A continuous spectrophotometric enzyme-coupled assay for deoxynucleoside triphosphate triphosphohydrolases

We describe a continuous, spectrophotometric, enzyme-coupled assay useful to monitor reactions catalyzed by nucleoside triphosphohydrolases. In particular, using Escherichia coli deoxynucleoside triphosphohydrolase (Dgt), which hydrolyzes dGTP to deoxyguanosine and tripolyphosphate (PPP_i) as the enzyme to be tested, we devised a procedure relying on purine nucleoside phosphorylase (PNPase) and xanthine oxidase (XOD) as the auxiliary enzymes. The deoxyguanosine released by Dgt can indeed be conveniently subjected to phosphorolysis by PNPase, yielding deoxyribose-1-phosphate and guanine, which in turn can be oxidized to 8-oxoguanine by XOD. By this means, it was possible to continuously detect Dgt activity at 297 nm, at which wavelength the difference between the molar extinction coefficients of 8-oxoguanine (8000 M⁻¹ cm⁻¹) and guanine (1090 M⁻¹ cm⁻¹) is maximal. The initial velocities of Dgt-catalyzed reactions were then determined in parallel with the enzyme-coupled assay and with a discontinuous high-performance liquid chromatography (HPLC) method able to selectively detect deoxyguanosine. Under appropriate conditions of excess auxiliary enzymes, the activities determined with our continuous enzyme-coupled assay were quantitatively comparable to those observed with the HPLC method. Moreover, the enzyme-coupled assay proved to be more sensitive than the chromatographic procedure, permitting reliable detection of Dgt activity at low dGTP substrate concentrations.     

A dual-step fluorescence resonance energy transfer-based quenching assay for screening of caspase-3 inhibitors

The control of cell death is an intricate process involving a multitude of intracellular modulators. Among these molecules, the caspases have a central role and have become an interesting group of enzymes in the current pharmaceutical industry. We have developed a novel dual-step fluorescence energy transfer-based separation-free assay method for the primary screening of caspase-3 inhibitors in vitro. This method relies on fluorescent europium(III)-chelate-doped nanoparticle donors coated with streptavidin in conjunction with a dual-labeled (N-terminal Alexa Fluor 680 fluorescent acceptor and C-terminal BlackBerry Quencher 650) caspase-3-specific peptide substrate modified with a biotinyl moiety. In the assay, the nanoparticle donor excites the fluorescent acceptor, whose emission is monitored with time-resolved measurements. The intensity of the acceptor reflects the activity of the enzyme because the intensity is controlled by the proximity of the quencher. Owing to the dual-step fluorescence resonance energy transfer, this method enables a sensitized fluorescence signal directly proportional to the extent of enzymatic activity with relatively background fluorescence-free measurements in the event of complete enzyme inhibition. The generic nanoparticle donors further promote versatility and cost-efficiency of the method. The performance evaluated as the inhibitor (Z-DEVD-FMK) dose-response curve (IC(50) value of approximately 12 nM) was in good agreement with that of the recent methods found in literature. This assay serves as a model application proving the feasibility of the europium-chelate-doped nanoparticle labels in a homogeneous assay for proteolytic activity.     

Evaluating the utility of the HTRF Transcreener ADP assay technology: A comparison with the standard HTRF assay technology

The HTRF (homogeneous time-resolved fluorescence) Transcreener ADP assay is a new kinase assay technology marketed by Cis-Bio International (Bagnols-Cèze, France). It measures kinase activity by detecting the formation of ADP using a monoclonal antibody and HTRF detection principles. In this article, we compare this technology with a standard HTRF kinase assay using EGFR [L858R/T790M] mutant enzyme as a case study. We demonstrate that the HTRF Transcreener ADP assay generated similar kinetic constants and inhibitor potency compared with the standard HTRF assay. However, the smaller dynamic window and lower Z′ factor of the HTRF Transcreener ADP assay make this format less preferable for high-throughput screening. Based on the assay principle, the HTRF Transcreener ADP assay can detect both kinase and ATPase activities simultaneously. The ability to probe ATPase activity opens up new avenues for assaying kinases with intrinsic ATPase activity without the need to identify substrates, and this can speed up the drug discovery process. However, caution must be exercised because any contaminating ATPase activity will result in an invalid assay. The inability to tolerate high concentrations of ATP in the assay will also limit the application of this technology, especially in compound mechanistic studies such as ATP competition. Overall, the HTRF Transcreener ADP assay provides a new alternative tool to complement existing assay technologies for drug discovery.     

