Figures & data
Figure 1. Physiological and biochemical identification of Xut1, a C. albicans xanthine-uric acid transporter. (a) Growth of C. albicans on purines as sole N sources, (b) Time course of uptake of 0.5 µM [3H]-xanthine or [3H]-uric acid, (c) Inhibition of [3H]-xanthine (0.5 µM) or [3H]-uric acid (0.5 µM) uptake by increasing concentrations of unlabelled xanthine or uric acid, respectively, (d)% inhibition of [3H]-xanthine (0.5 µM) uptake by increasing concentrations of unlabelled uric acid, and% inhibition of [3H]-uric acid (0.5 µM) uptake by increasing concentrations of unlabelled xanthine, (e) Energetic dependence of xanthine uptake. For details see Materials and methods. Results represent mean values from three independent determinations with standard deviations shown.
![Figure 1. Physiological and biochemical identification of Xut1, a C. albicans xanthine-uric acid transporter. (a) Growth of C. albicans on purines as sole N sources, (b) Time course of uptake of 0.5 µM [3H]-xanthine or [3H]-uric acid, (c) Inhibition of [3H]-xanthine (0.5 µM) or [3H]-uric acid (0.5 µM) uptake by increasing concentrations of unlabelled xanthine or uric acid, respectively, (d)% inhibition of [3H]-xanthine (0.5 µM) uptake by increasing concentrations of unlabelled uric acid, and% inhibition of [3H]-uric acid (0.5 µM) uptake by increasing concentrations of unlabelled xanthine, (e) Energetic dependence of xanthine uptake. For details see Materials and methods. Results represent mean values from three independent determinations with standard deviations shown.](/cms/asset/11a5a299-d0ef-47f2-8d62-4870fc356a4a/imbc_a_109284_uf0001_b.jpg)
Figure 2. Regulation of expression of Xut1. (a) Regulation of xut1 mRNA steady-state levels in response to the N source, purine availability, pH or T. The nitrogen sources for each lane shown were: Ur, urea; NH4, ammonium chloride; Ua, uric acid; Ad, adenine; -Nst, no nitrogen source; pH:8, pH:4 and 30°C were also in urea. Approximately equal RNA loading, except from the lane corresponding to uric acid (Ua) which was 50% of the rest, was controlled by estimating the amount of rRNA as previously described (Sambrook et al. [Citation1989]; not shown), (b) Xut1 transport activity in response to the N source, purine availability, or pH, (c) Xut1 transport activity at different developmental stages. For details see Materials and methods.
![Figure 2. Regulation of expression of Xut1. (a) Regulation of xut1 mRNA steady-state levels in response to the N source, purine availability, pH or T. The nitrogen sources for each lane shown were: Ur, urea; NH4, ammonium chloride; Ua, uric acid; Ad, adenine; -Nst, no nitrogen source; pH:8, pH:4 and 30°C were also in urea. Approximately equal RNA loading, except from the lane corresponding to uric acid (Ua) which was 50% of the rest, was controlled by estimating the amount of rRNA as previously described (Sambrook et al. [Citation1989]; not shown), (b) Xut1 transport activity in response to the N source, purine availability, or pH, (c) Xut1 transport activity at different developmental stages. For details see Materials and methods.](/cms/asset/7ecba07a-7195-4db4-879d-504a63968364/imbc_a_109284_uf0002_b.jpg)
Table I. Ki (µM) values and ΔG0 binding energies (kJ/mol) of UapA, Xut1 and YgfO. n.d., not determined, see text. Significant differences are highlighted in bold. All nucleobase analogues, among those listed in Materials and methods, not present in the table, were shown not to compete UapA-, Xut1- or YgfO-mediated xanthine uptake at concentrations as high as 2 mM.
Figure 3. Speculative models for the possible interactions of UapA, Xut1 and Ygfo with their substrates discussed in the text. ΔG0obs is the ΔG0 calculated directly from the Ki of the relevant substrate. ∑(ΔG0) is the sum of the differences in ΔG0 values obtained from binary comparisons of the relevant substrate and selected analogues. For oxypurinol only ΔG0obs values are shown. In that case, the contribution of different positions is based on xanthine binding (for the pyrimidine ring) and the difference between the ΔG0obs and the contribution of the pyrimidine part (for the imidazol ring). Estimated apparent ΔG0 values are in kJ/mol. R stands for an amino acid in the transporter interacting, most probably via H-bonds, with a specific purine position. Shaded areas depict positions of interactions with R. Preferred tautomers under conditions used in this work are shown (Hernandez et al. [Citation1996a], [Citation1996b]; Stoychev et al. [Citation2002], Kulikowska et al. [Citation2004]).
![Figure 3. Speculative models for the possible interactions of UapA, Xut1 and Ygfo with their substrates discussed in the text. ΔG0obs is the ΔG0 calculated directly from the Ki of the relevant substrate. ∑(ΔG0) is the sum of the differences in ΔG0 values obtained from binary comparisons of the relevant substrate and selected analogues. For oxypurinol only ΔG0obs values are shown. In that case, the contribution of different positions is based on xanthine binding (for the pyrimidine ring) and the difference between the ΔG0obs and the contribution of the pyrimidine part (for the imidazol ring). Estimated apparent ΔG0 values are in kJ/mol. R stands for an amino acid in the transporter interacting, most probably via H-bonds, with a specific purine position. Shaded areas depict positions of interactions with R. Preferred tautomers under conditions used in this work are shown (Hernandez et al. [Citation1996a], [Citation1996b]; Stoychev et al. [Citation2002], Kulikowska et al. [Citation2004]).](/cms/asset/2cb341ba-6b12-44e0-90f7-81a53d7eb44c/imbc_a_109284_uf0003_b.jpg)