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.     

New and highly sensitive assay for l-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography—voltammetry

This paper describes a new, inexpensive and highly sensitive assay for aromatic l-amino acid decarboxylase (AADC) activity, using l-5-hydroxytryptophan (l-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. l-5-HTP was used as substrate and d-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from l-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using l-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.     

New and highly sensitive assay for l-5-hydroxytryptophan decarboxylase activity by high-performance liquid chromatography—voltammetry

This paper describes a new, inexpensive and highly sensitive assay for aromatic l-amino acid decarboxylase (AADC) activity, using l-5-hydroxytryptophan (l-5-HTP) as substrate, in rat and human brains and serum by high-performance liquid chromatography (HPLC) with voltammetric detection. l-5-HTP was used as substrate and d-5-HTP for the blank. After isolating serotonin (5-HT) formed enzymatically from l-5-HTP on a small Amberlite CG-50 column, the 5-HT was eluted with hydrochloric acid and assayed by HPLC with a voltammetric detector. N-Methyldopamine was added to each incubation mixture as an internal standard. This method is sensitive enough to measure 5-HT, formed by the enzyme, 100 fmol to 140 pmol or more. An advantage of this method is that one can incubate the enzyme for longer time (up to 150 min), as compared with AADC assay using l-DOPA as substrate, resulting in a very high sensitivity. By using this new method, AADC activity was discovered in rat serum.     

Highly Sensitive Assay for Dopamine-β-Hydroxylase Activity in Human Cerebrospinal Fluid by High Performance Liquid Chromatography-Electrochemical Detection: Properties of the Enzyme

Abstract: This paper describes a new, sensitive assay for dopamine-β-hydroxylase (DBH) activity in human cerebrospinal fluid (CSF), serum and brain tissues by high performance liquid chromatography (HPLC) with electrochemical detection (ED). Dopamine (DA) was used as a substrate and was incubated under optimal conditions. Norepinephrine (NE) formed enzymatically from DA was isolated by a double-column procedure, the first column of Dowex-50-H⁺ and the second column of aluminum oxide. NE was adsorbed on the second aluminum oxide column and then eluted with 0.5 M-hydrochloric acid and assayed by HPLC-ED. Epinephrine (EN) was added to each incubation mixture as an internal standard, and this assay was therefore highly reproducible. The peak height in HPLC was linear from 500 fmol to 100 pmol of NE and EN. The lower limit of detection for NE formed enzymatically was about 30 pmol, which indicated that the sensitivity of this procedure was comparable to that of radioassay procedures. We applied the method to measurement of the activity of and examination of some of the characteristics of DBH in human CSF. DBH activity in CSF of Parkinsonian patients was lower than that of control patients. The properties of DBH in human CSF were similar to those in serum and adrenal medulla.     

The vasopressin precursor in the Brattleboro (di/di) rat

The vasopressin precursor in the rat hypothalamus has been studied, using trypsin to release desglycinamide vasopressin and coupling it to glycinamide (T & G treatment). The resulting amidated nonapeptide was detected and measured with a radioimmunoassay for vasopressin. The "vasopressin" produced in this way had the full immunoreactivity of the authentic peptide but eluted from an hplc column 1 min earlier and appeared to have a larger molecular weight. It was found that T&G treatment generated vasopressin immunoreactivity in extracts of the supraoptic nucleus (SON) of the Brattleboro rat in just the same way as it did in normal animals. Furthermore, this procedure produced vasopressin immunoreactivity in those hplc fractions from Brattleboro SON extracts that corresponded with the elution time of vasopressin precursor. Similar amounts of "vasopressin" could be generated from Brattleboro and normal SONs. These results support the suggestion that the Brattleboro SON synthesizes an aberrant vasopressin precursor which is not processed by the cell.     

A sensitive method for the determination of cytolytic activity

A new method is described for the determination of the cytolytic activity of extremely low levels of stable as well as very labile cytotoxins. The method involves the application of the cytotoxin to a column of immunobilized erythrocytes or other suitable cells and a continuous monitoring of the column eluate for the presence of hemoglobin or other cell constituents. The cytotoxic activity of horseradish peroxidase at concentrations as low as 10^?12, m can be measured with this technique. The column hemolytic assay is compared with a static (batch) hemolytic assay with respect to sensitivity and reproducibility. Furthermore, a method is described to determine the true rates of lysis, i.e., the number of cells lysed per minute.     

