Characterization and molecular cloning of a glutathione S-transferase from the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)

Glutathione S-transferases (GST) catalyzing the conjugation of reduced glutathione to a vast range of xenobiotics including insecticides were characterized in the whitefly Bemisia tabaci. GST activities were determined in susceptible and resistant strains of B. tabaci towards artificial substrates, i.e. 1-chloro-2,4-dinitrobenzene (CDNB) in a photometric microplate assay and monochlorobimane (MCB) in a fluoroemtric microplate assay and characterized by their Michaelis-Menten kinetics. The inhibitory potential of ethacrynic acid was very effective with IC50-values between 0.9 and 5.8 microM depending on substrate and strain. The inhibitory effect of dicumarol was 10 times lower. Glutathione-affinity chromatography purified GST enzymes of two different B. tabaci strains appeared as a single band on SDS-PAGE and had a molecular mass of 23.5 kDa determined by MALDI mass spectrometry. The N-terminus of the purified enzyme was sequenced by Edman degradation. The nearly full-length cDNA of the enzyme was isolated by RT-PCR using a degenerate primer derived from the N-terminal amino acid sequence and contained an open reading frame encoding a 194-amino-acid protein. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to insect class sigma GSTs.     

微孔比色法在猪胰腺PLA_2制备中的应用

微孔比色法采用合成的磷脂类似物２－硫代十六酰乙基磷酸胆碱作底物,在多孔聚苯乙烯板的小孔中反应,并用酶联免疫检测器连续测定和记录吸收值．同时应用此法及滴定法检测酶活力,从猪胰腺中制备了一种分子量低（１４．３ｋＤ）,对热、酸稳定,活性依赖Ｃａ２＋的ＰＬＡ２．两种方法检测结果具有可比性,而微孔比色法同时可测多个样品,有节约样品,灵敏度较高等优点．微孔比色法特别适用于大量的样品测定,如拮抗剂筛选、临床样品及制备酶时层析级分的检测等．     

Microplate enzyme assay for screening lipoxygenases to degrade wood extractives

Lipoxygenases are non-heme iron-containing dioxygenases, capable of catalyzing the oxidation of unsaturated fatty acids. The enzyme has the potential to degrade problematic wood extractives in the paper-making process. However, commercially available lipoxygenase is currently too expensive for this application. A 96-well UV microplate assay was developed to screen enzymes from fungal sources for a more cost-effective alternative lipoxygenase. The substrate used for this assay was linoleic acid, a predominant fatty acid in wood. The enzyme activity and reaction kinetics determined by this microplate assay were compared to those obtained from a conventional bench scale assay. A number of hydrolytic enzymes and other oxidases were also tested using this protocol, to examine the specificity of the assay. The results show that the microplate assay developed can provide an inexpensive method for accelerated screening of a large number of enzymes to identify potential oxidative enzymes with specific action in degrading wood extractives.     

Microplate enzyme assay for screening lipoxygenases to degrade wood extractives

Lipoxygenases are non-heme iron-containing dioxygenases, capable of catalyzing the oxidation of unsaturated fatty acids. The enzyme has the potential to degrade problematic wood extractives in the paper-making process. However, commercially available lipoxygenase is currently too expensive for this application. A 96-well UV microplate assay was developed to screen enzymes from fungal sources for a more cost-effective alternative lipoxygenase. The substrate used for this assay was linoleic acid, a predominant fatty acid in wood. The enzyme activity and reaction kinetics determined by this microplate assay were compared to those obtained from a conventional bench scale assay. A number of hydrolytic enzymes and other oxidases were also tested using this protocol, to examine the specificity of the assay. The results show that the microplate assay developed can provide an inexpensive method for accelerated screening of a large number of enzymes to identify potential oxidative enzymes with specific action in degrading wood extractives.     

A rapid, quantitative microplate assay for NAD-linked d-mannitol dehydrogenase

A 96-well microtitre plate assay for NAD-linked D-mannitol dehydrogenase based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by reduced NAD is described. The assay allows rapid measurement of D-mannitol dehydrogenase in crude bacterial extracts derived by sonic disruption, in acetone permeabilized cells and in column eluates during enzyme purification. The absorbance of reaction mixtures in a microtitre plate is measured at 620 nm over a 3-4 min period using a programmable microplate reader. The rate of increase in absorbance is directly proportional to the amount of enzyme present and there is excellent correlation between activities derived using the microplate assay with those determined using conventional spectrophotometric methods.     

