Department of Chemistry, Chemistry of Life Processes Institute, and Center for Molecular Innovation and Drug Discovery, Northwestern University, Evanston, IL 60208, USA.
Anal Biochem. 2013 Sep 15;440(2):145-9. doi: 10.1016/j.ab.2013.05.025. Epub 2013 Jun 5.
We have developed two new continuous coupled assays for ornithine-δ-aminotransferase (OAT) that are more sensitive than previous methods, measure activity in real time, and can be carried out in multiwell plates for convenience and high throughput. The first assay is based on the reduction of Δ(1)-pyrroline-5-carboxylate (P5C), generated from ornithine by OAT, using human pyrroline 5-carboxylate reductase 1 (PYCR1), which results in the concomitant oxidation of NADH (nicotinamide adenine dinucleotide, reduced form) to NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). This procedure was found to be three times more sensitive than previous methods and is suitable for the study of small molecules as inhibitors or inactivators of OAT or as a method to determine OAT activity in unknown samples. The second method involves the detection of L-glutamate, produced during the regeneration of the cofactor pyridoxal 5'-phosphate (PLP) of OAT by an unamplified modification of the commercially available Amplex Red L-glutamate detection kit (Life Technologies). This assay is recommended for the determination of the substrate activity of small molecules against OAT; measuring the transformation of L-ornithine at high concentrations by this assay is complicated by the fact that it also acts as a substrate for the L-glutamate oxidase (GluOx) reporter enzyme.
我们开发了两种新的连续偶联测定法来测定鸟氨酸 δ-氨基转移酶（OAT），与之前的方法相比，这两种方法更加灵敏，可实时测量活性，并且可以在多孔板中进行，以便于进行高通量操作。第一种测定法基于 OAT 从鸟氨酸生成的 Δ(1)-吡咯啉-5-羧酸（P5C），利用人吡咯啉 5-羧酸还原酶 1（PYCR1）将其还原，同时将 NADH（烟酰胺腺嘌呤二核苷酸，还原形式）氧化为 NAD⁺（烟酰胺腺嘌呤二核苷酸，氧化形式）。该方法比之前的方法灵敏三倍，适用于研究小分子作为 OAT 的抑制剂或失活剂，或作为测定未知样品中 OAT 活性的方法。第二种方法涉及检测 L-谷氨酸，该物质是在 OAT 的辅酶吡哆醛 5′-磷酸（PLP）再生过程中产生的，该方法是对市售的 Amplex Red L-谷氨酸检测试剂盒（Life Technologies）进行未经放大的修饰而得到的。该测定法推荐用于测定小分子对 OAT 的底物活性；通过该测定法测量高浓度 L-鸟氨酸的转化时，由于它也作为 L-谷氨酸氧化酶（GluOx）报告酶的底物，因此会变得复杂。