Determination of pyridoxal 5′-phosphate as the semicarbazone derivative using high-performance liquid chromatography

A high-performance liquid chromatographic (hplc) procedure is described for the determination of pyridoxal 5′-phosphate (PLP) in animal tissues. The procedure is based on extraction with perchloric acid and treatment with semicarbazide to form PLP-semicarbazone. This derivative is quantitatively determined using hplc with an octylsilica column, an acidic phosphate mobile phase buffer, and fluorometric detection. The validity of the method was confirmed by inspection of the fluorescence spectra of the PLP-semicarbazone hplc peaks of semicarbazide-treated standards and tissue extracts. Preparative chromatography for extract purification is not required because of the hplc efficiency and detection specificity. This method provides a simple technique for the rapid, direct assay of PLP in animal tissues.     

MPP+ toxicity in rat striatal slices: Relationship between non-selective effects and free radical production

Incubations of rat striatal slices have been used to assay MPP⁺ neurotoxicity. MPP⁺, at concentrations of 1 mM or higher, caused a marked increase in hydroxyl radicals, measured as malondialdehyde (MDA) accumulation, but not in nitric oxide production. At these doses, MPP⁺ showed an effect on dopamine terminals, causing a massive dopamine decrease, and on non-neuronal glial cells, where a marked reduction in glutamine synthetase activity was detected. At lower concentrations (25 μM), the toxic effect on dopaminergic endings was maintained without increasing malondialdehyde concentrations or inhibiting glutamine synthetase activity. The effect on glutamine synthetase was prevented by the addition to the medium of 0.5% dimethyl sulfoxide, a hydroxyl-radical scavenger, but this did not protect the effect of dopamine depletion. We propose that non-selective effects of MPP⁺, at doses of 1 mM or higher, are mediated by extracellular overproduction of hydroxyl radicals. The main factor responsible for this overproduction would not be the released dopamine but rather the MPP⁺ itself, through non selective inhibition of the mitochondrial respiratory chain or through a redox cycling that can trigger oxygen radical production.     

Alcohol dehydrogenase-coupled spectrophotometric assay of epoxide hydratase activity

A coupled assay was devised for the assay of liver microsomal epoxide hydratase using the ability of alcohol dehydrogenase to transfer electrons from diols to NAD⁺: epoxide hydratase activity was continuously monitored at 340 nm. Rates of hydrolysis of octene-1,2-oxide and styrene-7,8-oxide measured utilizing this assay were similar to those determined using gas-liquid chromatography and radiometric thin-layer chromatography, respectively. The assay was used to examine the ability of rat liver microsomes and highly purified rat liver microsomal epoxide hydratase fractions to hydrolyze 15 other epoxides.     

Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum

Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP-sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a coupling enzyme. Glucuronokinase was partially purified on a DEAE-sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is used by UDP-sugar pyrophosphorylase and further converted to UDP-glucuronic acid. This UDP-sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange column or a reversed phase C18 column at a wavelength of 260 nm. This assay is adaptive to different kinases and sugars because of the broad substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min^-1. Furthermore, it can be used for determination of pure kinases as well as crude or partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.     

High-pressure liquid chromatographic method for the determination of 25-hydroxycholecalciferol in cow plasma

A high-pressure liquid chromatographic (hplc) procedure was developed for the determination of 25-hydroxycholecalciferol (25-OH-D₃) in cow plasma or serum. The procedure involved extraction with an ethanol-ethyl ether mixture, separation of the aqueous phase, solvent partitions, column chromatography on silica gel, and, finally, determination by reversed phase hplc on a C₁₈-bonded microparticulate silica column. The identity of the drug in the extract was confirmed by comparison with a standard by liquid, thin-layer, and gas-liquid chromatography as the free steroid and the heptafluorobutyrate and by the uv spectra and also from the mass spectrum of the heptafluorobutyrate. Twenty-four samples from cows on normal diet (dry, lactating, and pregnant) were analyzed. The normal circulating levels of 25-OH-D₃ ranged from 40 to 58 ng/g; mean 48 ± 5.0 ng/g. The procedure was used to analyze a limited number of human and hog samples. Human serum contained 10–20 ng/g which was in agreement with literature values. Hog serum contained 18 ng/g.     

Automated nonisotopic assay for protein-tyrosine kinase and protein-tyrosine phosphatase activities.

A sensitive, automated, and nonisotopic assay for protein-tyrosine kinases and phosphatases has been developed. The assay uses commercially available antiphosphotyrosine monoclonal antibodies and the recently developed particle concentration immunofluorescence immunoassay technology. The assay is specific for phosphotyrosine residues, can be performed faster, and is at least 100-fold more sensitive than the current standard filter type radioassay. Myelin basic protein and a synthetic peptide corresponding to the autophosphorylation site of p56lck performed equally well in the detection of p56lck kinase activity. Myelin basic protein phosphorylated on tyrosine residues by p56lck was successfully used as substrate in the detection of phosphatase activity and vanadate or molybdate were shown to inhibit the phosphatase activity. The assay is particularly useful for the rapid detection of enzyme activities in column fractions from biochemical procedures steps and also for screening of large numbers of potential inhibitors or activators of protein-tyrosine kinases and phosphatases.     

