592 G. SCHMIDT 



Speci^city. The specificity of bull semen phosphatase was thoroughly- 

 investigated by Heppel and Hilmoe, who found that 5'-adenylic acid, 5'- 

 uridylic acid, and nicotinamide-5-nucleotide, as well as ribose-5-phosphate, 

 were rapidly hydrolyzed by the enzyme, whereas all other phosphoric acid 

 esters tested, in particular, the 2'- and 3'-ribonucleotides and the adenyl 

 pyrophosphates, were resistant toward the enzyme. Of particular interest 

 is the fact that ribose-5-phosphate is hydrolyzed at a very considerable rate 

 whereas glucose-6-phosphate, phosphogluconate, and 1,6-fructose diphos- 

 phate are practically resistant against the enzyme. 



Michaelis-Menten constants. The affinity of seminal 5'-nucleotidase for 

 the purine and pyrimidine-5'-nucleotides (iv^^deoyUc <l^~\ A'jfuridyiic 

 <10~'*, i^Afcytidyiic = 2 X 10~*) Is much higher than those for ribose-5'- 



phOSphate (/Vjifrlbose-S-phosphate — 2.6 X 10~ ; ivAfnicotinamide-S-nucIeotide ~ 1-6 



X 10-3). 



Activators and inhibitors. Magnesium ions in approximately 0.01 M con- 

 centration have considerable activating effect; they cannot be replaced by 

 manganese ions. The extent of the magnesium activation depends on the 

 nature of the buffer : Heppel and Hilmoe found that in tris(hydroxymethyl) 

 aminomethane buffer the enzyme has 75 % of its full activity without the 

 addition of magnesium ions, whereas, in glycine buffer the activity without 

 magnesium ions amounts only to 30% of the optimal activity. ^^ 



Fluorides in 0.01 M concentration inhibit the enzyme activity by 70%, 

 in 0.1 M concentration completely. Borate in 0.08 M concentration inhibi- 

 ted the activity 85 % in comparison with glycine buffer at the same pH. 



Stability. Crude as well as purified preparations of seminal 5'-nucleotidase 

 can be preserved in active form for a considerable time in the frozen state, 

 before or after lyophilization. 



b. 3' -Nucleotidase (formerly b -nucleotidase) 



Shuster and Kaplan^ ^^ discovered the presence in germinating rye grass 

 and germinating barley of a phosphatase which specifically catalyzes the 

 hydrolysis of 3 '-nucleotides to nucleosides and inorganic phosphate. En- 

 zymes of similar specificity occur in other vegetable materials, such as wheat 

 and corn leaves, soy bean leaves, and lawn grass leaves. Takadiastase like- 

 wise dephosphorylates 3'-nucleotides much faster than 2'- or 5'-nucleotides. 

 So far, the most powerful 3 '-nucleotidase activity was found in germinating 

 rye grass. 2'-Nucleotides, 5'-nucleotides, and pyrophosphates were not 

 hydrolyzed by purified preparations of the enzyme. The fact that one of 

 the phosphoryl groups of coenzyme A is hydrolyzed by 3'-nucleotidase is 

 interpreted by assigning to this phosphoryl group the 3'-position of the 



1" L. Shuster and N. O. Kaplan, J. Biol. Chem. 201, 535 (1953). 



