Development of a Fluorescent Quenching Based High Throughput Assay to Screen for Calcineurin Inhibitors
Fig 1
Characterization of enzyme activity.
(a) In order to test the effect of pH on CaN activity, CaN assay was performed at different pH. Our data suggested that pH 7 is optimum for CaN activity. To study the effect of bivalent metal ions on CaN activity, enzyme activity assays were performed at different MnCl2 (b) and MgCl2 (c) concentrations. The data indicated that bivalent manganese increased CaN activity almost two times (b) whereas magnesium had not effect (c). Note that (a), (b) and (c) were performed in absence of CM. (d) The concentration of CM which produces 50% activation (IC50 CM) of CaN was determined by plotting initial reaction velocities at different Ca2+ and CM concentrations. Our data indicated that CM produces half maximum CaN activity at 1:1 molecular ratio. (e) Enzyme Km was determined by plotting initial velocity at different substrate (RIIP) concentrations. The Km was 213 μM. (f) DMOS tolerance of the enzyme was assayed by performing the assay at different DMSO concentration ranging from, 0.125 to 4%. Our data indicate that at ≤0.5% DMSO did not alter enzyme activity. The assays were done in duplicate (except for experiment in Fig 1d) and the data were expressed as means and standard error. The fittings were done using Prism software.