A new colorimetric assay for glutathione transferase-catalyzed halogen ion release for high-throughput screening

Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes catalyzing the conjugation of a broad range of toxicologically important halogenated compounds to the tripeptide glutathione (GSH) with concomitant halogen ion release. In the present work, a rapid quantitative screening method for GSTs based on colorimetric measurement of halogen ions released from halogenated xenobiotics was developed. The assay is based on the color formation resulting from the reaction of Hg(SCN)₂ with the released halogen ion of the substrate in the presence of Fe³⁺. The color intensity is proportional to the extent of the catalytic reaction, allowing a quantitative measurement of the GST catalytic activity. The assay can be performed using purified recombinant enzyme (the isoenzyme GmGSTU4-4 from Glycine max) or crude recombinant Escherichia coli cell lysates in 96-well microtiter plates. The suitability of the colorimetric assay for screening mutant GST variants derived from a directed evolution library was successfully evaluated. In addition, the assay was also used for screening GST synthetic inhibitors. It was concluded that the proposed colorimetric assay is selective and sensitive and allows the screening of large numbers of samples within a few minutes.     

Crude and purified proteasome activity assays are affected by type of microplate

Measurement of proteasome activity is fast becoming a commonly used assay in many laboratories. The most common method to measure proteasome activity involves measuring the release of fluorescent tags from peptide substrates in black microplates. Comparisons of black plates used for measuring fluorescence with different properties show that the microplate properties significantly affect the measured activities of the proteasome. The microplate that gave the highest reading of trypsin-like activity of the purified 20S proteasome gave the lowest reading of chymotrypsin-like activity of the 20S proteasome. Plates with medium binding surfaces from two different companies showed an approximately 2-fold difference in caspase-like activity for purified 20S proteasomes. Even standard curves generated using free 7-amino-4-methylcoumarin (AMC) were affected by the microplate used. As such, significantly different proteasome activities, as measured in nmol AMC released/mg/min, were obtained for purified 20S proteasomes as well as crude heart and liver samples when using different microplates. The naturally occurring molecule betulinic acid activated the chymotrypsin-like proteasome activity in three different plates but did not affect the proteasome activity in the nonbinding surface microplate. These findings suggest that the type of proteasome activity being measured and sample type are important when selecting a microplate.     

SOX11结合p53并上调其转录活性

目的:研究SOX11对p53转录活性的影响,并检测二者的体外相互作用。方法:在H1299(p53缺失)和H460(含野生型p53)2种细胞中分别过表达SOX11和p53,用双萤光素酶方法测定p53的转录活性;用大肠杆菌DH5α表达GST和GST-p53融合蛋白并将其纯化,用GST pull-down实验检测SOX11与p53在体外是否存在相互作用。结果:萤光素酶实验结果表明,在H1299和H460细胞中,过表达SOX11分别能促进外源p53和内源p53的转录活性;GST pull-down实验表明SOX11能在体外与p53发生相互作用。结论:SOX11能在体外与p53发生相互作用并促进p53的转录活性,为进一步研究p53的功能提供了新的线索。     

High-throughput mechanobiology: Force modulation of ensemble biochemical and cell-based assays

Mechanobiology is focused on how the physical forces and mechanical properties of proteins, cells, and tissues contribute to physiology and disease. Although the response of proteins and cells to mechanical stimuli is critical for function, the tools to probe these activities are typically restricted to single-molecule manipulations. Here, we have developed a novel microplate reader assay to encompass mechanical measurements with ensemble biochemical and cellular assays, using a microplate lid modified with magnets. This configuration enables multiple static magnetic tweezers to function simultaneously across the microplate, thereby greatly increasing throughput. We demonstrate the broad applicability and versatility through in vitro and in cellulo approaches. Overall, our methodology allows, for the first time (to our knowledge), ensemble biochemical and cell-based assays to be performed under force in high-throughput format. This approach substantially increases the availability of mechanobiology measurements.     

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase ^{1, 2}. Manual FV assays have been described ^{3, 4}, but they are time consuming and subjective. Automated FV assays have been reported ^5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput ^{8, 9}. Microplate assays have been reported for clot lysis ¹⁰, platelet aggregation ¹¹, and coagulation Factors ¹², but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) ¹³. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections ¹⁴. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology ¹⁵. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.     

Toxicity,inheritance and biochemistry of clofentezine resistance in Tetranychus urticae

Abstract This study evaluates the toxicological and biochemical response of two‐spotted spider mites to clofentezine selection pressure. The mortality rate of Tetranychus urticae in adult females depends on increased clofentezine concentration and clofentezine was found to be effective against females. The resistance rate of the CUM strain selected 12 times once per generation with clofentezine was increased from 1.28‐ to 105.27‐fold. The interaction of some synergists with clofentezine was analyzed in the clofentezine‐resistant CLOF 12 strain. Synergists had no effect on clofentezine toxicity. The clofentezine‐resistant CLOF 12 strain showed resistance against chlorpyrifos, abamectin, propargite, fenpyroximate and amitraz. The modes of inheritance of resistance to clofentezine were found to be incompletely dominant and not sex‐linked. Esterase enzyme activity was detected both by gel electrophoresis and microplate reader methods, while glutathione S‐transferase (GST) and monooxygenase (P450) activity were detected only by the microplate reader method. During the selection period the esterase, the GST and the P450 enzymes activities were raised from 7.69, 7.09 and 0.003 3 to 18.40, 13.11 and 0.003 7 milli‐optical density/min/mg proteins, respectively. An increase was observed in the band intensity of esterases and esterase enzymes may play a role in clofentezine resistance in T. urticae.     

