Alteration of Cerebral Taurine Biosynthesis in Spontaneously Hypertensive Rats

Abstract: Cerebral taurine biosynthesis in a spontaneously hypertensive rat (SHR) has been studied. Cysteine sulfinic acid (CSA) and cysteic acid (CA), possible key intermediates in taurine biosynthesis, were found in the rat brain, whereas no cysteamine-cystamine was detected. In the brain of SHR, a statistically significant decrease in the contents of CSA, CA, and taurine was noted in the cerebellum, hypothalamus, and striatum as compared with normotensive Wistar Kyoto rats. Similarly, it was demonstrated that the activity of cysteine dioxygenase, the enzyme catalyzing cysteine to CSA, was attenuated significantly in the same brain areas of SHR. In contrast, no alteration in the activity of CSA decarboxylase, the enzyme converting CSA to hypotaurine or CA to taurine, was observed. A decline in the percent conversion of [¹⁴C]cysteine to [¹⁴C]taurine was found also in tissue homogenates from the cerebellum, hypothalamus, and striatum of SHR, indicating that the declines in taurine content may be due to an attenuation of taurine biosynthesis, possibly at the step involving cysteine dioxygenase.     

A chromogenic assay of substrate depletion by thiol dioxygenases

A fast and easy method for enzyme activity assays using the chromogenic Ellman reagent, 5,5′-dithiobis(2-nitrobenzoic acid), was developed. The method was used to measure the activity of the nonheme mono-iron enzyme cysteine dioxygenase. Quantifying the depletion of the substrate, cysteine, allowed standard kinetic parameters to be determined for the enzyme from Rattus norvegicus. The assay was also used to quickly test the effects of ionic strength, pH, enzyme storage conditions, and potential inhibitors and activators. This assay facilitates a higher throughput than available HPLC-based assays, as it enjoys the advantages of fewer sample handling steps, implementation in a 96-well format, and speed. In addition, the relative specificity of Ellman’s reagent, coupled with its reaction with a wide range of thiols, means that this assay is applicable to many enzymes. Finally, the use of readily available reagents and instrumentation means that this assay can be used by practically any research group to compare results with those of other groups.     

Expression and localization of cysteine dioxygenase mRNA in the liver, lung, and kidney of the rat

Summary The expressions of cysteine dioxygenase (CDO) gene in the liver, lung, skeletal muscle, and kidney were studied byin situ hybridization with a cDNA probe from rat liver CDO under normal conditions. Significant expression of the CDO gene was detected in the liver, lung, and kidney, but not skeletal muscle. In the liver, the signal was confined to the cytoplasm of the hepatocytes. Furthermore, the signal was stronger in the periportal than that in the perivenous areas. In the lung, an intensive signal was found in the bronchiolar epithelium. As to the kidney, an intensive signal was observed in the distal convoluted tubules, while no signal was found in the proximal convultions.     

Production of a recombinant carotenoid cleavage dioxygenase from grape and enzyme assay in water-miscible organic solvents

A recombinant carotenoid cleavage dioxygenase from Vitis vinifera L. was produced by Escherichia coli as a fusion with the glutathione-S-transferase (GST) protein under different bacterial growth conditions. The enzyme production was monitored by a GST assay. Addition of Triton X-100 prior to bacterial cell disruption doubled the release of soluble protein. A simple spectrophotometric enzyme assay was developed to measure carotenoid cleavage activity using lutein as substrate. Enzyme activity showed a 26-fold increase with the addition of 10% (v/v) acetone in the reaction mixture.     

A capillary electrophoresis method to assay catalytic activity of botulinum neurotoxin serotypes: implications for substrate specificity

The potent botulinum neurotoxin inhibits neurotransmitter release at cholinergic nerve terminals, causing a descending flaccid paralysis characteristic of the disease botulism. The currently expanding medical use of the neurotoxin to treat several disorders, as well as the potential misuse of the neurotoxin as an agent in biowarfare, has made understanding of the nature of the toxin's catalytic activity and development of inhibitors critical. To study the catalytic activity of botulinum neurotoxin more thoroughly and characterize potential inhibitors, we have developed a capillary electrophoresis method to measure catalytic activity of different serotypes of botulinum neurotoxin using peptides derived from the native substrates. This assay requires only a minute amount of sample (25 nl), is relatively rapid (15 min/sample), and allows the determination of enzyme kinetic constants for a more sophisticated characterization of inhibitors and neurotoxin catalytic activity. Using this method, we can measure activity of five of the seven serotypes of botulinum neurotoxin (A, B, E, F, and G) with two peptide substrates. Botulinum neurotoxin serotypes C and D did not cleave our peptides, lending insight into potential substrate requirements among the serotypes.     

