A novel method for the synthesis of nucleoside triphosphates labelled with inorganic [32P]phosphate specifically in the β-position

Abstract

A method was developed for the introduction of [32p]Pi specifically into the beta-position of ATP and GTP. The method is based on two separate reactions involving (a) phosphorolysis of poly(A) or poly(G) [Soreq, Nudel, Salomon, Revel & Littauer (1974) J. Mol Biol. 88, 233-245] in the presence of [32P]Pi and (b) conversion of the labelled diphosphate into the corresponding triphosphate by transferring the active phosphate group from 1,3-diphosphoglycerate in a coupled reaction as decribed by Glynn & Chappell [(1964) Biochem. J. 90, 147-149]. Radioactivity in the beta- and gamma-phosphate groups of the labelled triphosphate was measured by using polynucleotide kinase. No detectable radioactivity was found in the gamma-phosphate group. The suitability of this method for the synthesis of other nucleoside triphosphates specifically labelled in the beta-position is discussed.