With Ga as being a model PMT, the authors demonstrated that the strategy is extremely quantitative and suitable for characterizing the kinetics of PMT catalyzed reactions. PRMTs generate three kinds of arginine methylation products . To distinguish the three varieties of products, SAM labeled substrate samples may be subjected to acid hydrolysis to yield MMA, ADMA and SDMA amino acids, which could be even further characterized by column thin layer chromatography or MS evaluation. With all the acid hydrolysis technique, Branscombe et. al. and Lee et. al. had been capable to detect the SDMA goods of PRMT and PRMT, and categorized the 2 enzymes as Kind II PRMTs Using the exact same approach, the Frankel laboratory was able to experimentally define PRMT as being a Kind I PRMT. The Wang laboratory even more demonstrated a MALDITOF MS MS method to differentiate MMA, ADMA and SDMA on the peptidic degree. The MMA , ADMA and SDMA containing peptides showed characteristic neutral losses of , and , respectively.
Direct Quantification of SAH with MS or ANTI SAH antibody MS and antibody based mostly approaches have also been put to use to measure the byproduct SAH in PMT catalyzed reactions . The Frankel lab reported a tandem SB 415286 MS MS method to quantify SAH. With this assay, they have been capable of quantify the sources resulting in SAH background in PRMT catalyzed reactions and concluded that, in addition to the SAH through the contamination in industrial SAM and from SAM?s nonenzymatic decomposition , automethylation of PRMT accounts for any portion on the observed SAH background. The byproduct SAH in PMT catalyzed reactions may also be quantified by antibody based mostly competitive assays . Capdevila et. al. primary reported a aggressive immunoassay employing SAH BSA conjugate and anti SAH antibody to quantify SAH in plasma.
Within this assay, SAH competes with microplate coated SAH BSA to bind anti SAH antibody and hence decreases Hematoxylin ELISA signal from the microplate immobilized antibody. Graves et. al. developed a comparable aggressive assay with fluorescein SAH and anti SAH antibody. In Graves?s method, SAH is quantified by competing fluorescein SAH to bind the antibody and thus result in the loss of fluorescence polarization signal. The assay has demonstrated its feasibility for catechol Omethyltransferase and is most likely applicable to PMTs, provided their shared byproduct SAH. Nonetheless, one particular need to be cautious to utilize the SAH primarily based fluorescence polarization as the readout is linear only within a narrow variety of SAH concentration . PMT action assays as a result of SAH derivatives Quite a few SAH primarily based quantification assays were formulated for modest molecule methyltransferases similar to salicylic acid methyltransferase and catechol Omethyltransferase.
The Zhou laboratory reported an enzyme coupled chromogenic assay for salicylic acid methyltransferase. This assay relied on two coupling enzymes MTAN and LuxS to convert SAH into homocysteine . Homocysteine can then be quantified with Ellman?s reagent . The Hrycyna laboratory reported a comparable fluorogenic assay for catechol Omethyltransferase .