Highly sensitive assay for phenylethanolamine N-methyl-transferase activity in rat brain by high-performance liquid chromatography with electrochemical detection

This paper describes a new and highly sensitive assay for phenylethanolamine N-methyl-transferase (PNMT) activity with noradrenaline as substrate in various rat brain regions by high-performance liquid chromatography with electrochemical detection. Commercially available noradrenaline contained about 0.27% of contaminating adrenaline, which was removed to reduce the blank value. Enzymatically formed adrenaline was adsorbed on an aluminium oxide column, eluted with 0.5 M hydrochloric acid, separated by high-performance reversed-phase paired-ion chromatography and measured with electrochemical detection. 3,4-Dihydroxybenzylamine was added to the incubation mixture as an internal standard after the reaction. This assay was very sensitive and 0.5 pmol of adrenaline formed enzymatically could be detected. This assay method was applied to measure PNMT activity in various rat brain regions. The highest activity was observed in the hypothalamus, pons plus medulla oblongata, septum, lower brain stem, and cerebral cortex; the lowest activity was in the striatum, hippocampus, cerebellum, and limbic brain.     

The effects of dopamine on Na+K+-ATPase activity in nerve ending membranes are prevented by N-ethylmaleimide

The activity of Na⁺K⁺-ATPase in the membranes of nerve endings isolated from rat cerebral cortex was inhibited by dopamine. On the contrary, when the soluble fraction from cortical homogenates was added, dopamine stimulated enzyme activity. By varying the concentration of the soluble fraction present in the incubation medium for Na⁺K⁺-ATPase assay, it was possible to establish that this fraction modulates those effects of dopamine on Na⁺K⁺-ATPase.The preincubation of the membranes with N-ethylmaleimide under conditions in which the Na⁺K⁺-ATPase activity was not inhibited (5 × 10^?5 M for 10 min at 37°C), prevented both the inhibitory and the stimulatory effects of dopamine observed without or with the soluble fraction from brain respectively.These results suggest that dopamine probably acts on regions of the protein containing -SH groups, different from those sites responsible for the catalytic activity of the enzyme.     

Mechanism of the effect of urethane on the secretion of prolactin in the male rat

A subcutaneous injection of urethane (200 mg/100 g body wt.) into adult male rats resulted in a significant increase in serum prolactin (PRL) at 30 minutes. Subsequent measurements at 60, 90 and 120 minutes postinjection revealed a marked and rapid decrease in serum PRL to levels significantly lower than those of unanesthetized animals. The administration of the dopamine antagonist pimozide (8, 40 or 200 μg) 30 minutes after urethane injection elevated serum PRL levels in a dose-dependent manner and thus prevented the urethane-induced depression in serum PRL observed at 60 minutes postinjection. Hypothalamic synthesis of ¹⁴C-dopamine from its precursor ¹⁴C-tyrosine was measured in both urethane-anesthetized and unanesthetized rats. The synthesis of hypothalamic dopamine was dramatically increased in the urethane-anesthetized animals as compared to newly synthesized hypothalamic dopamine levels in the unanesthetized controls. These results indicate that the PRL-inhibitory effects of urethane anesthesia in the rat may be exerted through increased dopaminergic activity.     

A radioisotopic method for the determination of acetoacetyl Coenzyme A with 3-hydroxy-3-methylglutaryl Coenzyme A synthase

A simple and sensitive assay for the quantitative determination of acetoacetyl-CoA (AcAc-CoA) in liver and heart is described. The method is based on incorporation of [¹⁴C]acetyl-CoA into acid-stable nonvolatile material in the presence of avian HMG-CoA synthase. The specificity of this procedure for the measurement of AcAc-CoA was demonstrated by pretreating tissue extracts with 3-hydroxyacyl-CoA dehydrogenase or CoA transferase from Escherichia coli to deplete. AcAc-CoA prior to assay. Acid-stable nonvolatile ¹⁴C activity measured in the assay was proportional to the amount of tissue extract added. Satisfactory recovery of AcAc-CoA added at the initial extraction step further validated this procedure. This radioactive assay for acetoacetyl-CoA using a highly purified avian 3-hydroxy-3-methylglutaryl-CoA synthase has the advantages of both extreme specificity for AcAc-CoA as substrate and high sensitivity, facilitating the determination of this metabolite under a variety of physiological conditions.     

