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The Activity of Rat Pineal and Brain Tyrosine Hydroxylase During the Daily Cycle of Light and Darkness as Determined by the Modified 14 CO 2 Assay Method
Author(s) -
Watanabe Shuzo,
Tom Michio,
Ichiyama Arata,
Kataoka Tohru
Publication year - 1981
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.1471-4159.1981.tb02403.x
Subject(s) - darkness , tyrosine hydroxylase , pineal gland , tyrosine , biology , endocrinology , medicine , chemistry , zoology , biochemistry , circadian rhythm , botany , dopamine
A previous published assay method for tyrosine hydroxylase by the evolution of 14 CO 2 was modified to a two‐step procedure to allow reliable measurement of large numbers of samples containing low tyrosine hydroxylase activity. The reliability of the method was examined in detail. Properties of rat brain and pineal tyrosine hydroxylase solubilized with 0.2% Triton X‐100 were as follows. The apparent K m values of the brain enzyme for L‐tyrosine with 1 mM‐(6‐ dl )‐5,6,7,8‐tetrahydro‐L‐erythro‐biopterin (BPH 4 ) as cofactor and for BPH 4 with 62 μM‐L‐tyrosine as substrate were approximately 25 μM and 85 μM, respectively. The K m for L‐tyrosine with 1 mM‐(6‐ dl )‐5,6,7,8‐tetra‐hydro‐6‐methylpterin (6MPH 4 ) as cofactor and for 6MPH, with 210 μM‐L‐tyrosine as substrate were 68 μM and 270 μM, respectively. The marked substrate inhibition by high concentrations of L‐tyrosine was observed only when BPH 4 was used as cofactor. High concentrations of BPH 4 inhibited the reaction slightly. The kinetic properties of tyrosine hydroxylase in the pineal extract were similar to those of the brain enzyme, except that a Lineweaver‐Burk plot of reciprocal velocity versus the reciprocal concentration of BPH 4 with 62 μM‐L‐tyrosine as substrate deviated downward at a BPH 4 concentration of about 100 μM. Analyses of the plot indicated that the peculiar kinetic property may represent either the reaction occurring at two independent sites or with two forms (61.‐and 6 d ‐isomers) of the tetrahydrobiopterin cofactor, with apparent K m for BPH 4 of 23 μM and 1025 μM, respectively, or the negatively cooperative ligand binding with a Hill coefficient of 0.72. Based on the results obtained as reported above the standard assay conditions of tyrosine hydroxylase in tissue extracts were established. Using the assay method and conditions, the absence of the daily rhythmicity of tyrosine hydroxylase in rat pineal glands and three discrete brain areas was demonstrated. The findings, especially on pineal tyrosine hydroxylase, are discussed in relation to the daily change of noradrenaline turnover.