Adp luciferase assay

The T. brucei gene encoding IPMK was cloned into pET-29a (Novagen) and expressed in Escherichia coli DE3 pLysS Rosetta cells. Recombinant protein was purified using nickel-resin (Cestari and Stuart, 2015 (link)). Protein activity was assayed using the ADP luciferase assay (Promega). IPs and diC8 PIs were purchased from Echelon Biosciences. Assays were performed in 25 µl reactions in 96 well-plates using 20 mM HEPES, 150 mM NaCl, 2 mM MgCl₂ at pH 7.5 at 37°C and 270 nM rTbIPMK. Substrate an alysis was performed for 60 min. with 50µM of each substrate, and dose-response assays were performed with 1–200 µM of Ins(1,4,5)P3 or Ins(1,2,4,5)P4 for 30 min. For inhibition assays all compounds were diluted in water and reaction mixes incubated for 30 min. Ins(1,4,5)P3, Ins(1,3,4,5)P4 and ATP were used at 30, 40 and 50 µM, respectively. For K_i analysis and mechanisms of enzyme inhibition, reactions were prepared with 10, 30 and 60 µM of Ins(1,4,5)P3 and C44 (0, 1, 5, 10 and 25 µM) or C52 (2, 10, 20 and 40 µM). Kinetic analyses were calculated by non-linear regression using GraphPad Prism for Windows 5.03 (GraphPad Software, Inc). Compound analogs were identified using an atom pair method and Tanimoto coefficients using ChemmineR (Cao, et al., 2008 (link)).