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Studies on Tyrosine Hydroxylase System in Rat Brain Slices Using High‐Performance Liquid Chromatography with Electrochemical Detection
Author(s) -
Hirata Yoko,
Togari Akifumi,
Nagatsu Toshiharu
Publication year - 1983
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1983.tb08130.x
Subject(s) - tyrosine hydroxylase , tetrahydrobiopterin , tyrosine , tyrosine 3 monooxygenase , high performance liquid chromatography , aromatic l amino acid decarboxylase , chemistry , enzyme , chromatography , hydroxylation , endogeny , dihydroxyphenylalanine , biochemistry , cofactor , endocrinology , biology , dopamine
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°C, and increased by incubation with 20 m M 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.