Real-time monitoring of tyrosine hydroxylase activity using a plate reader assay.

Tyrosine hydroxylase (TH) is the rate-limiting step in dopamine (DA) synthesis, oxidizing tyrosine to l-DOPA, which is further metabolized to DA. Current assays for monitoring activity of this enzyme require extensive work-up, require long analysis time, and measure end points, thereby lacking real-time kinetics. This work presents the development of the first real-time colorimetric assay for determining the activity of TH using a plate reader. The production of l-DOPA is followed using sodium periodate to oxidize l-DOPA to the chromophore dopachrome, which can be monitored at 475 nm. Advantages to this method include decreased sample analysis time, shorter assay work-up, and the ability to run a large number of samples at one time. Furthermore, the assay was adapted for high-throughput screening and demonstrated an excellent Z-factor (> 0.8), indicating suitability of this assay for high-throughput analysis. Overall, this novel assay reduces analysis time, increases sample number, and allows for the study of activity using real-time kinetics.

Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli.

Tyrosine hydroxylase is the rate-limiting step in the synthesis of dopamine and is tightly regulated. Previous studies have shown it to be covalently modified and potently inhibited by 3,4-dihydroxyphenylacetaldehyde (DOPAL), an endogenous neurotoxin via dopamine catabolism which is relevant to Parkinson's disease. In order to elucidate the mechanism of enzyme inhibition, a source of pure, active tyrosine hydroxylase was necessary. The cloning and novel purification of human recombinant TH from Escherichia coli is described here. This procedure led to the recovery of ~23 mg of pure, active and stable enzyme exhibiting a specific activity of ~17 nmol/min/mg. The enzyme produced with this procedure can be used to delineate the tyrosine hydroxylase inhibition by DOPAL and its relationship to Parkinson's disease. This procedure improves upon previous methods because the fusion protein gives rise to high expression and convenient affinity-capture, and the cleaved and highly purified hTH makes the product useful for a wider variety of applications.

Specificity determinants of allosteric modulation in the neuronal nicotinic acetylcholine receptor: a fine line between inhibition and potentiation.

We are interested in the allosteric modulation of neuronal nicotinic acetylcholine receptors (nAChRs). We have postulated that the anthelmintic morantel (Mor) positively modulates (potentiates) rat α3ß2 receptors through a site located at the ß(+)/α(-) interface that is homologous to the canonical agonist site (J Neurosci 29:8734-8742, 2009). On this basis, we aimed to determine the site specificity by studying differences in modulation between α3ß2 and α4ß2 receptors. We also compared modulation by Mor with that of the related compound oxantel (Oxa). Whereas Mor and Oxa each potentiated α3ß2 receptors 2-fold at saturating acetylcholine (ACh) concentrations, Mor had no effect on α4ß2 receptors, and Oxa inhibited ACh-evoked responses. The inhibition was noncompetitive, but not due to open channel block. Furthermore, the nature and extent of modulation did not depend on subunit stoichiometry. We studied six positions at the α(-) interface that differ between α3 and α4. Two positions (α3Ile57 and α3Thr115) help mediate the effects of the modulators but do not seem to contribute to specificity. Mutations in two others (α3Leu107 and α3Ile117) yielded receptors with appreciable α4-character; that is, Mor potentiation was reduced compared with wild-type α3ß2 control and Oxa inhibition was evident. A fifth position (α3Glu113) was unique in that it discriminated between the two compounds, showing no change in Mor potentiation from control but substantial Oxa inhibition. Our work has implications for rational drug design for nicotinic receptors and sheds light on mechanisms of allosteric modulation in nAChRs, especially the subtle differences between potentiation and inhibition.