Protein arginine N-methyltransferase activity determination with filter binding and phosphor screening (FBAPS) assay

Abstract

We developed a cost-effective assay to measure protein arginine N-methyltransferase (PRMT) activity in a medium-throughput manner by combining P81 filter binding and phosphor screening (FBAPS). Recombinantly-expressed PRMT1 and coactivator-associated arginine methyltransferase 1 (CARM1) were used to develop the FBAPS assay using GST fusions of glycine- and arginine-rich (GAR) protein and polyA binding protein 1 (PABP1(437-488)) as substrates, respectively, and radiolabelled S-adenosyl-L-[methyl-14C]-methionine as cofactor. Methylation reactions were spotted onto P81 filter paper in a dot blot apparatus and radioactive signals were measured both by phosphor imaging and liquid scintillation counting. Kinetic parameters (KM, kcat) for enzymes and substrates were determined, and IC50 values were obtained for well-characterized inhibitors. FBAPS yielded kinetic parameters with no statistically significant difference to what was obtained using liquid scintillation counting. The IC50 values obtained by the FBAPS assay for PRMT1 and CARM1 were comparable to values reported in literature. The FBAPS assay is a modification to the P81 filter binding assay with a dot blot apparatus that allows for processing of samples in a multi-well format, moderately increasing throughput. Signal detection by phosphor imaging offers an affordable and quantitative method that can be used to screen several inhibitors simultaneously against PRMT enzymes with high accuracy.