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Figure 2

From: Inosine triphosphate pyrophosphatase from Trypanosoma brucei cleanses cytosolic pools from deaminated nucleotides

Figure 2

TbITPA exhibits NTP pyrophosphatase activity over non-canonical NTP substrates. (A) Purified protein was separated on SDS-PAGE gel and stained with Coomassie. M, molecular weight markers; F6-F9, fractions containing purified TbITPA were obtained after affinity and anion exchange chromatography. (B) A sample of purified TbITPA protein was applied to a Superdex 200 10/300 GL column (GE Healthcare) in 50 mM phosphate, 0.15 M NaCl, pH 7.2 and eluted at 0.5 mL/min in the same buffer. The molecular weight (MW) determination is extrapolated by comparing the Ve/Vo ratio for TbITPA to the Ve/Vo of protein standards of known molecular weight, Ve = Elution volume of the protein; Vo = Void volume of the column. (C) Substrate saturation curves obtained for ITP, (D) dITP and (E) XTP. Each data point is the mean (± SD) of at least three independent determinations. Reactions were performed and analyzed as described in Materials and Methods. The data were fitted to a non-linear kinetic model for substrate inhibition and analyzed with GraphPad Prism 5 software.

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