Hirata Y, Togari A, Nagatsu T
J Neurochem. 1983 Jun;40(6):1585-9. doi: 10.1111/j.1471-4159.1983.tb08130.x.
A new method for the measurement of tyrosine hydroxylase (TH; EC 1.14.16.2) activity in brain slices was developed by using high-performance liquid chromatography (HPLC) with electrochemical detection (ED). To estimate TH activity in brain slices containing all of the components of the enzyme system, tetrahydrobiopterin, dihydropteridine reductase, and TH itself, slices were incubated with NSD-1055, an inhibitor of aromatic L-amino acid decarboxylase, and 3,4-dihydroxyphenylalanine (DOPA) formed from endogenous tyrosine was measured using HPLC-ED. Hydroxylation of endogenous tyrosine to DOPA in striatal slices was linear up to 90 min at 37 degrees C, and increased by incubation with 20 mM K+ to depolarize the nerve cells. Furthermore, the formation of DOPA could be detected in all parts of brain regions examined, and the activity in this slice system was nearly parallel to the maximal velocity of the homogenate from the slices as enzyme in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin as cofactor. This assay system should be useful to study the regulatory mechanisms of TH in relatively intact tissue preparations.
通过使用高效液相色谱（HPLC）结合电化学检测（ED），开发了一种测量脑切片中酪氨酸羟化酶（TH；EC 1.14.16.2）活性的新方法。为了估计包含酶系统所有成分（四氢生物蝶呤、二氢蝶啶还原酶和TH本身）的脑切片中的TH活性，将切片与芳香族L-氨基酸脱羧酶抑制剂NSD-1055一起孵育，并使用HPLC-ED测量由内源性酪氨酸形成的3,4-二羟基苯丙氨酸（DOPA）。在37℃下，纹状体切片中内源性酪氨酸向DOPA的羟基化在90分钟内呈线性，并且通过与20 mM K +孵育使神经细胞去极化而增加。此外，在所检查的脑区的所有部位都可以检测到DOPA的形成，并且在该切片系统中的活性几乎与切片匀浆作为酶在酪氨酸饱和浓度和6-甲基四氢蝶呤作为辅因子存在下的最大速度平行。该检测系统对于研究相对完整组织制剂中TH的调节机制应该是有用的。