Single-label time-resolved luminescence assay for estrogen receptor--ligand binding

Homogeneous luminescence-based microplate assays are desirable in high-throughput screening of new nuclear receptor regulators. Time-resolved fluorescence resonance energy transfer (TR–FRET) assays provide high sensitivity due to low background signal. The TR–FRET concept requires labeling of both ligand and receptor, making the assay format and its development relatively expensive and complex compared with single-label methods. To overcome the limitations of the multilabel methods, we have developed a single-label method for estrogen receptor (ER)–ligand binding based on quenching resonance energy transfer (QRET), where estradiol labeled with luminescent europium(III) chelate (Eu–E₂) is quenched using soluble quencher molecules. The luminescence signal of Eu–E₂ on binding to full-length ER is protected from quenching while increasing competitor concentrations displace Eu–E₂ from the receptor, reducing the signal. The QRET method was paralleled with a commercial fluorescence polarization (FP) assay. The measured signal-to-background (S/B) values for estradiol, estrone, fulvestrant, and tamoxifen obtained for the QRET assay (5.8–9.2) were clearly higher than the S/B values for the FP assay (1.3–1.5). A K_d value of 30 nM was calculated for binding of Eu–E₂ to ER from a saturation binding isotherm. The QRET method provides an attractive new single-label assay format for nuclear receptor ligand screening.     

A fluorescence-based glycosyltransferase assay for high-throughput screening

Glycosyltransferases catalyze the transfer of a monosaccharide unit from a nucleotide or lipid sugar donor to polysaccharides, lipids, and proteins in a stereospecific manner. Considerable effort has been invested in engineering glycosyltransferases to diversify sugar-containing drugs. An important requirement for glycosyltransferase engineering is the availability of a glycosyltransferase assay system for high-throughput screening of glycosyltransferase mutants. In this study, a general glycosyltransferase assay system was developed based on an ATP sensor. This system showed submicromolar sensitivity and compatibility with both purified enzymes and crude cell extracts. The assay system will be useful for glycosyltransferase engineering based on high-throughput screening, as well as for general glycosyltransferase assays and kinetics.     

Design of fluorogenic substrates for continuous assay of sialyltransferase by resonance energy transfer

Glycosyltransferases are important synthetic enzymes for the construction of naturally occurring glycoconjugates as well as for the design of neoglycoconjugates. The assay methods currently available for these enzymes require tedious and time-consuming procedures for separation of products and do not permit continual assay of enzyme activities. As a set of convenient fluorogenic substrates for continuous monitoring of sialyltransferase activities, we designed and synthesized a novel CMP-Neu5Ac derivative with a naphthylmethyl group at the C-9 position and N-acetyllactosamine derivative containing a dansyl group at the terminal position of aglycon. In such substrates, the emission peak of the naphthylmethyl group (lambdaem = 340 nm) of the glycosyl donor is successfully overlapped with the excitation peak due to the dansyl group (lambdaex = 335 nm) of the glycosyl acceptor. A coupling reaction of these two substrates catalyzed by rat liver 2,6-sialyltransferase caused an increase of dansyl fluorescence (lambdaem = 525 nm) and a decrease of naphthylmethyl fluorescence on the basis of resonance energy transfer between two fluorescence probes. The substrates presented here permit continuous fluorescent monitoring of enzymatic sugar combining reactions. Actually, using this time course of enzymatic reactions, kinetic constants of rat liver 2,6-sialyltransferase against glycosyl donor substrates were estimated to be Km = 4.85 microM and Vmax. = 0.119 micromol/min, respectively. This strategy allows precise and efficient analyses of enzyme kinetics not possible with the conventional assay methods for the glycosyltransferases that usually require separation of products from the reaction mixture.     

