Figure 3.

Thymidine uptake in trophozoites of T. vaginalis. A. Time course of 1 µM [³H]-thymidine, displaying a rate of 0.139 ± 0.008 pmol(10⁷ cells)⁻¹s⁻¹ (r² =0.98; not significantly non-linear, P = 0.20; significantly different from zero, P < 0.0001). In the presence of 1 mM thymidine, the rate was reduced by 90.6% to 0.013 ± 0.0018 pmol(10⁷ cells)⁻¹s⁻¹ (r² =0.98; not significantly non-linear, P = 0.67; significantly different from zero, P = 0.02). B. Time course of 0.125 µM [³H]-thymidine. Data are presented as the average of 4 experiments over 60 s. Rate was 0.0045 ± 0.0012 pmol(10⁷ cells)⁻¹s⁻¹ (n=4), inhibited 75% by 1 mM unlabelled thymidine. C. Dose-dependent inhibition of 0.125 µM [³H]-thymidine uptake by thymidine and uridine. Thymidine: Hill slope = −0.89, EC₅₀ = 11.7 µM, r² = 0.996; Uridine: Hill slope = −0.95, EC₅₀ = 15.3 µM, r² = 0.993. Inset: conversion to Michaelis-Menten saturation plot, K_m = 15.1 µM, V_max = 0.245 pmol(10⁷ cells)⁻¹s⁻¹ for this experiment. Incubation time was 30 s. D. Dose-dependent inhibition of 0.125 µM [³H]-thymidine transport by guanosine and cytidine (Hill slopes of −1.09 and −0.54, respectively). EC₅₀ (guo) = 51.2 µM in this experiment (r² 0.98). 2-transporter plot for cytidine (dashed line) yielded EC₅₀ values of 2.86 µM and 322 µM (r² 0.99). E. Inhibition plots with 1 µM [³H]-thymidine as radiotracer. Hill slopes for thymidine and adenosine were −0.61 and −1.09, respectively. When plotted to a 2-site equation (dashed line), the thymidine inhibition curve yielded EC₅₀ values of 10.7 µM and 529 µM (r² = 0.99); adenosine EC₅₀ = 89.8 µM in this experiment. Inset: conversion to saturation curve, yielding K_m = 394 µM, V_max = 12.9 pmol(10⁷ cells)⁻¹s⁻¹ (r² = 0.98).F. Lineweaver-Burk double reciprocal plot from the thymidine dose-response data in C. Low affinity component: K_m 340 µM, V_max 12.0 pmol(10⁷ cells)⁻¹s⁻¹, r² 0.98; Higher affinity K_m 23.6 µM, V_max 2.2 pmol(10⁷ cells)⁻¹s⁻¹, r² 0.99.