In vitro processing of the adenosine analog formycin A to the mono-, di-, and triphosphate by a soluble multienzyme system from mouse liver

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Abstract
The adenosine kinase activity present in a soluble preparation from mouse liver was investigated using formycin A (FoA), a fluorescent analog of adenosine as the phosphoryl acceptor and ATP as the donor. Reversed-phase high-performance liquid chromatography (h.p.l.c.) was used to separate substrate from product, and the progress of the phosphorylation reaction was followed by monitoring fluorometrically the amount of formycin 5''-monophosphate (FoMP), the AMP analog, that was formed. While FoMP was formed during the reaction indicating that an adenosine kinase activity was present, both formycin 5''-di- and triphosphate (FoDP and FoTP respectively), the corresponding analogs of ADP and ATP, were also formed, suggesting that an adenylate kinase activity was present. This result was confirmed with FoMP as the substrate and showing the formation of FoDP and FoTP. Other experiments carried out with FoMP as the substrate revealed the formation of FoA. Taken together, these results indicated that a 5''-nucleotidase activity as well as an adenylate kinase was present. Using this analog and h.p.l.c., the complete salvage of a nucleoside to the triphosphate level was demonstrated for the first time in an in vitro system.