312 F. SCHLENK 



and co-workers* had already observed that a- or j8-methyl-D-ribosides are 

 not spht. The restricted action of nucleosidases from various sources, and 

 the importance of the base in determining the susceptibility to the enzjones 

 of this type, have been amply confirmed since Dixon and Lemberg's in- 

 vestigation; some nucleosides A\ath nonbiological bases, however, are an 

 exception.^ 



The studies of the Thannhauser schooP''"^^ resulted in significant ad- 

 vances. W. Klein^^ extracted and purified purine nucleosidase from spleen, 

 lung, liver, and heart tissue. He found that the enzyme was inactivated by 

 dialysis and reactivated by the addition of phosphate or arsenate. No ex- 

 planation of the phenomenon was given, and it is not surprising that, with- 

 out the precedent of any monosaccharide 1-phosphate ester, the nature of 

 the reaction product was not recognized. The method of deproteinizing split 

 most or all of the labile ester, thus allo^ving determination of the reducing 

 group of the ribose by iodimetry. It remained for H. M. Kalckar'^ to draw 

 the correct conclusions in his continuation of Klein's experiments. With the 

 help of greatly improved and new analytical techniques, which were de- 

 veloped especially for this study, the following reactions were observed to 

 occur with rat liver enzyme: 



Guanosine + phosphate ;=i guanine + ribose-1 -phosphate (3) 



Inosine + phosphate ;=i hypoxanthine + ribose-l -phosphate (4) 



The name nucleoside phosphorylase was suggested for this enzyme. The 

 dialysis experiments of Klein'^ could be confirmed with the purified enzyme. 

 With an improved method for the determination of phosphate, which leaves 

 sensitive phosphoric acid esters intact, it was found that inorganic phos- 

 phate was esterified to the same extent as the purine base was set free. The 

 structure of ribose- 1-phosphate was assigned to this compound. The ester 

 could be isolated and, using it in combination with guanine or hypoxanthine, 

 nucleoside synthesis was achieved (Fig. 1). With equimolar amounts of 

 ribose- 1-phosphate and hypoxanthine the equilibrium is 85 to 90% in favor 

 of nucleoside formation. Therefore, if the preparation of ribose- 1-phos- 

 phate in quantity from inosine or guanosine is required, a high concentra- 

 tion of phosphate is used. Xanthine oxidase, or, if guanosine is used, 

 guanase, is added to deaminate and oxidize the base which is liberated from 



8 P. A. Levene, W. A. Jacobs, and F. Medigreceanu, J. Biol. Chem. 11, 371 (1912). 



8 M. Friedkin, J. Cellular Comp. Physiol. 41,Suppl. 1, 216 (1953); M. Friedkin and 



D. Roberts, J. Biol. Chem. 207, 245 (1954). 

 ^0 S. J. Thannhauser and M. Angermann, Z. physiol. Chem. 186, 13 (1929). 

 " W. Deutsch and R. Laser, Z. physiol. Chem. 186, 1 (1929). 

 12 W. Klein, Z. physiol. Chem. 231, 125 (1935). 

 1' H. M. Kalckar, J. Biol. Chem. 158, 723 (1945); 167, 477 (1947). 