Glycosyltransferases:key players involved in the modification of plant secondary metabolites

Glycosyltransferases are members of the multigene superfamily in plants that can transfer single or multiple activated sugars to a range of plant molecules,resulting in the glycosylation of plant compounds.Although the activities of many glycosyltransferases and their products have been recognized for a long time,only in recent years were some glycosyltransferase genes identified and a few functionally characterized in detail.Glycosylation is thought to be one of the most important modification reactions towards plant secondary metabolites,and plays a key role in maintaining cell homeostasis,thus likely participating in the regulation of plant growth,development and in defense responses to stress environments.With advances in plant genome projects and the development of novel technologies in analyzing gene function,significant progress could be made in gaining new insights into the properties and precise biological roles of plant secondary product glycosyltransferases,and the new knowledge will have extensive application prospects in the catalytic synthesis of glycoconjugates and metabolic engineering of crops.In this review,we summarize the current research,highlighting the possible biological roles,of plant secondary metabolite glycosyltransferases and discuss their potential applications as well as aspects to be further studied in the near future.     

Glycosyltransferases: key players involved in the modification of plant secondary metabolites

Glycosyltransferases are members of the multigene superfamily in plants that can transfer single or multiple activated sugars to a range of plant molecules, resulting in the glycosylation of plant compounds. Although the activities of many glycosyltransferases and their products have been recognized for a long time, only in recent years were some glycosyltransferase genes identified and a few functionally characterized in detail. Glycosylation is thought to be one of the most important modification reactions towards plant secondary metabolites, and plays a key role in maintaining cell homeostasis, thus likely participating in the regulation of plant growth, development and in defense responses to stress environments. With advances in plant genome projects and the development of novel technologies in analyzing gene function, significant progress could be made in gaining new insights into the properties and precise biological roles of plant secondary product glycosyltransferases, and the new knowledge will have extensive application prospects in the catalytic synthesis of glycoconjugates and metabolic engineering of crops. In this review, we summarize the current research, highlighting the possible biological roles, of plant secondary metabolite glycosyltransferases and discuss their potential applications as well as aspects to be further studied in the near future.     

Nanoliter scale PCR with TaqMan detection.

We monitored PCR in volumes of the order of 10 nl in glass microcapillaries using a fluorescence energy transfer assay in which fluorescence increases if product is made due to template-dependent nucleolytic degradation of an internally quenched probe (TaqMan assay). This assay detected single starting template molecules in dilutions of genomic DNA. The results suggest that it may be feasible to determine the number of template molecules in a sample by counting the number of positive PCRs in a set of replicate reactions using terminally diluted sample. Since the assay system is closed and potentially automatable, it has promise for clinical applications.     

Studies on the anaerobic degradation of crystalline cellulose by Clostridium thermocellum using a new assay

Abstract The anaerobic degradation of microcrystalline cellulose by thermostable cellulolytic enzyme complexes from Clostridium thermocellum JW20 (ATCC 31449) was monitored. For quantitative investigations as enzyme-coupled spectrophotometric assay has been developed. The assay allows for the evaluation of the release of cellubiose-/glucose-units from native cellulose. Kinetic studies revealed that the anaerobic breakdown of crystalline cellulose (CC) at 60°C follows Michaelis-Menten kinetics K _m CC values have been determined for different aggregation states of the cellulolytic complex. The presented assay seems well suited to screen for CC-degrading enzymes of various sources, and to further explore the mechanism of CC-breakdown.     

Conserved domains of glycosyltransferases.

