Inhibition of DNA synthesis in permeabilized mouse P815 mast cells by arabinofuranosyladenine 5'-triphosphate

P815 mast cells (mouse leukemic cells) have been successfully permeabilized to 9-beta-arabinofuranosyladenine 5'-triphosphate (araATP) using lysolecithin. The permeabilized cells remained viable according to the following criteria: (a) exclusion of trypan blue after replacing the lysolecithin with culture medium; (b) cell growth rate identical to control cultures, and (c) intact DNA-synthesizing complex in permeable cells. It was shown that permeabilized cells incorporate [3H]thymidine and [3H]deoxyadenosine-triphosphate (dATP) into DNA when the suspension is supplied with the appropriate substrates and cofactors. Permeable cells break down araATP to 9-beta-D-arabinofuranosyladenine 5'-diphosphate only slightly after incubation for 60 min; no further dephosphorylation to 9-beta-D-arabinofuranosyladenine 5'-monophosphate or 9-beta-D-arabinofuranosyladenine occurs. In kinetic experiments it was shown that the DNA-replicating complex of permeabilized cells is competitively inhibited by araATP with respect to dATP; Ki(araATP) was determined to be 0.6 microM and the Km(dATP), 0.9 microM.