Metabolism of tubercidin and formycin to their 3':5'-cyclic nucleotides in mammalian cells.

High performance anion exchange chromatography was utilized to demonstrate extensive metabolism of tubercidin and formycin to their corresponding 5’-triphosphates in mouse leukemia EL4 cells and a concomitant decrease in cellular ATP. Efficient anabolism of formycin required the presence of an inhibitor (erythro9-(2-hydroxy-3-nonyl)adenine) of adenosine deaminase in the cellular incubations. Acid-soluble extracts from cells treated with these adenosine analogues were chromatographed on sequential columns of aluminum oxide and an appropriate ion exchange resin, and fractions from the latter columns were monitored for material cross-reactive in a radioimmunoassay specific for adenosine 3’:5’-monophosphate (CAMP). Extracts of the tubercidinand formycin-treated cells were found to contain unique immunoreactive substances, sensitive to CAMP phosphodiesterase, whose chromatographic properties were consistent with their tentative identification as the 3’:5’-monophosphates of these two purine analogues. The cellular formation of these two presumptive CAMP analogues was both timeand (analogue) dosedependent. Formation of putative formycin 3’:5’-monophosphate was enhanced when cells were incubated with formycin in the presence of erythro-9-(2-hydroxy3-nonyl)adenine. Treatment of cells with these adenosine analogues resulted in a marked decrease in CAMP levels, presumably due in part to the decreased cellular levels of ATP. Prostaglandin El and cholera toxin, in the presence of 1-methyl-3-isobutylxanthine, each increased the concentration of these analogue cyclic nucleotides severalfold in cells preloaded with nucleotides of tubercidin or formycin. Evidence is also presented for the metabolism of tubercidin to its 3’:5’-monophosphate in mouse leukemia L1210 cells, mouse cytotoxic lymphocytes, and human leukocytes. Tubercidin (7-9) and formycin (10-12) are metabolized to their corresponding 5’-mono-, -di-, and -triphosphates in a variety of cell types; however, formycin requires inhibition of adenosine deaminase in order for its conversion to nucleotides to proceed extensively in tissues (12). Both tubercidin (7, 13, 14) and formycin (15-18) are incorporated into the nucleic acids of microbial and mammalian cells. This paper describes evidence for yet another metabolic fate of these two antibiotics in mammalian cells, conversion of their 5’-triphosphates to their corresponding 3’:5’-monophosphates via adenylate cyclase.