An optimised assay for quantitative, high-throughput analysis of polysialyltransferase activity

Abstract

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Exploring and Exploiting Acceptor Preferences of the Human Polysialyltransferases as a Basis for an Inhibitor Screen

“…We conclude that CMP‐Neu5Cyclo binds to the donor binding site of ST8SiaII, thus directly competing with CMP‐Sia. The level of inhibition is comparable to that observed for CMP, thus indicating that Neu5Cyclo does not make a strong contribution to binding affinity. However, as this substrate is unable to be transferred to polySia, it serves as a useful lead compound in the search for new anticancer therapeutics.…”

“…For investigating the preference for specific acceptor structures, it was essential to measure only the first transfer onto the acceptor, as each transfer generated a new acceptor species with potentially different acceptor properties. We applied optimised reaction conditions for predominantly single transfers, thus obtaining DMB‐DP4 and DMB‐DP13 as the major products . Preliminary tests using equal amounts of the two polySTs showed that the specific activity of ST8SiaIV was considerably lower than that of ST8SiaII.…”

An efficient assay for identification and quantitative evaluation of potential polysialyltransferase inhibitors

“…DMB-DP3 synthesis was conducted as described previously. 20 Briefly, DP3 (10 mg ml −1 ) was dissolved in a labelling solution containing DMB (20 mM), sodium hydrosulfite (40 mM) and β-mercaptoethanol (1 M). The mixture was then added to an equal volume of ice-cold trifluoroacetic acid (40 mM), followed by incubation at 4°C overnight.…”

“…1). 20 The fluorescent acceptor and product in the enzyme reaction are separated by reversed-phase chromatography over a short timeframe (10 min), while being simultaneously visualised by fluorescence detection (Fig. 1, inset), allowing rapid analysis.…”

“…competitive or non-competitive). 20,21 Herein we report a cell-free polyST assay that meets these demands. We have systematically investigated the assay parameters that are predicated to influence the underlying enzyme catalysis with a view to providing a sound basis on which to reliably determine mode of inhibition, IC 50 and K i values for putative inhibitors, the latter through both prediction and experiment.…”