DiFMUP phosphatase assay

The phosphatase activities of Rtr1 and RPAP2, as well as their mutation variants, were characterized by detecting the hydrolysis of DiFMUP. DiFMUP generates a fluorescent signal once the associated phosphate is removed. Florescent DiFMU, the hydrolysis product of DiFMUP, was monitored at excitation/emission wavelengths of 358/455 nm using a TECAN plate reader (Infinite 200). A standard curve was generated using various concentrations of DiFMU. Both DiFMU and DiFMUP were dissolved in 100% DMSO to make 10 mM stock. For each experiment, fresh substrate was prepared by diluting compounds in buffer containing 20 mM tris-acetate (pH 6.0), 150 mM NaCl, and BSA (0.1 mg/ml) to desired concentrations. Background activity in the presence of BSA alone was subtracted from all values. Assays were carried out in 384-well amber plates in a 20-μl reaction volume. Michaelis-Menten kinetic parameters were determined by measuring initial reaction rates at various DiFMUP concentrations in assay buffer with 7 μM Rtr1 protein. Initial rates were calculated using the Magellan 6 software (TECAN). The datawere fit to the Michaelis-Menten equation using nonlinear curve fit in KaleidaGraph (Synergy), and k_cat and K_m were determined.