What are the spatial 

 limits of dependability of the phos- 

 phatase procedures? Two references 

 on this debated topic are: Martin, B. 

 F. and Jacoby, F., J. Anat., 1949, 83, 

 351-363, and Leduc, E. H. and Demp- 

 sey, E. W., J. Anat., 1951, in press. 



Finally attention should be called to 

 the excellent review on phosphatases 

 by Lison, L., Bull. d'HistoL, 1949, 25, 

 23-41. 

 Phosphate Ion. A capillary colorimetric 

 technique has been described by 

 Walker, A. M., J. Biol. Chem., 1933, 

 101, 239-254. He employed it for 

 glomerular urine. See discussion by 

 Sumner, J. B., Science, 100, 413-414. 

 The technique is suitable for even 

 0.08 jttl containing less than 1 nm gm. 

 phosphate phosphorus with a mean 

 error approximately 0.1% (Glick, p. 