An assay method for ganglioside synthase using anion-exchange chromatography

A rapid procedure which is based on combined ion-exchange chromatography and solubility was established for determination of the activity of ganglioside synthases and cerebroside sulfotransferase. The procedure consists of selective elution of radiolabeled reaction products (acidic glycolipids) freed from labeled precursors and breakdown products on a DEAE-Sephadex column and of direct radioassay of the products in the eluate. Monosialogangliosides were eluted from the column with 40 mM ammonium acetate (AcONH4) in methanol, cerebroside sulfate with 90 mM AcONH4 in methanol, and disialogangliosides with 40 mM AcONH4 in isopropanol/n-hexane/water (55/20/19, v/v/v). The established procedure is simple, reproducible, and economical. Using rat Golgi membrane as enzyme source the recovery rate of the products was over 95%.     

Factors influencing the recovery of dopamine sulfate in the assay of phenol sulfotransferase

Phenol sulfotransferase activity is often measured by incubating dopamine with a source of the enzyme and the sulfate donor 35S-3'-phosphoadensine-5'-phosphosulfate and then separating the product, 35S-dopamine sulfate, from the substrates using precipitation with barium hydroxide and zinc sulfate. Using dopamine sulfate standards and high performance liquid chromatography with dual-electrode electrochemical detection, we investigated the effects of several parameters on product recovery obtained with this procedure. Amounts of precipitants needed to produce maximal sample-to-blank ratios were determined using crude enzyme preparations from rat brain and human platelets incubated under conditions generating approximately 3 nM dopamine sulfate. Under these conditions, recovery of 3H-DAS standards was 70-80%. Increasing either the concentration of dopamine sulfate or the amounts of precipitants used resulted in concentration-dependent decreases in dopamine sulfate recovery. These data indicate that the recovery of product in this assay may vary with assay conditions, and should be carefully monitored and optimized. Caution should be exercised when using substrates other than dopamine in this assay procedure, since similar factors might also contribute to variable recovery of other products.     

Reconstitution and purification of the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain

The sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain has been highly purified. Synaptic plasma membranes from rat brain were extracted with cholate in the presence of 10% ammonium sulfate. The soluble extract was incorporated into liposomes consisting of asolectin and crude brain lipids. Brain lipids markedly enhanced the transport activity. The resulting proteoliposomes catalyzed sodium- and chloride-coupled gamma-aminobutyric acid transport which, in the presence of internal potassium, was greatly (up to 20-fold) stimulated by valinomycin. Using this transport of the reconstituted system as an assay, the transporter was purified by the following steps. The cholate extract was fractionated by ammonium sulfate. The activity was not precipitated by 50% but could be precipitated by 70% ammonium sulfate. The cholate and ammonium sulfate were removed on a Sephadex G-50 column. Subsequently, the transporter was partially purified on DEAE-cellulose in a mixture of Triton X-100 and octyl glucoside. The active fractions were chromatographed on a hydroxylapatite column in the presence of Triton X-100. Although the increase in specific activity was only up to 100-fold, this was due to partial inactivation. The actual purification was at least 1000-fold. The purified transporter exhibited the same features of the synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogenicity, and a similar affinity. The sodium dodecyl sulfate gel pattern indicated that a major protein ran as a 24-kDa band. This band may represent the gamma-aminobutyric acid transporter.     

In Vivo Stimulation of D1 Receptors Increases the Phosphorylation of Proteins in the Striatum

In vivo changes in levels of DARPP-32 [dopamine (DA)- and cyclic AMP-regulated phosphoprotein, Mr = 32,000] protein phosphorylation in response to DA agonists in the rat striatum were measured using a novel assay that combines the benefits of rapid quenching of enzyme activity by focused microwave irradiation with a back-phosphorylation assay. The basal level of phospho-DARPP-32 was 5.6% of total DARPP-32. Injections of L-3,4-dihydroxyphenylalanine (100 mg/kg) increased this level to 44.4%. This effect was not as great if focused microwave irradiation was not used. The D1-specific agonist SKF 38393 (10 mg/kg) increased the level of phospho-DARPP-32 to 36.4%. A further modification of the back-phosphorylation assay was used to detect other phosphoproteins that appear to be regulated by DA. These results establish an assay for in vivo studies of postsynaptic responses involving second messengers in the DA system and provide direct in vivo evidence for the hypothesis that stimulation of D1 receptors increases the phosphorylation of DARPP-32, as well as several other proteins.