The Uptake of Pyrimidine Nucleosides in Tetrahymena. I. Uridine*

Abstract

SYNOPSIS. Uridine uptake was examined in Tetrahymena pyriformis GL‐7 in defined medium under conditions where food vacuole formation is not a significant factor in solute acquisition by the cell. The results indicate the presence of a saturable mechanism which follows Michaelis‐Menten kinetics. When corrected for diffusion the apparent Km for the carrier is 2.3 ± 0.6 μM and the Vmax is 7.3 ± 0.2 × 10−7 nmoles/cell/min. It is evident from nucleotide pool analysis that most of the radioactivity of externally supplied [3H]uridine appears in UMP with the remainder in UTP. Uridine is apparently phosphorylated immediately upon entry into the cell and neither uridine‐cytidine kinase activity nor RNA synthesis are rate‐limiting in the uptake process. Uridine transport is competitively inhibited by a variety of ribo‐ and deoxyribonucleosides as well as several nucleoside analogs. Neither uracil nor ribose or deoxyribose are effective inhibitors of uridine transport indicating the carrier is specific for the nucleoside. There is little difference between the Ki values for ribo‐ as opposed to deoxyribonucleosides except in the case of deoxyguanosine which is much less effective as an inhibitor under the conditions of this study, than all the other nucleosides, including guanosine.

Effects of six pyrimidine analogs on the growth ofTetrahymena thermophilaand their implications in pyrimidine metabolism

“…Several early studies in T. pyriformis suggested that their transports might involve the use of a nucleoside transporter system [16,25,26]. Transporter proteins in this system have the ability to move one or more types of nucleotides across membranes, with functional redundancies [16,26]. Indeed, results derived from recent bioinformatic analyses suggest that there are more than 10 equilibrative nucleoside transporter genes present in the T. thermophila genome [27,28], indirectly confirming those early observations.…”

“…Specifically how nucleobases and nucleosides are transported into Tetrahymena remains unknown. Several early studies in T. pyriformis suggested that their transports might involve the use of a nucleoside transporter system [16,25,26]. Transporter proteins in this system have the ability to move one or more types of nucleotides across membranes, with functional redundancies [16,26].…”

Effects of six pyrimidine analogs on the growth of Tetrahymena thermophila and their implications in pyrimidine metabolism

“…pyriformis suggested that the transport of these compounds might involve a nucleoside transporter system [ 16 , 25 , 26 ]. Transporter proteins in this system have the ability to move one or more types of nucleotides across membranes, with functional redundancies [ 16 , 26 ]. Indeed, results derived from recent bioinformatic analyses suggest that there are more than 10 equilibrative nucleoside transporter genes present in the T .…”

“…Freeman and Moner fed T . pyriformis GL-7 with [ 3 H]uridine in a short period of time before a new food vacuole could be formed and quantified the amount of radioactive uridine present in different forms in the cell [ 16 ]. They concluded that uridine could enter through the cell surface and was immediately phosphorylated.…”

The evaluation of initial rates in the presence of background noise

“…Some authors chose short labelling times in order'to be in linear range' of time-dependent pool saturation. The linear range, however, is scarcely observed but assumed to occur between to and the shortest pulse possible (Freemann & Moner, 1976;Wolfe, 1975;Dall'asta et al, 1978). In some instances a background due to incomplete removal of the label is considered and subtracted from the data, with reference to inulin experiments (Reiser & Christiansen, 1972;Winter & Christensen, 1964).…”