异亮氨酸tRNA合成酶(IleRS),亮氨酰-tRNA合成酶(LeuRS),和缬氨酸-tRNA合成酶(ValRS)是具有测定l-异亮氨酸潜力的酶,l-亮氨酸,食品和血浆中的L-缬氨酸。然而,这些酶的缺点是它们的特异性和敏感性。这里,我们检查了IleRS的底物特异性,LeuRS,和ValRS在各种条件下进行焦磷酸扩增,以提高其特异性和灵敏度。IleRS中产生的焦磷酸盐的量,LeuRS,加入过量的腺苷-5'-三磷酸和镁离子后,ValRS反应被放大,大约是9-,8-,高出7倍,分别,对于每个初始1-氨基酸底物(50μM)。然而,除了它们的目标氨基酸,IleRS,LeuRS,ValRS也与L-缬氨酸反应,l-赖氨酸,和l-苏氨酸,分别。通过使反应pH更酸性和通过增加镁离子浓度来克服这种底物误识别。IleRS中的焦磷酸扩增,LeuRS,和ValRS反应导致p1,p4-二(腺苷)5'-四磷酸的产生。我们还观察到产生的焦磷酸盐的量与具有5和50μMl-异亮氨酸的l-氨基酸的初始浓度之间存在很强的正相关性(R=0.99)。l-亮氨酸,还有L-缬氨酸.我们的结果表明,使用IleRS的氨基酸测定,LeuRS,和ValRS是准确测量L-缬氨酸的有前途的方法,l-异亮氨酸,食品和血浆中的l-亮氨酸。
Isoleucyl-tRNA synthetase (IleRS), leucyl-tRNA synthetase (LeuRS), and valyl-tRNA synthetase (ValRS) are enzymes that have potential for the determination of l-isoleucine, l-leucine, and l-valine in food products and plasma. However, the disadvantages of these enzymes are their specificity and sensitivity. Here, we examined the substrate specificity of IleRS, LeuRS, and ValRS under various conditions of pyrophosphate amplification to improve their specificity and sensitivity. The amount of pyrophosphate produced in IleRS, LeuRS, and ValRS reactions was amplified after the addition of excess adenosine-5\'-triphosphate and magnesium ions, and was approximately 9-, 8-, and 7-fold higher, respectively, for each of the initial l-amino acid substrates (50 μM). However, in addition to their target amino acids, IleRS, LeuRS, and ValRS also reacted with l-valine, l-lysine, and l-threonine, respectively. This substrate misrecognition was overcome by making the reaction pH more acidic and by increasing the magnesium ion concentration. The pyrophosphate amplification in IleRS, LeuRS, and ValRS reactions resulted in the production of p1, p4-di (adenosine) 5\'-tetraphosphate. We also observed a strong positive correlation (R = 0.99) between the amount of pyrophosphate produced and the initial concentration of l-amino acid with 5 and 50 μM l-isoleucine, l-leucine, and l-valine. Our results suggest that amino acid assays using IleRS, LeuRS, and ValRS are promising methods to accurately measure l-valine, l-isoleucine, and l-leucine in food products and plasma.