A high-throughput competitive scintillation proximity aminoacyl-tRNA synthetase charging assay to measure amino acid concentration.
An enhanced method to measure the concentration of individual naturally occurring free amino acids in solution is described. This relatively simple but robust method combines two previously reported procedures: the use of scintillation proximity assay (SPA) technology to measure aminoacyl-tRNA synthetase (aaRS) activity and the use of aaRS activity to measure amino acid concentration using the enzymatic isotope dilution technique. The format described is called an aaRS competitive scintillation proximity assay (cSPA). This cSPA takes advantage of competition between a fixed concentration of radiolabeled amino acid and an unknown concentration of the same nonradiolabeled amino acid for its cognate tRNA catalyzed by the aaRS specific for that amino acid. Under equilibrium conditions, in the case of limiting tRNA, the rate of the enzyme-catalyzed reaction relative to substrate concentration becomes irrelevant and the enzymatic isotopic dilution technique becomes the simple isotopic dilution technique. Due to the exquisite specificity of the reaction, a crude mixture of tRNAs and aaRSs can be used to detect the concentration of a particular amino acid without interference from noncognate amino acids. When used to monitor aminopeptidase M activity, this assay produced similar results in time course and inhibition experiments as compared with a traditional fluorescent assay. High-throughput compatibility was demonstrated by screening 12,000 compounds against aminopeptidase M in 384-well microtiter plates with Z factors ranging from 0.53 to 0.70. This competitive assay can be used as a general method to detect amino acids at concentrations less than 100 nM and to monitor enzyme activity in biological samples, and it is amenable to high-throughput screening.
Forbes
Chris D
CD
Bayer Pharmaceuticals, West Haven, CT 06516, USA.
Myung
Jayhyuk
J
Szewczak
Alexander A
AA
Landro
James A
JA
D000596:Amino Acids / Q000032:analysis* / Q000378:metabolism; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000378:metabolism*; D000626:Aminopeptidases / Q000378:metabolism; D007201:Indicator Dilution Techniques; D063208:Methionyl Aminopeptidases; D012343:RNA, Transfer / Q000378:metabolism; D015203:Reproducibility of Results; D012588:Scintillation Counting / Q000379:methods
D000596:Amino Acids; D012343:RNA, Transfer; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; D000604:Amino Acyl-tRNA Synthetases