188 S. S. COHEN 



The reaction, as shown in formula (XXXIII), wiU proceed with each of the 

 nucleoside diphosphates, with the exception of guanosine diphosphate, or 

 with a mixture of all the diphosphates, includmg that of guanosine. In the 

 former instance, the synthetic product is a high molecular polynucleotide 

 comprised of a single type of nucleotide, bound in the chain by 3'5' phospho- 

 diester hnkages (Heppel et al., 1957a). In the latter case, the synthetic poly- 

 nucleotide product contains a mixture of all of the bases, as in natural RNA 

 (Heppel et al., 1957b). Synthetic polynucleotides containing a smgle base are 

 rapidly phosphorylyzed in the presence of the enzyme; natural RNA and 

 synthetic polynucleotides containmg four bases are slowly phosphorylyzed 

 (Ochoa, 1957). In the latter case it has been suggested that the enzymatic 

 resistance of these substances arise from their existence in solution as multi- 

 stranded chains. 



The ready reversibility of the polynucleotide phosphorylase reaction 

 revealed that the free energy change (AF) in hydrolysis of the pyrophosphate 

 of the nucleoside diphosphate is of the same order as the AF of cleavage of 

 the phosphodiester hnkage in RNA. The existence of the hydroxyl at C 2 of 

 ribose presumably determmes this property of the polyribotides, since the 

 enzyme is inactive with DNA or deoxynucleoside diphosphates. In addition 

 to its presence in bacteria, the enzyme has been found in small amounts in 

 spinach leaf, and yeast (Heppel and Rabinowitz, 1958) and a suggestion of 

 its presence in the nuclei of guinea pig liver has been reported (HUmoe and 

 Heppel, 1957). In the latter instance the phosphorolysis of adenine poly- 

 nucleotide was detected but, starting with ADP, a net synthesis was not 

 observed. 



The purification of the enzyme has revealed that the addition of a nucleo- 

 side diphosphate to form polynucleotide requires the presence of a primer 

 (Mii and Ochoa, 1957; Singer et al, 1957). Thus, the polymerization of HDP 

 showed a lag, abolished almost specifically by polyuridylic acid. Polymeriza- 

 tion of ADP and CDP required polyadenylic acid and polycytidylic acid, 

 respectively. The primer must be at least a dinucleotide in length and appears 

 to be incorporated into the polymer that is produced. The primer provides 

 the terminal nucleotide ending in a 5 '-phosphate. Thus: 



adenylyladenosine-5'pliosphate -j- n HDP > polyuridylyl adenylyIadenosine-5'- 



phosphate -f- nP 



In a comparable phenomenon, polynucleotides were degraded by the enzyme 

 to "hmit polyimcleotides" consisting of a dinucleotide or a duiucleoside 

 monophosphate, depending on the substitution on the terminal nucleoside. 

 Physical measurements of the synthetic polynucleotides have revealed 

 particle weights in the range of 10^ to 10^. However, end-group analyses have 

 indicated markedly lower weights for the same polymers. This appears to 

 arise as a result of aggregation in solution, a result confirmed by study of the 



