Measurement of OGT and OGA Activity OGT and OGA are the key enzymes for the addition and removal of O-GlcNAc modifications; thus, their levels and activity are important for sensing nutrient availability and metabolic, proteotoxic, and oxidative stress. These enzymes then modulate O-GlcNAcylation of target proteins to control the activities of proteins in response to changing nutrients, changing metabolites, and accumulations of toxic proteins and oxidants. Methods to assess the levels and localization of OGT and OGA proteins and other posttranslational modifications (e.g., phosphorylation) are now standard using western blots and immunocytochemistry and, thus, not includ in this review.
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Instead, we focus on assessing OGT and OGA activities in cells and tissues (Table 1). Sources for key chemicals are also indicat where appropriate. Table 1 frontiersin TABLE 1. OGT and OGA activity assays. OGT enzymatic activity is st measur using standard biochemical assays. These assays quantify products in a time-dependent manner Germany Email Database using appropriate substrates. The traditional method uses radioisotope-lal UDP-[3H]GlcNAc and a known OGT target peptide CKII aa340-352. UDP-[3H]GlcNAc incorporat into the CKII peptide can measur, and OGT activity can calculat. Negative controls containing a mimic of the CKII peptide with Ser/Thr replac with Ala and a negative control without a lysate ne to includ.
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Nonradioactive OGT assays have also en develop using either a chemosensor or a ligand displacement method (Kim, 2011). The chemosensor method is bas on the higher affinity of the sensor for binding UDP vs. UDP-GlcNAc; thus, a chemosensor-detect increase in UDP represents a measure of OGT activity. The ligand displacement method is bas on fluorescent UDP-GlcNAc analogs, which have stronger fluorescence signals when binding to OGT. Neither of the nonradioactive methods matches the specificity and applicability of the radioactive assay (Kim, 2011). In vitro activity assays using recombinant OGT (Alteen et al., 2020) have en TH Lists develop for screening OGT inhibitors, and these studies are not the focus of this review.