Glycosyltransferases catalyze the synthesis of glycoconjugates by transferring a properly activated sugar residue to an appropriate acceptor molecule or aglycone for chain initiation and elongation. The acceptor can be a lipid, a protein, a heterocyclic compound, or another carbohydrate residue. A catalytic reaction is believed to involve the recognition of both the donor and acceptor by suitable domains, as well as the catalytic site of the enzyme. To elucidate the structural requirements for substrate recognition and catalytic reactions of glycosyltransferases, we have searched the databases for homologous sequences, identified conserved amino acid residues, and proposed potential domain motifs for these enzymes. Depending on the configuration of the anomeric functional group of the glycosyl donor molecule and of the resulting glycoconjugate, all known glycosyltransferases can be divided into two major types: retaining glycosyltransferases, which transfer sugar residue with the retention of anomeric configuration, and inverting glycosyltransferases, which transfer sugar residue with the inversion of anomeric configuration. One conserved domain of the inverting glycosyltransferases identified in the database is responsible for the recognition of a pyrimidine nucleotide, which is either the UDP or the TDP portion of a donor sugar-nucleotide molecule. This domain is termed "Nucleotide Recognition Domain 1 beta," or NRD1 beta, since the type of nucleotide is the only common structure among the sugar donors and acceptors. NRD1 beta is present in 140 glycosyltransferases. The central portion of the NRD1 beta domain is very similar to the domain that is present in one family of retaining glycosyltransferases. This family is termed NRD1 alpha to designate the similarity and stereochemistry of sugar transfer, and it consists of 77 glycosyltransferases identified thus far. In the central portion there is a homologous region for these two families and this region probably has a catalytic function. A third conserved domain is found exclusively in membrane-bound glycosyltransferases and is termed NRD2; this domain is present in 98 glycosyltransferases. All three identified NRDs are present in archaebacterial, eubacterial, viral, and eukaryotic glycosyltransferases. The present article presents the alignment of conserved NRD domains and also presents a brief overview of the analyzed glycosyltransferases which comprise about 65% of all known sugar-nucleotide dependent (Leloir-type) and putative glycosyltransferases in different databases. A potential mechanism for the catalytic reaction is also proposed. This proposed mechanism should facilitate the design of experiments to elucidate the regulatory mechanisms of glycosylation reactions. Amino acid sequence information within the conserved domain may be utilized to design degenerate primers for identifying DNA encoding new glycosyltransferases.     

Immunocapture and microplate-based activity measurement of mammalian pyruvate dehydrogenase complex

Altered pyruvate dehydrogenase (PDH) functioning occurs in primary PDH deficiencies and in diabetes, starvation, sepsis, and possibly Alzheimer's disease. Currently, the activity of the enzyme complex is difficult to measure in a rapid high-throughput format. Here we describe the use of a monoclonal antibody raised against the E2 subunit to immunocapture the intact PDH complex still active when bound to 96-well plates. Enzyme turnover was measured by following NADH production spectrophotometrically or by a fluorescence assay on mitochondrial protein preparations in the range of 0.4 to 5.0 micro g per well. Activity is sensitive to known PDH inhibitors and remains regulated by phosphorylation and dephosphorylation after immunopurification because of the presence of bound PDH kinase(s) and phosphatase(s). It is shown that the immunocapture assay can be used to detect PDH deficiency in cell extracts of cultured fibroblasts from patients, making it useful in patient screens, as well as in the high-throughput format for discovery of new modulators of PDH functioning.     

Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores

Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3′ ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5′ phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5′ extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido–pyrimidine–DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions).     

High-throughput, homogeneous, fluorescence intensity-based measurement of adenosine diphosphate and other ribonucleoside diphosphates with nanomolar sensitivity

A new, homogeneous, high-throughput-compatible assay method is described for the fluorescence-based quantitation of nanomolar concentrations of ribonucleoside diphosphates (rNDPs). The principle of the method is the conversion of the rNDPs to RNA by the enzyme polynucleotide phosphorylase (EC 2.7.7.8) and detection of the RNA by the increased fluorescence of a commercial nucleic acid detection dye. A commercial RNA homopolymer complementary to the RNA product is included to increase the sensitivity for ADP and UDP. Standard curves for nanomolar concentrations of ADP, UDP, GDP, and CDP are shown. The assay detected 75 nM ADP produced by the pyruvate kinase-catalyzed phosphorylation of pyruvate with a signal-to-baseline ratio of 2.8. The assay may be used in either a continuous or a discontinuous mode.     