Homogeneous assay for real-time and simultaneous detection of thymidine kinase 1 and deoxycytidine kinase activities

Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37 °C. Complementation, for producing dCTP and TTP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5′ exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90 min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R² values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples.     

Effects of mitochondria and o-methoxybenzoylalanine on 3-hydroxyanthranilic acid dioxygenase activity and quinolinic acid synthesis

The use of o-methoxybenzoylalanine, a selective kynureninase inhibitor, has been proposed with the aim of reducing brain synthesis of quinolinic acid, an excitotoxic tryptophan metabolite. In liver homogenates, however, this compound caused unexpected accumulation of 3-hydroxyanthranilic acid, the product of kynureninase activity and the precursor of quinolinic acid. To explain this observation, we investigated the interaction(s) of o-methoxybenzoylalanine with 3-hydroxyanthranilic acid dioxygenase, the enzyme responsible for quinolinic acid formation. When the purified enzyme or partially purified cytosol preparations were used, o-methoxybenzoylalanine did not affect 3-hydroxyanthranilic acid dioxygenase activity. However, a significant reduction of this enzymatic activity did occur when o-methoxybenzoylalanine was tested in the presence of mitochondria. It is interesting that addition of purified mitochondria to 3-hydroxyanthranilic acid dioxygenase preparations reduced the enzymatic activity and the synthesis of quinolinic acid. In vivo, administration of o-methoxybenzoylalanine significantly reduced quinolinic acid synthesis and content in both blood and brain of mice. Our results suggest that mitochondrial protein(s) interact(s) with soluble 3-hydroxyanthranilic acid dioxygenase and cause(s) modifications in the enzyme resulting in a decrease in its activity. These modifications also allow the enzyme to interact with o-methoxybenzoylalanine, thus leading to a further reduction in quinolinic acid synthesis.     

Regulation of Escherichia coli IscS desulfurase activity by ferrous iron and cysteine

IscS plays a principal role in the synthesis of sulfur-containing biomolecules. It is known that the expression of iscS can be negatively regulated by IscR, the first gene product of iscRSUA-hscBA-fdx. What governs the regulation of cysteine desulfurase activity, however, is unknown. Here, we report that IscS from Escherichia coli is able to bind iron with an association constant of 1.6 × 10¹⁷ M⁻¹ to form an IscS-iron complex. IscS is also capable of binding both iron and sulfide to form an IscS-iron-sulfide complex with a higher affinity. The desulfurase activity is gradually inhibited as the amount of iron and sulfide bound to IscS increases. When 2Fe-2S binds IscS, about 20% of the activity is inhibited; when 8Fe-8S adheres to IscS, about 70% of the activity is inhibited. Thus, the cell is able to modulate its desulfurase activity with the formation of an IscS-iron-sulfide complex.     

Development and validation of a fluorogenic assay to measure separase enzyme activity

Separase, an endopeptidase, plays a pivotal role in the separation of sister chromatids at anaphase by cleaving its substrate cohesin Rad21. Recent study suggests that separase is an oncogene. Overexpression of separase induces aneuploidy and mammary tumorigenesis in mice. Separase is also overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma. Currently, there is no quantitative assay to measure separase enzymatic activity. To quantify separase enzymatic activity, we have designed a fluorogenic assay in which 7-amido-4-methyl coumaric acid (AMC)-conjugated Rad21 mitotic cleavage site peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) is used as the substrate of separase. We used this assay to quantify separase activity during cell cycle progression and in a panel of human tumor cell lines as well as leukemia patient samples.     

Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening

The role that heparanase plays during metastasis and angiogenesis in tumors makes it an attractive target for cancer therapeutics. Despite this enzyme’s significance, most of the assays developed to measure its activity are complex. Moreover, they usually rely on labeling variable preparations of the natural substrate heparan sulfate, making comparisons across studies precarious. To overcome these problems, we have developed a convenient assay based on the cleavage of the synthetic heparin oligosaccharide fondaparinux. The assay measures the appearance of the disaccharide product of heparanase-catalyzed fondaparinux cleavage colorimetrically using the tetrazolium salt WST-1. Because this assay has a homogeneous substrate with a single point of cleavage, the kinetics of the enzyme can be reliably characterized, giving a K_m of 46 μM and a k_cat of 3.5 s⁻¹ with fondaparinux as substrate. The inhibition of heparanase by the published inhibitor, PI-88, was also studied, and a K_i of 7.9 nM was determined. The simplicity and robustness of this method, should, not only greatly assist routine assay of heparanase activity but also could be adapted for high-throughput screening of compound libraries, with the data generated being directly comparable across studies.     

Comparative quantitation of abscisic acid in plant extracts by gas-liquid chromatography and an enzyme-linked immunosorbent assay using the avidin-biotin system

In this report we describe an enzyme-linked immunosorbent assay (ELISA) for the quantitation of abscisic acid (ABA) in plant extracts. A microtitration plate is coated with an ABA-protein complex. The ABA, standard or sample, is then added to each well with a limiting quantity of rabbit anti-ABA antibodies. During the following incubation period, antibodies bind either to free or to bound ABA on the plates. After washing, bound antibodies are indirectly labelled in two steps by the means of biotinylated goat antirabbit immunoglobulin-G antibodies which act as a link between rabbit anti-ABA antibodies and an avidin-alkaline phosphatase complex. The relative enzyme activity bound is measured spectrophotometrically. The detection limit of this method is 5 pg ABA and the measuring range extends to 10 ng. Gas-liquid-chromatography controls, with an electron capture detector, show a good correlation with ELISA results obtained using extracts of Lycopersicon esculentum, Nicotiana tabacum and Pseudotsuga menziesii samples purified by high-performance liquid chromatography. This provides a good argument for the accuracy of the immunoenzymatic method. The indirect labelling of antibodies, with the avidin-biotin amplifying system, should make this technique suitable for the quantitation of other plant growth substances against which specific antibodies are available.Abbreviations ABA abscisic acid - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - GLC gas liquid chromatography - HPLC high-performance liquid chromatography - IgG Immunoglobulin G - PBS phosphate-buffered saline     

Comparative characterization of direct and indirect substrate probes for on-chip transamidating activity assay of transglutaminases

The development of molecular probes is a prerequisite for activity-based protein profiling. This strategy helps in characterizing the catalytic activity and function of proteins, and how these proteins and protein complexes control biological processes of interest. These probes are composed of a reactive functional group and a reporter tag. The reactive group of these substrate probes has been considered to be important to their design, while the significance of the reporter tag is relatively underestimated. In this study we compare TAMRA-cadaverine and biotin-cadaverine, two substrate probes that have different reporter tags but an identical reactive functional group. We assess the on-chip transamidating activity of two transglutaminases; transglutaminase 2 and blood coagulation factor XIII. Activity assays were more easily executed when using the direct probe TAMRA-cadaverine. However the indirect probe, biotin-cadaverine, provided a wider dynamic range, higher signal-to-noise ratio, and lower limit of detection compared to TAMRA-cadaverine. Additionally, we successfully used the on-chip activity assay using the indirect probe to determine TG2 and FXIII activities in Hela cell lysates and human plasma samples, respectively. These results demonstrate that the reporter tag of the substrate probe is critical for protocol execution, sensitivity, and dynamic range of enzyme activity assays. Furthermore, this study provides a helpful guide for development of new probes, which is necessary for the identification of potential biomarkers and therapeutic targets for treating enzyme-related diseases.     

