A simple and sensitive fluorescence assay for tyrosine hydroxylase activity.

A simple and sensitive fluorescence assay for tyrosine hydroxylase (TH) activity wasdevised based on rapid isolation of enzymatically formed dopa by a double-column procedure fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminum oxide). Interfering substances were removed by the first Amberlite CG-50 column. Dopa was adsorbed on the second aluminum oxide column, then eluted with 0.5 m acetic acid, and assayed by the highly sensitive hydroxyindole method of Johnson et al. (1973, Anal. Biochem.54, 129–136). The standard incubation mixture (total volume, 0.5 ml) contained 0.3 mm l-tyrosine, 1.0 mm 6-methyl-5,6,7,8-tetrahydropterin, 100 mm mercaptoethanol, and an optimal concentration of ferrous ion. d-Tyrosine was used for the blank incubation. Recovery of dopa added to the standard incubation mixture as internal standard was about 70% and was reproducible. The fluorescence characteristics of the product were the same as those of authentic dopa. Blank fluorescence was very low even with crude enzyme preparations. The limit of sensitivity was 100 pmol of dopa formed, which is close to the sensitivity of radioassays. TH activity in homogenates of rat brain stem or human putamen could be assayed in the standard incubation system containing ferrous ion. The validity of this fluorescence assay has been shown by the agreement between the values obtained by this method and by radioassay using l-[U-¹⁴C]tyrosine as substrate. In the rapid assay procedure dopa in the eluate from aluminum oxide was assayed directly by native fluorescence. Although the sensitivity was about 1 nmol, this rapid assay procedure was found to be particularly useful for the purification of TH.     

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. d-Tyrosine was used for the control. α-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.     

Rapid assay for hormone-sensitive lipase activity of adipose tissue

A highly specific and rapid assay for hormone-sensitive lipase activity of rat adipose tissue is described. The method employs emulsified 2,3-di-O-oleyl-[9,10-(3)H(2)]oleoyl glycerol as a substrate; it is very sensitive and is suitable for serial sampling.     

Rapid assay for catechol-O-methyltransferase activity by high-performance liquid chromatography-fluorescence detection

A rapid assay for measuring the activities of catechol-O-methyltransferase (COMT) is described. The method is based on high-performance liquid chromatography (HPLC)-fluorescence detection, and includes on-line extraction of catecholamines with a precolumn, separation of norepinephrine (NE) and normetanephrine (NMN) on an ODS column, electrochemical oxidation, and post-column fluorogenic derivatization using ethylenediamine. The method took less than 25 min for one sample, which is half that of the previous method and the sensitivity was similar. The intra-day assay precisions were 0.52-1.6%, and the inter-day assay precisions were 3.6-5.8% for rat liver and cerebral cortex (n = 5). The method is suitable for the rapid measurement of COMT activities of many biological samples.     

A rapid assay for collagen proline hydroxylase

A method for the assay of collagen proline hydroxylase is presented. Hydroxyproline-deficient protein, which serves as substrate, is prepared by incubating minces of chick embryo with 3,4-T-l-proline in the presence of α,α′-dipyridyl. Following extraction and partial purification, the substrate is incubated with proline hydroxylase, which converts some of the 3,4-T-l-proline residues to 3-T-l-hydroxyproline. The tritium atom which is released from carbon 4 equilibrates with water and the resulting tritiated water is assayed following vacuum distillation. It was shown that tritiated water release is entirely dependent on enzymic hydroxylation and that equivalent amounts of 3-T-l-hydroxyproline and tritiated water are formed. This method is much more rapid and accurate than previously described methods.     

A specific kinetic assay for phenylalanine hydroxylase

An assay procedure is given which is speedy, accurate, and specific, permitting direct recording of velocities, and obviating the use of reagents other than those necessary for the enzymatic reaction itself. The method is suitable for the study of enzyme mechanism and inhibition and also offers distinct advantages when used for other purposes, e.g., assay during purification of enzymes or for measurement of phenylalanine hydroxylase activity in the liver of hyperphenylalaninemics.The method is based on the phenylalanine-dependent change in absorbance of the tetrahydropteridine cofactor as it is oxidized to the dihydro form. The reaction rate measured by this procedure is linear over a wide range of enzyme concentration. The K_m and V for both tetrahydropteridine and for phenylalanine were the same as the values determined by the old procedure. Measurement of the stoichiometry of the reaction showed that one dihydropteridine is formed per tyrosine formed, or per DPNH consumed. The rate of reaction was identical to that measured by a coupled assay using DPNH and purified dihydropteridine reductase.     

Tyrosine hydroxylase assay for detection of low levels of enzyme activity in peripheral tissues

A nonisotopic assay for tyrosine hydroxylase, with optimized signal-to-noise ratios, enables determination of low levels of enzyme activity in peripheral tissues. DOPA produced by the enzyme is measured using HPLC with electrochemical detection. Increased signal-to-noise ratios are obtained by including in the reaction mixture glycerol for reduction of blank values and dihydropteridine reductase and NADPH for regeneration of the tetrahydropteridine cofactor. With this method, tyrosine hydroxylase activity can be detected in as few as 200 PC12 cells and in peripheral tissues at levels as low as 4.5 fmol/min/mg wet weight. The assay permits activity to be assessed in a variety of peripheral tissues.     

