In vitro processing of the adenosine analog formycin A to the mono-, di-, and triphosphate by a soluble multienzyme system from mouse liver.
The adenosine kinase activity present in a soluble preparation from rat 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), and the AMP analog, that was formed. The results showed that 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 than 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., it has been possible to demonstrate for the first time in an in vitro system the complete salvage of a nucleoside to the triphosphate level.
D015720:5'-Nucleotidase; D000248:Adenosine Kinase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000378:metabolism*; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005573:Formycins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D009708:Nucleotidases / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D012265:Ribonucleotides / Q000302:isolation & purification / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D005573:Formycins; D012265:Ribonucleotides; C034550:formycin; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000248:Adenosine Kinase; D000263:Adenylate Kinase; D009708:Nucleotidases; D015720:5'-Nucleotidase