An efficient synthesis of polyguanylic acid by a thermophilic polynucleotide phosphorylase.
Polyguanylic acid (poly(G)) was synthesized from GDP in a yield of 60-75% by Thermus thermophilus polynucleotide phosphorylase (polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) at 70 degrees C, pH 8.5 in the presence of Mg2+. The yield was dependent on the ratio of GDP to Mg2+, but was independent of the concentrations of enzyme or substrate. The maximal rate of GDP polymerization was obtained when the ratio of GDP to Mg2+ was 3:1. However, by prolonged incubation, the higher initial ratio of over 4:1 was preferred because of the rapid consumption of GDP in the reaction mixture. Poly(G) prepared by 1 h incubation was heterogeneous in size from 5 S to over 23 S, but by prolonged incubation of 19 h the size of product converged to 9 S as judged by sucrose density gradient centrifugation. Its chain length was determined by terminal nucleoside analysis to be 200 nucleotides long.
D001419:Bacteria / Q000201:enzymology*; D006150:Guanine Nucleotides; D006358:Hot Temperature; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011068:Poly G / Q000096:biosynthesis*; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism*; D011131:Polyribonucleotides / Q000096:biosynthesis*
D006150:Guanine Nucleotides; D011131:Polyribonucleotides; D011068:Poly G; D011117:Polyribonucleotide Nucleotidyltransferase; D008274:Magnesium