The differential reaction of histamine and N tau-methylhistamine with Pauly's diazo reagent: application to assay of histamine N-methyltransferase activity

A simple nonradioisotopic fluorescent method for assay of histamine N-methyltransferase (HMT) activity was developed. After termination of the HMT reaction, the remaining excess substrate, histamine, was degraded by Pauly 's diazo reagent, whereas the product, N tau-methylhistamine (N- MeHA ), was not degraded by the reagent. Then the mixture was applied to high-performance liquid chromatography under conditions in which N- MeHA was not separated from histamine, and N- MeHA was measured fluorometrically by condensation with o-phthalaldehyde. The method would be convenient for measurement of HMT activity during enzyme purification.     

Methods for rapid screening and isolation of bacteria producing acidic lipase: feasibility studies and novel activity assay protocols

Acidic lipase finds its commercial values in medical applications and bioremediation of food wastes. In this work, approaches for rapid screening of lipase-producing bacteria were developed and the feasibility assessment of the screening methods was performed. From food waste samples, the proposed screening procedures allowed isolation of sixteen pure bacterial strains expressing higher lipase activity at acidic pH (pH 6.0) than at alkaline pH (pH 9.0). To enhance the accuracy of lipase activity determination under acidic conditions, a novel assay procedure was also developed by deactivating lipase activity by microwave treatment prior to back titration. This additional step could minimize interferences arising from residual lipase activity during conventional direct back-titration methods in measuring lipase activity at acidic pH. Using the four strategies proposed in this work, the best acidic-lipase-producing isolate was obtained by strategy C (SSC) and was identified as Aeromonas sp. C14, displaying an optimal lipase activity of 0.7 U/ml at an acidic pH of 6.0.     

A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc-dependent metalloprotease bearing both an epoxide hydrolase, producing the pro-inflammatory LTB4 leukotriene, and an aminopeptidase activity, whose physiological relevance has long been ignored. Distinct substrates are commonly used for each activity, although none is completely satisfactory; LTA4, substrate for the hydrolase activity, is unstable and inactivates the enzyme, whereas aminoacids β-naphthylamide and para-nitroanilide, used as aminopeptidase substrates, are poor and nonselective. Based on the three-dimensional structure of LTA4H, we describe a new, specific, and high-affinity fluorigenic substrate, PL553 [l-(4-benzoyl)phenylalanyl-β-naphthylamide], with both in vitro and in vivo applications. PL553 possesses a catalytic efficiency (k_cat/K_m) of 3.8 ± 0.5 × 10⁴ M⁻¹ s⁻¹ using human recombinant LTA4H and a limit of detection and quantification of less than 1 to 2 ng. The PL553 assay was validated by measuring the inhibitory potency of known LTA4H inhibitors and used to characterize new specific amino-phosphinic inhibitors. The LTA4H inhibition measured with PL553 in mouse tissues, after intravenous administration of inhibitors, was also correlated with a reduction in LTB4 levels. This authenticates the assay as the first allowing the easy measurement of endogenous LTA4H activity and in vitro specific screening of new LTA4H inhibitors.     

Reaction of Trigonopsis variabilis d-amino acid oxidase with 2,6-dichloroindophenol: kinetic characterisation and development of an oxygen-independent assay of the enzyme activity

2,6-Dichloroindophenol (DCIP) is shown to be utilised efficiently as electron acceptor replacing dioxygen in the reaction of Trigonopsis variabilis d-amino acid oxidase (TvDAO) with d-methionine as the substrate. The specificity constant for DCIP reduction at 30 °C is one-twelfth that of oxygen conversion into hydrogen peroxide. Time course analysis of simultaneous consumption of DCIP and dioxygen, recorded on-line by absorption and non-invasive fluorescence quenching, respectively, pinpoints the preferential utilisation of dioxygen; and reveals a maximum DCIP conversion rate that is independent of the initial concentration of dioxygen. A robust direct assay of TvDAO activity has been developed that does not require anaerobic reaction conditions. It was down-scaled to microtitre plate format and overcomes practical limitations of other assays due to the low affinity of TvDAO for dioxygen (K_m ≈ 0.7 mmol L⁻¹).     

