Plasmodium berghei: In vitro incorporation of purine derivatives into nucleic acids

Uptake of radioactive adenosine by Plasmodium berghei parasitized erythrocytes is sensitive to competition for uptake over a wide range of concentrations of nonradioactive adenosine. Infection points in the uptake curve for adenosine-8- 3H occur between 10 −4 to 10 −5 and 10 −9 to 10 −10 m nonradioactive adenosine. This finding prompted study of the optimal conditions for the in vitro incorporation of radioactivity from AMP-8- 3H into DNA and RNA of erythrocyte-free parasites. These conditions include: incubation temperature (37C), pH (7.4), amount of tritiated precursor (2.5 μCi), and stability of free parasite preparations upon storage in the cold (up to 4 hr at 4C). Whether a deoxyribose (deoxyadenosine-8- 3H) or ribose (adenosine-8- 3H) form of purine nucleoside is used, radioactivity associates primarily with parasite RNA (95% of total incorporation) but only slightly with parasite DNA (5% of total incorporation). The ratio is similar for incorporation of radioactivity from other precursors (purine nucleotides: AMP-8- 3H, ADP-8- 3H, ATP-8- 3H, GTP-8- 3H). During the first 20 min of incubation, radioequivalent amounts (2.5 μCi) of exogenous adenine derivatives incorporate into parasite nucleic acids in decreasing order as follows: AMP-8- 3H > ADP-8- 3H > ATP-8- 3H > adenosine-8- 3H > adenine8- 3H > cyclic-3′,5′-AMP-8- 3H. No detectable incorporation into free parasite nucleic acids occurs when either ATP-α- 32P or ATP-β-γ- 32P is added as substrate. Incorporation into either RNA or DNA is greatest when exogenous AMP-8- 3H is used as substrate. Incorporation of radioactivity from AMP-8- 3H into nucleic acids of erythrocyte-free parasites is twofold greater than into nucleic acids of the parasitizedblood equivalent. Incorporation from AMP-8- 3H into free parasite nucleic acids is sensitive to competition for incorporation by nonradioactive AMP (10 −5 m nonradioactive AMP decreases incorporation from AMP-8- 3H by 54%; 10 −3 m nonradioactive AMP decreases incorporation from AMP-8- 3H by 98%). Similarly, over a range of concentrations, 6-mercaptopurine (6-MP) competes for and decreases incorporation of label from exogenous AMP-8- 3H.