A Metazoan/Plant-like Capping Enzyme and Cap Modified Nucleotides in the Unicellular Eukaryote Trichomonas vaginalis
Figure 2
Triphosphatase activity of TvCE.
A. Activity at various pHs. B. Effect of MgCl2 and EDTA on activity. Products were resolved on TLC plates and detected by autoradiography. Arrows denote the origin of loading, substrate ([γ-32P]ATP) and product (32Pi). C. Detection of the TvCE-cysteinyl-S-phosphate formation. Labeling with [γ-32P]ATP (left panel) and [α-32P]ATP (right panel) compared from pH 3.0–5.0. 32P-labeled TvCE detected by SDS-PAGE and autoradiography and TvCE protein loading controls flanked by pre-stained molecular weight markers (PageRuler prestained protein ladder, Fermentas) are shown in top and bottom panels, respectively. D. Effect of iodine and NH2OH on thiophosphate linkage formation (left panels) and TvCE-cysteinyl-S-phosphate formation of TvCE wild-type (WT) and mutant (H125S; C126S; R526A; C126S & R526A) proteins (right panels). 32P-labeled TvCE detected by SDS-PAGE and autoradiography (top panels) and TvCE protein loading controls (bottom panels) are shown. The purified protein is observed as a single band of the predicted ∼71 kDa molecular mass in both C and D.