Chobotská K, Arnold M, Werner P, Pliska V
Department of Animal Science, Swiss Federal Institute of Technology, Zürich.
Biol Chem. 1998 Jan;379(1):59-63. doi: 10.1515/bchm.1998.379.1.59.
Tyrosine hydroxylase (TH) (EC 1.14.16.2) activity has been frequently employed as a marker of adrenomedullar catecholamine-synthesizing capacity and, thus, as an indicator of chronic stress exposure in various animal species. We have developed a thin layer chromatography (TLC) procedure for its assay in adrenal glands of rats and large animals that reduces some of the drawbacks of currently employed methods, thereby facilitating routine use. Preparation of tissue samples was adapted for rats and pigs. The activity of the enzyme is expressed as the rate of the TH-catalysed tyrosine hydroxylation to 3,4-dihydroxyphenyl-alanine (DOPA) using tritium-labeled tyrosine, in the presence of cofactors and a DOPA decarboxylase inhibitor. The subsequent separation of the radioactive product (DOPA) from the substrate (tyrosine) is accomplished by TLC on silicagel plates, in a n-butanol/acetic acid/water solvent system (4:1:1). Radioactivity in the scraped zones, in which DOPA has been detected by means of an internal standard, is measured by beta-counting. An advantage of this procedure is its simplicity, reliability, and convenience for routine assays. Levels of endogenous adrenal tyrosine (HPLC assay) are considerably higher in pig (2.5-5 nmol/mg protein) than in rat (0.15 nmol/mg protein); their effects upon assay results being, in both cases, negligible. Michaelis constants estimated by this procedure amounted to 0.9 mmol l(-1) (at 0.7 mM DMPH4) for pig, and 1.1 mmol l(-1) (at 1.5 mM DMPH4) for rat.
酪氨酸羟化酶（TH）（EC 1.14.16.2）活性常被用作肾上腺髓质儿茶酚胺合成能力的标志物，因此也作为各种动物慢性应激暴露的指标。我们开发了一种薄层色谱（TLC）方法，用于测定大鼠和大型动物肾上腺中的该酶活性，该方法减少了当前所用方法的一些缺点，从而便于常规使用。组织样品的制备适用于大鼠和猪。该酶的活性表示为在存在辅因子和多巴脱羧酶抑制剂的情况下，使用氚标记的酪氨酸，TH催化酪氨酸羟化生成3,4 - 二羟基苯丙氨酸（DOPA）的速率。随后通过硅胶板上的TLC在正丁醇/乙酸/水溶剂系统（4:1:1）中从底物（酪氨酸）中分离出放射性产物（DOPA）。通过内标检测到DOPA的刮取区域中的放射性通过β计数进行测量。该方法的一个优点是其简单性、可靠性以及便于常规测定。猪肾上腺内源性酪氨酸水平（高效液相色谱法测定）（2.5 - 5 nmol/mg蛋白质）比大鼠（0.15 nmol/mg蛋白质）高得多；在两种情况下，它们对测定结果的影响都可忽略不计。通过该方法估计的米氏常数，猪为0.9 mmol l(-1)（在0.7 mM DMPH4时），大鼠为1.1 mmol l(-1)（在1.5 mM DMPH4时）。
