A homogeneous method to measure aminoacyl-tRNA synthetase aminoacylation activity using scintillation proximity assay technology.
A new method to measure the aminoacylation of tRNA based upon the use of the scintillation proximity assay (SPA) technology has been developed. The assay detects incorporation of radiolabeled amino acids into cognate tRNA, catalyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic conditions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregates, while the radiolabeled amino acid substrate remains in solution, resulting in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by measurement of steady-state kinetic constants and inhibitor binding constants for a range of aaRS enzymes in comparison with data from standard, trichloroacetic acid-precipitation-based assays. In all cases, the data were quantitatively comparable. Although the radioisotopic counting efficiency of the SPA method was less than that of standard liquid scintillation counting, the statistical performance (i.e., signal to background, variability, stability) of the SPA assays was at least equivalent to the separation-based methods. The assay was also shown to work well in miniaturized 384-well microtiter plate formats, resulting in considerable reagent savings. In summary, a new method to characterize aaRS activity is described that is faster and more amenable to high-throughput screening than traditional methods.
Macarrón
R
R
Department of Molecular Screening Technologies, SmithKline Beecham S. A. Centro de Investigación Básica, Tres Cantos, Spain. Ricardo_Macarron@SBPHRD.com
Mensah
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L
Cid
C
C
Carranza
C
C
Benson
N
N
Pope
A J
AJ
Díez
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D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D016712:Mupirocin / Q000494:pharmacology; D011485:Protein Binding; D012588:Scintillation Counting; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D016712:Mupirocin; D000604:Amino Acyl-tRNA Synthetases