604 G. SCHMIDT 



enzyme were carried out at pH 7.5, but no systematic pH activity curves 

 have been reported. 



Influence of Substrate Concentration. The Michaelis-Menten constants of 

 the calf Hver enzyme are 1.7 X 10'^ M for ribonucleosides, 1.8 X 10"^ M 

 for deoxyribonucleosides, and 1.1 X 10"^ M for nicotinamide nucleoside. 

 According to Heppel and Hilmoe/^® the yeast enzyme has the higher Km 

 values of 1.1 X 10"^ M for guanosine, 6.5 X lO"" M for nicotinamide ribo- 

 side, and 8 X lO"" M for phosphate. 



Equilibrium Constant. The equilibrium of the phosphorolysis of purine 

 nucleosides is in favor of nucleoside formation. The equilibrium constant 

 for hypoxanthosine phosphorolysis at pH 7.4 is 0.03 according to deter- 

 minations with the yeast enzyme. The value for nicotinamide nucleoside 

 phosphorolysis in which H+ enters is 1 X lO-^^"' ^os 



Stability. Highly purified liver enzyme preparations can be stored at 

 -20° with very little loss, whereas the yeast nucleoside phosphorylase is 

 less stable, at least in highly purified conditions. 



Assaij Methods. In the earlier investigations of the period preceding the 

 work of Kalckar.i^" ^hg activity of nucleosidases was assayed reducto- 

 metrically. The liberation of reducing groups after the action of nucleo- 

 side phosphorylases is, however, a secondary reaction caused by acid 

 hydrolysis of the highly acid-labile ribose-1 -phosphate. 



In recent investigations, the action of the enzyme was measured either by 

 determination of inorganic phosphate or of the liberated purines according 

 to the sensitive and convenient procedures developed by Kalckar.^^* The 

 formation of deoxyribosides can also be followed with the microbiological 

 method of Hoff-j0rgensen2O9.2io which is based on the observation that 

 Thermobacterium acidophilus R26 (Orla Jensen Collection) requires deoxy- 

 ribosides for growth. 



b. Pyrimidine Nucleoside Phosphorylases 



The first observations suggesting the existence of specific phosphorylases 

 for pyrimidine nucleosides were made by Deutsch and Laser,!^^ ^y^Q dis- 

 covered the power of bone marrow extracts to cleave deoxyribonucleosides 

 of pyrimidines, and by Klein, ^^^ who demonstrated the phosphate— or 

 arsenate — requirement of these enzymes. He also found that kidney ex- 

 tracts were much more active toward uridine than toward deoxyriboguanine, 

 whereas the opposite behavior was found for spleen extracts. In Klein's ex- 

 periments guanine "inhibited" the cleavage of purine nucleosides, but not 

 that of uridine. The cleavage of cytidine was much slower than that of uri- 

 dine and thymidine. 



208 L. J. Zatman, N. O. Kaplan, and S. P. Colowick, J. Biol. Chem. 200, 197 (1953). 



203 E. Hoff-J0rgensen, J. Biol. Chem. 178, 525 (1949). 



210 E. Hoff-J0rgensen, M. Friedkin, and H. M. Kalckar, J. Biol. Chem. 184, 461 (1950). 



