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  • br Results and discussion The classical

    2024-07-10


    Results and discussion The classical radiochemical adenosine kinase assays developed to monitor AdK activity utilize [3H]adenosine or [14C]adenosine, respectively. The assays are terminated by spotting the incubation mixture onto diethylaminoethyl (DEAE) anion exchange filter disks or onto a polyethyleneimine-cellulose thin layer chromatography (TLC) plates.37, 38 Although AdK assays were developed using TLC and automatic plate scanning machines in order to increase the throughput, the time consuming handling requiring each sample to be processed separately before spotting it on polygram plates limits its usefulness for high-throughput screening. More recently a capillary electrophoresis (CE) assay was developed. Although a CE method was established in which the enzymatic reaction could be performed directly in the capillary leading to increased throughput, capillary electrophoresis has some drawbacks such as limited sensitivity. In addition it is not readily suitable for enzyme assays on intact cells. As radioactive assays are both extremely sensitive and specific, we decided to establish an improved, HTS-compatible radioactive enzyme assay using [3H]adenosine in analogy to a described method. The assay was miniaturized to a 96-well format and validated using standard AdK inhibitors. Human AdK was cloned and recombinantly expressed in an E. coli strain BL21[DE3] as described in the experimental section. After transformation of the E. coli host ghrelin receptor antagonist BL21[DE3] with the pASKhADK construct, overnight cultures were grown and protein expression was induced by addition of tetracycline (0.2μg/mL). To examine the optimal expression conditions, various induction times, namely 3, 4, 5, 6 h at 30°C or 16 h at 25°C, respectively were tested. AdK expression levels were monitored using SDS–PAGE. AdK could be detected as a 40kDa band, which is in agreement with other reports.41, 42 An increase in AdK protein was visible as early as 3h after induction. Comparison of the 3-h induction with the overnight induction revealed no difference in protein expression. Therefore the short 3-h induction was used to express AdK in a larger scale. The protein concentration determined by the method of Lowry using BSA as standard resulted in a protein concentration of 0.934mg/mL. AdK activity was assayed in a reaction mixture (final volume of 100μL) consisting of Tris–HCl, ATP, MgCl2, K2HPO4, [3H]adenosine (1μM) and adenosine kinase. We selected 50μg of protein per well with an incubation time of 15min. This represented the minimum amount of enzyme to produce a linear enzymatic response for the complete duration of the assay that also showed a suitable signal window (1:4) at the selected time point. The assays were conducted under initial velocity conditions where less than 10% of the substrate was converted. The formation of [3H]AMP increased linearly over time and with increasing enzyme concentration until saturation occurred at higher enzyme levels due to substrate depletion. The formed [3H]AMP was precipitated by adding a cold aqueous LaCl3 solution, the precipitate was collected by filtration through 96-well GF/B glass fiber filter plates, and the radioactivity was measured by liquid scintillation counting (see ‘Section 2’). This assay has the advantage of using 96-well plates. All steps, except for the initial pipetting of the test compound, can be performed with multichannel pipettes leading to a significant reduction in the number of pipetting steps thereby simplifying and accelerating the procedure. Another advantage is the requirement of only a single transfer step from the 96-well reaction plate to the 96-well filtration plate. The liquid scintillation counting can be conducted directly in the filtration plate after adding the scintillation cocktail. Moreover, the use of a 96-channel cell harvester allows a very fast separation (<30s). Due to the separate hot and cold waste lines of the harvester the radioactive waste can be kept very low. Altogether, the assay is simple and fast due to only few pipetting steps, and economic due to low sample consumption. After initial assay development, we evaluated the standard AdK inhibitor 5-iodotubercidin by using a range of concentrations from 0.01nM to 10,000nM. Eight separate experiments, each in triplicates, were performed, and Ki values were determined. 5 iodotubercidin potently inhibited the activity of the soluble enzyme (raw extract) in a concentration-dependent manner with a Ki value of 16.3±5.2nM (Fig. 1) which is in excellent agreement with published data (17nM, 26nM, 9.3nM).