Potent γ-secretase inhibitors/modulators interact with amyloid-β fibrils but do not inhibit fibrillation: A high-resolution NMR study

Recently, γ-secretase modulators (GSM) have been shown to interact directly with the amyloid precursor protein (APP) and simultaneously inhibit the activity of the Presenilin domain of γ-secretase. A clear understanding of the molecular recognition pathways by which GSM can target both γ-secretase and Aβ precursor protein can lead to the development of more effective inhibitors. To examine whether this direct interaction with APP affects the downstream Aβ fibril formation, we chose to investigate three different molecules in this study: Sulindac sulfide, Semagacestat and E2012 from the class of generation I GSMs, γ-secretase inhibitors (GSI), and generation II GSM molecules, respectively. Firstly, through NMR based ligand titration, we identified that Sulindac sulfide and Semagacestat interact strongly with Aβ40 monomers, whereas E2012 does not. Secondly, using saturation transfer difference (STD) NMR experiments, we found that all three molecules bind equally well with Aβ40 fibrils. To determine if these interactions with the monomer/fibril lead to a viable inhibition of the fibrillation process, we designed an NMR based time-dependent assay and accurately distinguished the inhibitors from the non-inhibitors within a short period of 12 h. Based on this pre-seeded fibril assay, we conclude that none of these molecules inhibit the ongoing fibrillation, rather ligands such as Semagacestat and E2012 accelerated the rate of aggregation.     

An enzyme-coupled assay measuring acetate production for profiling histone deacetylase specificity

Histone deacetylases catalyze the hydrolysis of an acetyl group from post-translationally modified acetyl-lysine residues in a wide variety of essential cellular proteins, including histones. Because these lysine modifications can alter the activity and properties of affected proteins, aberrant acetylation/deacetylation may contribute to disease states. Many fundamental questions regarding the substrate specificity and regulation of these enzymes have yet to be answered. Here, we optimize an enzyme-coupled assay to measure low micromolar concentrations of acetate, coupling acetate production to the formation of NADH (nicotinamide adenine dinucleotide, reduced form) that is measured by changes in either absorbance or fluorescence. Using this assay, we measured the steady-state kinetics of peptides representing the H4 histone tail and demonstrate that a C-terminally conjugated methylcoumarin enhances the catalytic efficiency of deacetylation catalyzed by cobalt(II)-bound histone deacetylase 8 [Co(II)–HDAC8] compared with peptide substrates containing a C-terminal carboxylate, amide, and tryptophan by 50-, 2.8-, and 2.3-fold, respectively. This assay can be adapted for a high-throughput screening format to identify HDAC substrates and inhibitors.     

Assays for mechanistic investigations of protein/histone acetyltransferases

Protein/histone acetyltransferases (PATs/HATs) have been implicated in a number of cellular functions including gene regulation, DNA synthesis, and repair. This paper reviews methods that can be used to quantitatively determine the activity and ultimately the catalytic/kinetic mechanism of PAT/HATs in vitro. Two methods will be described in detail. The first method is a filter-binding assay that measures the transfer of radiolabeled acetate from acetyl-CoA to protein. The second method is a continuous, spectroscopic, enzyme-coupled assay that links the PAT/HAT reaction to the reduction of NAD+ by pyruvate or alpha-ketoglutarate dehydrogenase. Both methods are highly applicable in determining steady-state reaction rates, and obtaining the kinetic constants Vmax, Km, and V/K from substrate saturation curves. We describe a new application of the filter-binding assay to determine the kinetic parameters for HATs using low concentrations of nucleosomal substrates.     

Homogeneous TR-FRET high-throughput screening assay for calcium-dependent multimerization of sorcin

A homogeneous high-throughput screening method based on time-resolved fluorescence resonance energy transfer (TR-FRET) for the measurement of calcium-dependent multimerization of an EF-hand protein, sorcin, is described. The assay is based on a specific sorcin binding peptide conjugated either with an intrinsically highly fluorescent europium chelate (donor) or an Alexa Fluor 700 fluorophore (acceptor). Addition of calcium results in multimerization of sorcin, allowing several peptides to bind simultaneously to the epitopes of the multimeric protein complex, and the proximity of peptides labeled either with donor or acceptor label results in fluorescence resonance energy transfer between the 2 labels. When no calcium is present, the protein remains in a monomer form, and thus no FRET can take place. In the optimized assay construct, the assay was performed in 45 min, and a more than 20-fold signal-to-background ratio was achieved. The reversibility of sorcin multimerization was shown by chelating free calcium with ethylenediamine tetraacetic acid (EDTA). The developed homogeneous assay can be used in screening molecules that either inhibit or enhance multimerization of sorcin, and the assay format is applicable to various noncompetitive high-throughput screening assays detecting protein multimerization reactions.     

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.