ZYMOHEXASE 87 



librium between the two. Together these two enzymes are referred 

 to as zymohexase. 



Allen and Bourne ( 1943) adapted the microscopic technique for 

 phosphatase (page 78) to this enzyme system, whose distribution 

 they studied in skeletal, heart, and smooth muscle tissue. By 

 incorporating iodoacetic acid into their substrate media, they 

 prevented further enzymatic breakdown of the triose phosphates. 

 The distinct difference in localization of zymohexase and alkaline 

 phosphatase precluded the possibility of confusing the two; however, 

 the phosphatase activity could be selectively blocked by fluoride. 

 Allen and Bourne utilized the fact that the triose phosphates formed 

 by the zymohexase action will spontaneously liberate inorganic 

 phosphate at room temperature in alkaline solution. The phosphate 

 could then be precipitated, and finally visualized in the manner of 

 Gomori (page 78). It was observed that sections which had been 

 infiltrated with paraffin lost their enzyme activity and, accordingly, 

 frozen sections were employed. 



Allen and Bourne Method for Zymohexase 



SPECIAL REAGENTS 



0.1 M Sodium lodoacetate. Neutralize 1.86 g. iodoacetic acid with 

 1 N sodium hydroxide to bromothymol blue and dilute to 100 ml. 



0.1 M Sodium Fluoride. 



2% Cobalt Chloride (C0CI2.6H0O). 



Ammonium Sulfide Solution. Dilute 1 ml. yellow ammonium sulfide 

 to 50 ml. with distilled water. Prepare fresh before use. 



Magnesia Mixture. Dissolve 5.5 g. magnesium chloride 

 (MgCl2.6H20) and 7.0 g. ammonium chloride in 35 ml. 5 N am- 

 monium hydroxide. Filter after 1 hr. and add 60 ml. 4 A^" am- 

 monium hydroxide to the filtrate. 



Purified Sodium Hexose Diphosphate. Formula I: mix 40 ml. 4% 

 sodium hexose diphosphate ( prepared by treating the calcium salt 

 with sodium oxalate) and 20 ml. magnesia mixture, and filter off 

 precipitated phosphate after 30 min. Formula II: mix 20 ml. of 

 4% sodium hexose diphosphate with 20 ml. of magnesia mixture 

 and 20 ml. of distilled water; filter after 30 min. as above. 



Substrate A. Combine 10 ml. of the purified salt soln. (Formula I) 

 with 1.7 ml. of 0.1 M sodium iodoacetate and 5 ml. distilled water. 



