31. SYNTHESIS OF POLYNUCLEOTIDES 



125 



+ nH 3 P0 4 (1) 



/ 

 a. Some Properties of Polynucleotide Phosphor ylase 



Good balance between the disappearance of nucleoside-5 '-diphosphates, 

 with concomitant formation of polynucleotides, and the release of inorganic 

 phosphate can be demonstrated 31 • 33 as would be expected for Eq. (1). The 

 reaction requires magnesium and the rate is sensitive to the ratio of its con- 

 centration to that of nucleoside diphosphate. (Optimal rates of reaction 

 with the E. coli enzyme 33 as well as with a highly purified Azotobacter en- 

 zyme 36 were obtained when the ratio of the nucleoside diphosphate to mag- 

 nesium ions was 1 : 1.5-2.) As regards the pH optimum, different results were 

 obtained with the Azotobacter enzyme using two different assays. 32 It is 

 possible that the different ratio of magnesium ion concentration to ADP 

 concentration used in the two assays was a major cause of the variation in 

 the result. 



Beers 35, 37 has also observed rather complex interrelationships between 

 the relative concentrations of ADP and magnesium chloride and the pH 

 optimum of the reaction for the M. lysodeikticus enzyme. He 35, 37 has also 

 noted the requirement for high salt concentration for this enzyme. No 

 such requirement was found (in fact, some inhibition by the high concentra- 

 tion of salt was observed) by Singer et al. s6 for the purified Azotobacter en- 

 zyme. 



The affinity of the enzyme for nucleoside-5 '-diphosphates is very low 32 ' 33 

 (Km's of the order of 10" 2 M) and the enzyme is apparently devoid of 

 specificity towards the purine or pyrimidine base in the ribonucloside-5'- 

 diphosphates. A notable exception is that of guanosine-5'-diphosphate 32, 33 

 which by itself does not polymerize (see below). At various stages of purifi- 

 cation (up to 300-fold) of the Azotobacter enzyme, the specific activity with 

 respect to different nucleoside diphosphates increased roughly to the same 

 extent. 38 These results as well as the formation of copolymeric chains of 



34 R. F. Beers, Jr., Nature 177, 790 (1956). 

 38 R. F. Beers, Jr., Biochem. J. 66, 686 (1957). 



36 M. F. Singer, L. A. Heppel, and R. J. Hilmoe, J. Biol. Chem. 235, 738 (1960). 



37 R. F. Beers, Jr., Arch. Biochem. Biophys. 75, 497 (1958). 



38 S. Ochoa, S. Mii, and M. C. Schneider, Proc. Intern. Symposium on Enzyme Chem. 

 p. 44 (1957). 



