Methylation and demethylation of cellular proteins and DNA/RNA plays a significant role in the regulation of a variety of signal transduction pathways including protein translation and complex energy transfer systems
Because the enzymes responsible for methylation and demethylation reactions are deeply intertwined with the systems they regulate, these enzymes can be especially tricky to study and understand. Many types of enzymatic assays rely on the target enzyme acting upon a modified assay substrate. This substrate can be a labeled peptide or protein fragment, or an extremely specific native or recombinant protein where the presence or absence of methyl groups at a specific site of interest can be detected via antibody binding. Both these options have their weaknesses.
In assays where modification is detected by a specific antibody, the enzymes of interest are acting upon either a native substrate, or a very similar recombinant protein. Even though the substrates are either immobilized or tagged for later pull-down, this type of assay allows for kinetics to be closer to what might be found in vivo. Substrate affinities and potential inhibitor or activator leads identified in this type of assay have a higher chance of being confirmed with later studies in vivo. Arbor Assays offers several antibodies targeted to a specific lysine methylation site on histone H3 appropriate for this type of work (A004, A005, A006, A007). A downside to this type of assay system is the potential isolation of a single modification site, which may over simplify some complex regulatory systems where potential modifications at many sites on numerous different proteins may work in concert to regulate the system. Assays of this type are also somewhat tricky develop and often must be customized to an individual’s research.
Small labeled peptide substrates are less expensive to produce than recombinant protein and modification specific antibody pairings, can often be used for more than one enzyme, and are more frequently commercially available. Unfortunately, they are also highly artificial. Enzyme kinetics with these substrates (often just a few amino acids in length) have little resemblance to real world kinetics with endogenous substrates and the behavior of potential activators or inhibitors is also frequently quite different. These assays may have utility in early high-throughput screening phases, but ultimately the information on how the target enzyme behaves in vivo is likely to be limited.
Fortunately, in some cases there is a third option. For some enzymes it is possible to detect a different non-protein product of the enzymatic reaction. For example, many demethylases produce formaldehyde as a product of the demethylase reaction, and formaldehyde is detectable by chemical means. Both Arbor Assays P450 Demethylation Fluorescent Activity Kit (K011-F1) and the Histone Demethylase Activity Kit (K010-F1) use this method to detect the formaldehyde byproduct of the demethylase reaction. A major advantage to this method is that the detection reaction takes place after the demethylase reaction has already happened, so any source of the demethylase enzyme and any appropriate substrate can be monitored. Also there are no “artificial” components such as antibodies or tagged proteins present during the enzymatic reaction to impact kinetics. This is especially important in cases like the P450s where there is a complicated energy transfer chain that can involve several enzymes with potential activation or inhibition anywhere in the chain. As long as formaldehyde is ultimately produced as a product of a demethylation reaction the reaction can be monitored, making these kits highly versatile tools in a variety of laboratory applications.
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