Add time:09/06/2019         Source:sciencedirect.com

The present study describes the synthesis of formycin 5′-triphosphate (FoTP), formycin 5′-monophosphate (FoMP), and formycin 3′,5′-cyclic monophosphate (cFoMP) from formycin A (FoA). These compounds, analogs of ATP, AMP, cAMP, and adenosine, respectively, are all fluorescent and differ chemically from the adenosine compounds by the reversal of the carbon atom at position 8 and the nitrogen at position 9 of the purine ring. Both FoMP and cFoMP were synthesized by chemical procedures from FoA while FoTP was made from FoMP enzymatically. All the analogs could be separated from each other using a high-performance liquid chromatographic (hplc) reverse-phase isocratic system that includes a μBondapak C-18 column as stationary phase and a solution of 0.01 m KH2PO4 adjusted to pH 5.5 with NaOH containing methanol as a mobile phase. At a flow rate of 2 ml/min, FoTP had a retention time of about 1 min followed by FoMP (2 min), cFoMP (3.5 min), and finally FoA (5.5 min). The analogs were detected by fluorometry using an excitation wavelength of 300 nm and an emission wavelength above 320 nm. This detection system proved to be more sensitive than absorbtion spectroscopy and as little as 2 pmol of the compounds could be measured.The analogs, together with the hplc system, were used to develop fluorometric (nonradioactive) assays for several enzymes including 3′5′-cyclic nucleotide phosphodiesterase (PDase), ATP pyrophosphohydrolase, and alkaline phosphatase. With these enzymes, the conversion of cFoMP to FoMP, FoTP to FoMP, and FoMP to FoA, respectively, could be followed. The conversion of FoA to formycin B (FoB), an analog of inosine, was also followed. The intracellular PDase activity isolated from the eukaryotic microorganism Dictyostelium discoideum was studied in some detail, and an apparent Km of 5 μm and Vmax of 0.1 nmol/min/mg protein were obtained for the enzyme at pH 7.5 and 30°. These values are compared to those in the literature.A number of advantages of this fluorometric-hplc assay procedure are discussed, including the facts that it offers an increase in sensitivity over other spectrophotometric assays and is at least equivalent to radiochemical assays currently in use.

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