Nakatsuka Tomoko, Aoki Hideyuki, Kida Mikiko, Kugimiya Akimitsu
a Research Laboratory, Ikeda Food Research Co., Ltd ., Fukuyama, Hiroshima , Japan.
b Department of Biomedical Information Sciences, Graduate School of Information Sciences, Hiroshima City University , Hiroshima , Japan.
Biosci Biotechnol Biochem. 2019 Sep;83(9):1616-1623. doi: 10.1080/09168451.2019.1608801. Epub 2019 Apr 27.
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 p, p-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). L-His: L-histidine; L-Tyr: L-tyrosine; aaRSs: aminoacyl-tRNA synthetases; ATP: adenosine-5'-triphosphate; aminoacyl-AMP-aaRS: aminoacyl-adenylate intermediate; ApA, P, P-di(adenosine) 5'-tetraphosphate; AMP: adenosine-5'-monophosphate; PAR: pyrophosphate amplification rate.
为了高灵敏度地测量氨基酸浓度，研究了组氨酰 - tRNA合成酶（HisRS）和酪氨酰 - tRNA合成酶（TyrRS）的焦磷酸扩增反应条件。在添加过量的腺苷 - 5'-三磷酸和镁离子后，于50°C碱性pH条件下孵育，与初始底物L - 氨基酸的量相比，涉及HisRS和TyrRS的反应产生的焦磷酸量被放大。HisRS和TyrRS反应中产生的焦磷酸量分别比L - His和L - Tyr的初始量高约24倍和16倍。涉及HisRS和TyrRS的焦磷酸扩增反应分别对L - His和L - Tyr表现出高底物特异性。使用高效液相色谱法将焦磷酸扩增产物鉴定为p，p - 二（腺苷）5'-四磷酸和腺苷 - 5'-单磷酸。在焦磷酸扩增反应中，对于0至50μM的L - His和L - Tyr观察到强正相关（R分别为0.98和1.00）。L - His：L - 组氨酸；L - Tyr：L - 酪氨酸；aaRSs：氨酰 - tRNA合成酶；ATP：腺苷 - 5'-三磷酸；氨酰 - AMP - aaRS：氨酰 - 腺苷酸中间体；ApA，P，P - 二（腺苷）5'-四磷酸；AMP：腺苷 - 5'-单磷酸；PAR：焦磷酸扩增率
