| Abstract: | A new set of bis(5'-nucleosidyl) tetraphosphates, the Bp4B' nucleotides (B and B' = C, G, or U not equal to A), are demonstrated in living cells. In exponentially growing Saccharomyces cerevisiae, cellular concentrations of Cp4U, Up4U, Gp4G, Cp4C, Gp4U, and Gp4C are 210, 200, 60, 50, 40, and 30 nM, respectively. It is likely that these nucleotides originate from the action of diadenosine-5',5"'-P1,P4-tetraphosphate alpha,beta-phosphorylase, an enzyme recently found in yeast. Upon temperature shift or exposure to cadmium, the Bp4B' nucleotides strongly accumulate in the yeast cells. In Escherichia coli, the same nucleotides occur, and similar effects of temperature shift or of cadmium are observed. However, in the bacterium, the origin of these nucleotides is not known. To quantitate these nucleotides in cellular extracts, specific procedures were developed. In the first step, after purification of the mixture of Np4N' (N and N' = A, C, G, or U) nucleotides, the Ap4N nucleotides are specifically removed by incubation with lysyl-tRNA synthetase. In the second step, the Bp4B' species are resolved with the help of anion-exchange high performance liquid chromatography. In the third step, the concentration of each Bp4B' is measured using three coupled enzymatic reactions to produce ATP and bioluminescence. With this strategy, 0.01 pmol of any Bp4B' nucleotide can be reliably detected. |
