Phosphoryl transfer assays

Until now, we have carried out phosphoryl transfer assays with 24 carboranyl thymidine analogs using recombinant TK1 and TK2. Various carborane-containing substituents were attached either to the N-3- or the C-5 position of the nucleoside scaffold. Most of the tested N-3 substituted carboranyl structures proved to be good substrates for TK1 while they were not or very poorly phosphorylated by TK2. For example, the phosphorylation velocity observed for N-4 with TK1 was 28% that of thymidine. None of the tested C-5 substituted carboranyl thymidine analogs were phosphorylated by TK1 and... 

Syntheses and results of biological studies of carborane-containing nucleosides have been reviewed several times for the last 10 years [50-56], Based on the results of phosphoryl transfer assays of a few series of carborane-containing nucleosides, three-dimensional quantitative structure-activity relationship has been developed [57], Structures of some carborane-containing nucleosides synthesized are illustrated in Figure 9.6. 

This assay principle has been successfully developed also to monitor enzymatic reactions that involve various types of ester [9,29,30] and hydantoin hydrolysis [30a], phosphoryl transfer [31], nucleotidyl transfer [32] and glycosyltransfer [33, 34], as well as decarboxylation reactions [35]. The advantages of pH-based assay methods are obvious pH indicators are inexpensive reagents, no auxiliary enzymes are required, initial reaction rates can be monitored continuously in real time, and the reaction principle should be easily adaptable for HTS in microtiter plate format. 

Selected entries from Methods in Enzymology [vol, page(s)] Acetate assay with, 3, 269 activation, 44, 889 activity assay, 44, 893, 894 alternative substrates, 87, 11 bridge-to-nonbridge transfer, 87, 19-20, 226, 232 chiral phosphoryl-ATP, 87, 211, 258, 300 cold denaturation, 63, 9 cysteine residues, 44, 887-889 equilibrium constant, 63, 5 exchange properties, 64, 9, 39, 87,... 

The activity of protein kinase is readily assayed by measuring the rate of transfer of P from [y- P]ATP to a protein substrate, such as histone, casein, or protamine [77,80,82]. The phosphorylated substrate is collected on a filter, eluted, and counted. 

In order to enhance affinity and selectivity for Brc-Abl, we modified the inhibitor methylating at positions I and II (Fig. 7.5d). The synthesis of the wrapping prototype recapitulates imatinib synthesis [38], as described in [39], To test whether the specificity and affinity for Brc-Abl improved, we conducted a spectrophotometric kinetic assay to measure the phosphorylation rate of peptide substrates in the presence of the kinase inhibitor at different concentrations. This assay couples production of adenosine diphosphate (ADP), the byproduct of downstream phosphorylation, with the concurrent detectable oxidation of reduced nicotinamide adenosine dinucleotide (NADH). The oxidation results upon transfer of phosphate from PEP (phospho-enolpyruvate) to ADP followed by the NADH-mediated reduction of PEP to lactate. Thus, phosphorylation activity is monitored by the decrease in 340 nm absorbance due to the oxidative conversion NADH->-NAD+ [34, 39]. 

Figure 37-24 Common probes and dyes for real-time PCR. f/J Double-stranded DNA dyes show a significant increase in fluorescence when bound to DNA (hv = excitation light). (2) Adjacent hybridization probes. Fluorescence resonance energy transfer (FRET) is illustrated between a donor and acceptor fluorophore.The x indicates phosphorylation of the 3 terminus of the probe to prevent polymerase extension. (3) FRET between a labeled primer and a single hybridization probe. (4) Hydrolysis probes are cleaved between the reporter and quencher, resulting in increased fluorescence. (5) Hairpin probes are quenched in the native conformation, but increase in fluorescence when hybridized. (6) Hairpin primers retain their native, quenched conformation until they are incorporated into a double-stranded product (Modified with permission of the publisher from Pritham GH, Wittwer CT Continuous f/uorescent monitoring of PCR.J Clin Ug Assay 1998, 21 404-412. 1998 Clinical Ligand Assay Society, Inc.)...

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