Measurement of PRPP availability, and purine and DNA synthesis

Cells were incubated in 12 well (PRPP availability and DNA synthesis) or 6 well plates (purine synthesis) for the indicated times.

For PRPP availability, [8-¹⁴C]-hypoxanthine (1 µCi, 58 mCi/mmol) was added during the last 20 min of incubation, and the cells were washed twice with PBS, extracted in 1 mM NaOH, and the extracts spotted onto squares of diethylaminoethyl cellulose paper⁶. The paper squares were washed three times with 1 mM ammonium formate, dried, and radioactivity on the squares was measured.

For purine synthesis, [¹⁴C]-formate (10 µCi, 56 mCi/mmol) was added during the last 90 min of incubation, and the cells were extracted in 0.4 N perchloric acid^{4, 6}. The extracts were boiled for 70 min to reduce all purine nucleotides and nucleosides to purine bases, and supernatants were applied to Dowex 50 columns eluted with 6 N HCl.

For DNA synthesis, [³H]-thymidine (1 µCi, 70 Ci/mmol) was added during the last 60 min of incubation, and the cells were washed once with PBS and extracted with 10% trichloroacetic acid^{4, 6}. The resulting precipitates were collected on glass fiber discs, which were washed with trichloroacetic acid, dried, and transferred to scintillation vials.

In all cases, radioactivity was measured by liquid scintillation counting using 2,5-diphenyloxazole and p-bis-[2-(5-phenyloxazolyl)]-benzene as primary and secondary fluorophores, respectively.