Tyrosine Hydroxylase Activity in Rat Brain Synaptosomes: Direct Measurement Using High Performance Liquid Chromatography

Abstract

By use of high performance liquid chromatography with electrochemical detection to measure dopamine production, tyrosine hydroxylase (EC 1.14.16.2) activity has been measured in rat brain synaptosomes from striatum and forebrain. Normal specific activities three- to fivefold higher than previously reported in the literature for radiochemical methods of assay were found. It is suggested that synaptosomes contain a significant amount of endogenous substrate for tyrosine hydroxylase, which causes dilution of the added labelled tyrosine and hence underestimation of the activity of this enzyme when radiochemical methods are used.

Dihydroxyphenylalanine Production in Rat Brain Striatal Synaptosomes: Stimulation by a Calcium Chelator

“…In agreement with the results of Messripour and Clark (1982), we find that the units of activity in the nonradioisotopic assay are somewhat higher than in the assay employing labeled tyrosine (Table I). This suggests that the labeled tyrosine in the medium in the radioisotopic assay does mix with the endogenous tyrosine and/or phenylalanine pools before being acted upon by tyrosine hydroxylase.…”

“…The two main conclusions of this study are (1) that a sensitive nonradioisotopic assay for tyrosine hydroxylase activity in synaptosomes can be performed utilizing HPLC-electrochemical detection of DOPA production in the presence of a decarboxylase inhibitor, and (2) that this assay can be especially useful in analyzing drug effects on synthesis where there is concern over possible drug-induced changes in precursor concentration, as is the case for EGTA. Messripour and Clark (1982) have recently reported the use of another nonradioisotopic procedure for measuring synaptosomal tyrosine hydroxylase activity; in their assay, the increase in dopamine content that occurs during incubation is measured. However, the sensitivity of the dopamine assay is limited by the fact that the baseline (blank) value of dopamine present in the unincubated samples represents a significant portion of the total dopamine present after incubation.…”

The Toxic Effect of Manganese on the Acetylcholinesterase Activity in Rat Brains

“…Fresh brains or brains that had been frozen in liquid nitrogen and stored at −75°C were homogenized in cold buffer phosphate, 28 mM (pH 7.8) containing Triton X100 as internal standard [17]. The supernatant following centrifugation at 13,000 ×g for 20 minutes was processed; the supernatant contained AChE that was used for enzyme assay [18].…”

Studies on Tyrosine Hydroxylase System in Rat Brain Slices Using High-Performance Liquid Chromatography with Electrochemical Detection

“…mine products from radioactive L-tyrosine or L-phenylalanine. Recently, the activity of TH in rat brain synaptosomes has been demonstrated by measuring dopamine production from endogenous tyrosine with high-performance liquid chromatography with electrochemical detection (HPLC-ED) (Messripour and Clark, 1982), but the reaction was carried out at pH 6.1, where TH activity is optimal. To study the physiological regulation of the TH system, it would be useful to determine the rate of the conversion of tyrosine to DOPA under physiological pH, 7.4, in a relatively intact tissue preparation, such as tissue slices, in which all of components of the enzyme system may be present at physiological levels.…”