Assay of adenosine 5′-P1-tetraphospho-P4-5′′′-adenosine and adenosine 5′-P1-tetraphospho-P4-5′′′-guanosine in Physarum polycephalum and other eukaryotes. An isocratic high-pressure liquid-chromatography method

Abstract

A5′pppp5′A has been proposed to serve as a molecular signal that triggers DNA replication. When published methods proved to be inadequate for the assay of A5′pppp5′A in Physarum polycephalum by h.p.l.c. (high-pressure liquid chromatography), a set of purification procedures was developed that allowed assay of as little as 2pmol of A5′pppp5′A. A5′pppp5′A was purified from cellular extract by covalent boronate chromatography, treated with alkaline phosphatase to hydrolyse residual mononucleotides and analysed by isocratic ion-exchange h.p.l.c. The analysis was facilitated by a pre-column switching procedure that allowed early-eluted species to be diverted from the analytical column. By using this procedure A5′pppp5′A has been detected in Physarum polycephalum (1.4 pmol/mg of protein), Saccharomyces cerevisiae (3.6 pmol/mg of protein) and rat liver (3.3 pmol/mg of protein). In each case a minor peak was also seen, which was identified as A5′pppp5′G. The identity of both peaks was confirmed by co-elution with standards on isocratic and gradient h.p.l.c. and treatment with enzymes, including a dinucleoside polyphosphate pyrophosphohydrolase from Physarum polycephalum.