126 H. GOBIND KHORANA 



adenylic and uridylic acids and also of adenylic, guanylic, uridylic, and 

 cytidylic acids point to there being a single enzyme. In contrast, Olmsted 39, 39a 

 working with M. lysodeikticus preparations has presented some evidence in 

 favor of the view that different enzymes may be involved in handling differ- 

 ent nucleoside diphosphates. 



As shown in Eq. (1) the reaction is reversible, although the point of 

 equilibrium may vary somewhat with different nucleoside diphosphates. 

 Under favorable conditions, the forward reaction (Eq. 1) may proceed so 

 far as to give 50-80% of the acid-labile phosphate as orthophosphate. The 

 reaction thus favors polynucleotide synthesis. 32, 33 



With the partially purified enzymes 32, 33, 35 linear rates of polynucleotide 

 synthesis were reported without the addition of any polynucleotide primers. 

 Further purification of the Azotobacter enzyme led to the recognition of a 

 lag in the reaction 40 and to the finding that a variety of polynucleotides 36, 

 40 • 41 abolished this lag (Section c, below). 



b. The Structure and Size of Ribopolynucleotides Synthesized by Polynucleo- 

 tide Phosphorylase 



All the enzymically synthesized polymers contain the repeating C 3 '-C & ' 

 interribonucleotide linkage which has previously been established to occur 

 in the ribonucleic acids (Volume I, Chapter 12). Thus, the products formed 

 upon hydrolysis of the synthetic products with alkali and with different 

 phosphodiesterases (venom and spleen phosphodiesterases and pancreatic 

 ribonuclease) are as expected from the established mode of action of these 

 agents. 42, 43 



The alkaline hydrolysis of some polymers gives in addition to a mixture 

 of nucleoside-2' and 3 '-phosphates, small amounts of free nucleosides and 

 nucleoside-2'(3') ,5'-diphosphate. 42, 44 The result indicates a polynucleotide 

 structure containing a 5'-phosphomonoester group at one end of the chain 

 and free 2'- and S'-hydroxyl groups at the other end. 45 The origin of the 

 o'-phosphomonoester group at the terminus cannot be uniquely explained 

 because of uncertainties regarding the mode of action of polynucleotide 

 phosphorylase. Firstly, if the chain formation begins at the 3'-hydroxyl end 



39 P. S. Olmsted, Biochim. et Biophys. Acta 27, 222 (1958). 



39a P. S. Olmsted and G. L. Lowe, J. Biol. Chem. 234, 2971 (1959). 



40 S. Mii and S. Ochoa, Biochim. et Biophys. Acta 26, 445 (1957). 



41 M. F. Singer, L. A. Heppel, and R. J. Hilmoe, Biochim. et Biophys. Acta 26, 447 

 (1957). 



41a M. F. Singer, R. J. Hilmoe, and L. A. Heppel, J. Biol. Chem. 236, 751 (1960). 



42 L. A. Heppel, P. J. Ortiz, and S. Ochoa, /. Biol. Chem. 229, 679 (1957). 



43 L. A. Heppel, P. J. Ortiz, and S. Ochoa, J. Biol. Chem. 229, 695 (1957). 



44 S. Ochoa, Federation Proc. 15, 832 (1956). 



45 R. Markham, R. E. F. Matthews, and J. D. Smith, Nature 173, 537 (1954). 