A simple radioisotope assay for microsomal aryl hydroxylase

A radioassay for liver microsomal aryl hydroxlase activity has been devised which depends on the liberation of tritiated water from generally tritiated benzo[α]pyrene during hydroxylation. The quantity of tritiated water has been shown to be proportional to the amount of 3-hydroxybenzo-[α]pyrene formed. Among the advantages of the radioassay are its speed, simplicity, and the fact that it essentially provides a cumulative measure of the hydroxylation of the benzo[α]pyrene ring. Investigation of a number of variables has made it possible to assay and obtain proportional results with as little as 3 μg of rat liver microsomes. Nucleic acids, but not their component mononucleotides, have been found capable of protecting the enzyme from product inhibition, presumably by interaction with benzo[α]-pyrene oxide, the primary product of benzo[α]pyrene hydroxylation.     

Rapid assay for bactericidal antibodies

[This corrects the article on p. 279 in vol. 20.].     

A rapid and simplified assay method for tyrosine hydroxylase

Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H2O with the hydroxyl proton of isoamyl alcohol. It is the [3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.     

Development of a convenient peptide-based assay for lysyl hydroxylase

Hydroxylysine (Hyl) is a posttranslationally modified amino acid found mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) catalyzes the hydroxylation of the C-5 position of a Lys residue, resulting in the production of Hyl. Mechanistically, LH incorporates one oxygen atom into both Lys and 2-oxoglutarate; the latter is decarboxylated to form succinate and CO(2). To develop a convenient, RP-HPLC based LH assay, we used Fmoc solid-phase methodology to synthesize three different peptides designed as LH substrates and one peptide corresponding to an LH product. Peptides were characterized by RP-HPLC, MALDI-TOF mass spectrometry and CD spectroscopy. Separation of peptides was examined under a variety of RP-HPLC conditions. The best results were achieved using peptide derivatization (1-anthroylnitrile for organic phase and dansyl chloride for aqueous phase) prior to RP-HPLC analysis. The products (di- and tetra-substituted Lys- and Hyl-containing peptides) were well resolved by RP-HPLC. The resolution of each peak allows for quantification of peak areas, which in turn, when examined as a function of time, can be utilized for studying the kinetics of LH catalyzed reactions. Most significantly, the RP-HPLC assay directly monitors the Hyl containing product. Prior LH assay methods are multi-step, require radio-labeled substrates, and/or measure depletion of 2-oxoglutarate or formation of CO(2). Since the LH reaction with 2-oxoglutarate is uncoupled from Lys hydroxylation, the most accurate assay of LH activity should monitor the formation of Hyl.     

Measurement of tyrosine hydroxylase activity in serve terminals of rat hippocampus,hypothalamus, and striatum using an improved assay for tyrosine hydroxylase

The formation of ³H₂O from L-4-³H-phenylalanine is used as an index of tyrosine hydroxylase activity in synaptosomes from rat hippocampus, hypothalamus, and striatum. The reactions are linear with respect to time (up to 20 min) and with respect to protein concentration (up to 0.2 mg/ml). Formation of ³H₂O from L-4-³H-phenylalanine is inhibited by standard tyrosine hydroxylase inhibitors (α-methyl-p-tyrosine, L-3-iodotyrosine, dopamine, L-norepinephrine, and L-apomorphine) and by the tyrosine hydroxylase substrate L-tyrosine as well as by synaptosomal lysis. The blank ³H₂O produced from L-4-³H-phenylalanine (0.02% of total DPM) is 10-fold less than the blank ³H₂O produced from L-3,5-³H-tyrosine. The K_m values of tyrosine hydroxylase for phenylalanine determined by the production of ³H₂O from L-4-³H-phenylalanine are 3.1, 1.3, and 1.2 μm in hippocampal, hypothalamic and striatal synaptosomes respectively. The results indicate that analysis of ³H₂O formed from L-4-³H-phenylalanine is a sensitive and reliable method for quantitating synaptosomal tyrosine hydroxylase activity from tissues with low levels of tyrosine hydroxylase such as synaptosomes from hippocampus and hypothalamus.     

Modification of the tyrosine hydroxylase assay

A modification of the tyrosine hydroxylase assay is described in which ascorbate, rather than 2-mercaptoethanol or dihydropteridine reductase with NADPH, is used as the reductant. Enzyme activity is 3–4 times higher with ascorbate than with the other reducing agents. Low blanks are obtained with the ascorbate system provided that catalase is also included. The tissue distribution and kinetic activation of the enzyme have been studied with the ascorbate assay. The results obtained are consistent with the biological and regulatory properties of the enzyme which have been determined with the other reducing systems.