Continuous assay measures methyltransferase activity: Defining the substrate specificity of rat Protein Arginine Methyltransferase 1 (PRMT1)

Since the discovery of protein arginine methyltransferases (PRMTs), the substrate specificity for all PRMT isoforms remains ill-defined due to the absence of a convenient activity assay. Research has shown that PRMT1 prefers to methylate arginine residues located in glycine/arginine rich (GAR) areas of a protein or RXR motifs. However, PRMT1 methylates Arg 3 of histone H4 in vivo, which does not contain a GAR or RXR motif. Thus far, PRMT1 activity has been assayed using labeled S-adenosyl methionine. We have developed a continuous spectrophotometric assay to measure methyltransferase activity that eliminates the cost and time of the radioactive method. The continuous assay utilizes two coupling enzymes to break down S-adenosylhomocysteine (SAH), a product and potent inhibitor of methyltransferases. SAH is converted into adenine and S-ribosyl homocysteine by MTA nucleosidase. Adenine is converted into hypoxanthine by adenine deaminase which is associated with a decrease in the absorbance at 265 nm. Kinetic parameters have been obtained for PRMT1 with peptide substrates derived from two in vivo substrates, fibrillarin and H4. The objective of this research is to characterize PRMT1 substrate specificity. A detailed knowledge of PRMT1 substrate specificity will help in the design of specific inhibitors, the creation of isoform specific antibodies, and the prediction of protein substrates. Funded by USU New Faculty Funds