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- Title
Pyrophosphate amplification reaction for measuring amino acid concentrations with high sensitivity using aminoacyl-tRNA synthetase from Escherichia coli.
- Authors
Nakatsuka, Tomoko; Aoki, Hideyuki; Kida, Mikiko; Kugimiya, Akimitsu
- Abstract
To measure amino acid concentrations with high sensitivity, the pyrophosphate amplification reaction conditions of histidyl-tRNA synthetase (HisRS) and tyrosyl-tRNA synthetase (TyrRS) were examined. The amount of pyrophosphate produced by reactions involving HisRS and TyrRS was amplified compared with the amount of the initial substrate L-amino acid after the addition of excess adenosine-5′-triphosphate and magnesium ions, with incubation at 50°C in an alkaline pH. The amount of pyrophosphate produced in the HisRS and TyrRS reactions was approximately 24- and 16-fold higher than the initial amount of L-His and L-Tyr, respectively. The pyrophosphate amplification reactions involving HisRS and TyrRS showed high substrate specificity for L-His and L-Tyr, respectively. Products of pyrophosphate amplification were identified as p1, p4-di(adenosine) 5′-tetraphosphate, and adenosine-5′-monophosphate using high-performance liquid chromatography. A strong positive correlation was observed for 0 to 50 μM of L-His and L-Tyr in the pyrophosphate amplification reaction (R = 0.98 and R = 1.00, respectively). Abbreviations: L-His: L-histidine; L-Tyr: L-tyrosine; aaRSs: aminoacyl-tRNA synthetases; ATP: adenosine-5′-triphosphate; aminoacyl-AMP-aaRS: aminoacyl-adenylate intermediate; Ap4A, P1, P4-di(adenosine) 5ʹ-tetraphosphate; AMP: adenosine-5′-monophosphate; PAR: pyrophosphate amplification rate Pyrophosphate amplification reaction for measuring amino acid concentrations with high sensitivity using aminoacyl-tRNA synthetase from Escherichia coli.
- Subjects
AMPLIFICATION reactions; AMINOACYL-tRNA; AMINO acids; ESCHERICHIA coli; AMINOACYL-tRNA synthetases; GLUTAMINE synthetase
- Publication
Bioscience, Biotechnology & Biochemistry, 2019, Vol 83, Issue 9, p1616
- ISSN
0916-8451
- Publication type
Article
- DOI
10.1080/09168451.2019.1608801