Unusual role of a cysteine residue in substrate binding and activity of human AP-endonuclease 1

The mammalian AP-endonuclease (APE1) repairs apurinic/apyrimidinic (AP) sites and strand breaks with 3′ blocks in the genome that are formed both endogenously and as intermediates during base excision repair. APE1 has an unrelated activity as a redox activator (and named Ref-1) for several trans-acting factors. In order to identify whether any of the seven cysteine residues in human APE1 affects its enzymatic function, we substituted these singly or multiply with serine. The repair activity is not affected in any of the mutants except those with C99S mutation. The Ser99-containing mutant lost affinity for DNA and its activity was inhibited by 10 mM Mg²⁺. However, the Ser99 mutant has normal activity in 2 mM Mg²⁺. Using crystallographic data and molecular dynamics simulation, we have provided a mechanistic basis for the altered properties of the C99S mutant. We earlier predicted that Mg²⁺, with potential binding sites A and B, binds at the B site of wild-type APE1-substrate complex and moves to the A site after cleavage occurs, as observed in the crystal structure. The APE1-substrate complex is stabilized by a H bond between His309 and the AP site. We now show that this bond is broken to destabilize the complex in the absence of the Mg²⁺. This effect due to the mutation of Cys99, ∼ 16 Å from the active site, on the DNA binding and activity is surprising. Mg²⁺ at the B site promotes stabilization of the C99S mutant complex. At higher Mg²⁺ concentration the A site is also filled, causing the B-site Mg²⁺ to shift together with the AP site. At the same time, the H bond between His309 and the AP site shifts toward the 5′ site of DNA. These shifts could explain the lower activity of the C99S mutant at higher [Mg²⁺]. The unexpected involvement of Cys99 in APE1's substrate binding and catalysis provides an example of involvement of a residue far from the active site.     

A newly developed assay for the quantitative determination of antimicrobial (anticyanobacterial) activity of both hydrophilic and lipophilic test compounds without any restriction

This paper reports about a newly developed assay which enables the user to quantify and compare the antimicrobial (anticyanobacterial) activity of both hydrophilic and lipophilic test compounds without any restriction. The assay is characterised by the application of a test compound solution on a porous matrix (silica gel) in well-defined concentrations per unit area, and by the coating of this matrix by a concentrated suspension of the tested living cyanobacterium (using a spraying or a dipping technique). The organism was only applied after the complete evaporation of the solvent used for preparation of the test compound solution to avert potential influences of this solvent on micro-organisms' viability. Toxic concentrations of a test compound caused a clearly contoured regional damage in the blue-green coloured uniform layer of the cyanobacterial test organism. The resulting regional decolourisation was readily identifiable within a species-dependent short time (e.g. for the species Arthrospira laxissima after 24h). This newly developed assay is applied for a patent in Germany.     

A high-throughput colorimetric assay to characterize the enzyme kinetic and cellular activity of spermidine/spermine N-acetyltransferase 1

Spermidine/spermine N¹-acetyltransferase 1 (SSAT1) is a key enzyme that catalyzes the catabolism of polyamines. SSAT1 is a very important enzyme because it not only maintains the homeostasis of polyamines but also is involved in many physiological and pathological events. As such, a rapid assay of SSAT1 activity is valuable in drug screening and clinical diagnostics. Here, we report a novel colorimetric assay for monitoring SSAT1 activity in zebrafish (zSSAT1). In comparison with the available SSAT1 assays, this new method is cost-effective and simple. The optimal zSSAT1 activity was obtained below 55 °C in a mild alkaline environment. The K_m values of zSSAT1 for spermidine and spermine are 55 and 182 μM, respectively, whereas putrescine is not a good substrate for zSSAT1. In addition to enzyme kinetic studies, the colorimetric assay was also used to detect the cellular activity of SSAT1. Thus, the current method is a reliable assay for determining SSAT1 activity with many potential applications in medical biology.     

Development of a simple and efficient method for assaying cytidine monophosphate sialic acid synthetase activity using an enzymatic reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide converting system

A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP+Sia-->CMP-Sia+pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia-->pyruvate+ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate+NADH-->lactate+oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay, relative activities toward Sia derivatives have been obtained. The preference of mouse CSS toward Neu5Ac and the ability of the rainbow trout enzyme to activate both KDN and Neu5Ac were confirmed. Thus, this simple and time-saving method is suitable for a systematic comparison of enzyme activity of structurally mutated enzymes based on the relative specific activity.