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A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity
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Lloyd, Adrian J., Thomann, H. U., Ibba, M. and Söll, D. (1995) A broadly applicable continuous spectrophotometric assay for measuring aminoacyl-tRNA synthetase activity. Nucleic Acids Research, 23 (15). pp. 2886-2892. doi:10.1093/nar/23.15.2886 ISSN 0305-1048.
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Official URL: https://doi.org/10.1093/nar/23.15.2886
Abstract
We describe a convenient, simple and novel continuous spectrophotometric method for the determination of aminoacyl-tRNA synthetase activity. The assay relies upon the measurement of inorganic pyrophosphate generated in the first step of the aminoacylation of a tRNA. Pyrophosphate release is coupled to inorganic pyrophosphatase, to generate phosphate, which in turn is used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methylpurine ribonucleoside. Of the reaction products, ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360 nm relative to the nucleoside and hence provides a spectrophotometric signal that can be continuously followed. The non-destructive nature of the spectrophotometric assay allowed the re-use of the tRNAs in question in successive experiments. The usefulness of this method was demonstrated for glutaminyl-tRNA synthetase (GlnRS) and tryptophanyl-tRNA synthetase. Initial velocities measured using this assay correlate closely with those assayed by quantitation of [3H]Gln-tRNA or [14C]Trp-tRNA formation respectively. In both cases amino acid transfer from the aminoacyl adenylate to the tRNA represents the rate determining step. In addition, aminoacyl adenylate formation by aspartyl-tRNA synthetase was followed and provided a more sensitive means of active site titration than existing techniques. Finally, this novel method was used to provide direct evidence for the cooperativity of tRNA and ATP binding to GlnRS.
| Item Type: | Journal Article | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||||
| Journal or Publication Title: | Nucleic Acids Research | ||||||||
| Publisher: | Oxford University Press | ||||||||
| ISSN: | 0305-1048 | ||||||||
| Official Date: | 11 August 1995 | ||||||||
| Dates: |
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| Volume: | 23 | ||||||||
| Number: | 15 | ||||||||
| Page Range: | pp. 2886-2892 | ||||||||
| DOI: | 10.1093/nar/23.15.2886 | ||||||||
| Status: | Peer Reviewed | ||||||||
| Publication Status: | Published | ||||||||
| Access rights to Published version: | Open Access (Creative Commons) |
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