Labeled adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')tetraphospho(5')nucleoside (Ap4N). Synthesis with firefly luciferase.
Labeled dinucleoside polyphosphates are not commercially available, in spite of being important molecules in metabolic regulation. Firefly luciferase (EC 1.13.12.7) is a useful enzyme for the synthesis of adenosine(5')tetraphospho(5')adenosine (Ap4A). As luciferase behaves as a nucleotidase at low ATP concentration, adequate concentrations (higher than 0.1 mM ATP) should be used to obtain a good yield of labeled Ap4A. [32P]Ap4A has also been synthesized from ATP and [32P]PPi. In a first step, [beta, gamma-32P]ATP is generated in a ATP-[32P]PPi exchange reaction catalyzed by luciferase. In a second step, the reaction is supplemented with pyrophosphatase and 32P labeled Ap4A is obtained. Radioactive adenosine(5')tetraphospho(5')nucleoside (Ap4N) can also be synthesized from ATP gamma S and labeled NTP or from low concentrations of labeled ATP and high concentrations of cold NTP. The syntheses of radioactive ApnA and pnA (n > 4) can also be approached with luciferase.
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001517:Coleoptera / Q000201:enzymology; D015226:Dinucleoside Phosphates / Q000138:chemical synthesis* / Q000737:chemistry; D011756:Diphosphates / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D008156:Luciferases / Q000737:chemistry*; D010761:Phosphorus Radioisotopes; D014316:Tritium
D015226:Dinucleoside Phosphates; D011756:Diphosphates; D010761:Phosphorus Radioisotopes; D014316:Tritium; C020733:diadenosine tetraphosphate; D000255:Adenosine Triphosphate; D008156:Luciferases