The Activity of Rat Pineal and Brain Tyrosine Hydroxylase During the Daily Cycle of Light and Darkness as Determined by the Modified 14CO2 Assay Method

Abstract

A previous published assay method for tyrosine hydroxylase by the evolution of 14CO2 was modified to a 2-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 Km values of the brain enzyme for L-tyrosine with 1 mM-(6-DL)-5,6,7,8-tetrahydro-L-erythro-biopterin (BPH4) as cofactor and for BPH4 with 62 .mu.M-L-tyrosine as substrate were .apprx. 25 and 85 .mu.M, respectively. The Km for L-tyrosine with 1 mM-(6-DL)-5,6,7,8-tetrahydro-6-methylpterin (6MPH4) as cofactor and for 6MPH4 with 210 .mu.M L-tyrosine as substrate were 68 and 270 .mu.M, respectively. The marked substrate inhibition by high concentrations of L-tyrosine was observed only when BPH4 was used as cofactor. High concentrations of BPH4 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 vs. the reciprocal concentration of BPH4 with 62 .mu.M L-tyrosine as substrate deviated downward at a BPH4 concentration of about 100 .mu.M. Analyses of the plot indicated that the peculiar kinetic property may represent either the reaction occurring at 2 independent sites or with 2 forms (6L- and 6D-isomers) of the tetrahydrobiopterin cofactor, with apparent Km for BPH4 of 23 and 1025 .mu.M, respectively, or the negatively cooperative ligand binding with a Hill coefficient of 0.72. 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 3 discrete brain areas was demonstrated. The findings, especially on pineal tyrosine hydroxylase, are discussed in relation to the daily change of noradrenaline [norepinephrine] turnover.