Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells.
Incubation of L1210 leukemia cells with 10 microM [3H]adenine in the absence of energy substrate results in a very rapid accumulation of 3H within the cells. By 20 s intracellular adenine is near steady-state; beyond this the rate of accumulation of intracellular 3H reflects nucleotide synthesis, predominantly the rate of ATP accumulation within the cell as determined by liquid chromatography. Adenine incorporation into the nucleotides proceeds via adenine-phosphoribosyl transferase, which is rate-limiting to AMP formation and subsequently the formation of ADP and ATP. Acceleration of this pathway by the addition of glucose and phosphate decreases the intracellular adenine level far below equilibrium as metabolism is increased relative to transport. Assessment of methodology to evaluate intracellular adenine and its metabolites indicates that (i) a 4 degree C wash removes the major portion of intracellular adenine and (ii) at 4 degree C, transport of adenine remains rapid and while nucleotide synthesis is decreased, ATP still accumulates within the cell. Hence, measurement of cellular uptake of radioactive label at 4 degree C after cells are washed free of adenine cannot be used as a measurement of adenine surface binding since this radioactive label represents, at least in part, phosphorylated derivatives of adenine within the cell. Unlabeled adenine and structurally related compounds were found to inhibit [3H]adenine net uptake under conditions where metabolism of adenine was reduced, suggesting that base transport is mediated by a facilitated diffusion mechanism. This is consistent with other studies from this laboratory that demonstrate exchange diffusion between adenine and other bases.
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000225:Adenine / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000494:pharmacology; D001692:Biological Transport; D005947:Glucose / Q000494:pharmacology; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000494:pharmacology; D007939:Leukemia L1210 / Q000378:metabolism*; D051379:Mice; D010710:Phosphates / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D001386:Azides; D007042:Hypoxanthines; D010710:Phosphates; D019271:Hypoxanthine; D005947:Glucose; D000225:Adenine; D015056:1-Methyl-3-isobutylxanthine