Optimization of a phagocyte microplate chemiluminescent assay

Chemiluminescent assays have been used to quantify phagocytic activity since 1972. In recent years these assays have been adapted to the 96-well microplate format as new luminometers have been developed. In this report we describe the optimization of a lucugenin enhanced phagocyte chemiluminescent assay using a Titertek Luminoskan. Factors such as cell concentration, serum concentration in the opsonization of the zymosan used and lucigenin concentration were all optimized in our assay. In addition we have found that some of the unique features of the Luminoskan, continuous microplate agitation during the assay and microplate temperature control up to 43°C, also significantly enhanced the chemiluminescent response.     

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.     

Coupling RNA annealing and strand displacement: a FRET-based microplate reader assay for RNA chaperone activity

Proteins with RNA chaperone activity help RNAs to obtain their native conformations, and many of them are active in the two basic reactions-RNA annealing and strand displacement. Therefore, we developed a time-saving in vitro assay that detects protein-facilitated annealing and strand displacement of fluorophore-labeled oligoribonucleotides in a microplate reader The two reactions are followed byfluorescence resonance energy transfer (FRET) in real-time, and the effect of the proteins on the reaction constants can be quantified. The high-throughput property of the fluorescence microplate reader the kinetic characterization, and the material-saving aspect of this assay enables a fast and convenient classification of proteins according to their RNA chaperone activity in annealing and strand displacement.     

A fluorimetric assay for on-line detection of DNA cleavage by restriction endonucleases

We have developed an assay for online detection of DNA cleavage by restriction endonucleases, suitable for the high throughput screening of the activity and flanking sequence preference of restriction endonuclease variants. For this purpose oligodeoxynucleotides were used, labeled with either 6-FAM or TAMRA whose fluorescence is quenched by a neighboring DABCYL group. After endonucleolytic cleavage the products are too short to remain double-stranded and the fluorophor labeled strand is released with concomitant increase in fluorescence which can be easily quantified. Employing this method, cleavage reactions can be monitored continuously, allowing for fast detection of specific activity as well as determination of kinetic parameters. To demonstrate the reliability of our assay we measured K(M) and k(cat) values for the restriction endonuclease EcoRV and obtained results similar to those obtained with established assays. Moreover, our method makes it possible to observe the cleavage of two different substrates differing in the sequences flanking the EcoRV site and labeled with different fluorophors in competition in a single experiment. This assay can be carried out in a microplate format, which allows for the analysis of many restriction endonuclease variants in parallel.     

Measurement of chemiluminescence from neutrophils in a 96-well microplate using Lumi Box U-800 II

We have developed a microplate photon counting system based on a cooled charge-coupled device (Lumi Box U-800 II) jointly with Maikurotekku Nition Company (Chiba, Japan). The system makes it possible to quantify chemiluminescence (CL) in a 96-well microplate automatically and simultaneously in a single experiment. We studied the measurement conditions for a luminol-dependent CL assay from neutrophils stimulated with opsonized zymosan (OZ) using this system. Parameters examined included the effect of OZ dose per well, mixing speed, mixing time and detection time on CL responses. The results indicated that this system allows the measurement of CL from phagocytes on a large number of samples using small amounts of sample and regents. © 1997 John Wiley & Sons, Ltd.     

钩虾胆碱酯酶（ChE）和谷胱甘肽转硫酶（GST）的敏感性和特异性比较研究

研究了有机磷农药甲地基嘧啶流磷,有机氯农药林丹,菊酯类农药氯菊酯,表面活性剂直链苯磺酸钠和重金属Zn对钩虾（Gammarus pulex L.)胆碱酯酶（ChE）和谷胱甘肽转硫酶（GST）活性变化以及毒性影响,结果表明,在暴露24h和48h后,仅有机磷农药甲基嘧硫磷显著抑制胆碱酯的酶的活性,在暴露48h后,有机氯农药林丹和菊酯类农药氯菊酯能显著提高谷胱甘肽转硫酶活性,在暴露24h后,仅梵在导致谷胱甘肽转硫酶明显升高,作为生物标志物,胆碱酯酶比谷胱甘肽转硫酶具有更高的特异性,这两种生物标志物较毒性试验方法具有更高的敏感性。     

New Insights into Butyrylcholinesterase Activity Assay: Serum Dilution Factor as a Crucial Parameter

Butyrylcholinesterase (BChE) activity assay and inhibitor phenotyping can help to identify patients at risk of prolonged paralysis following the administration of neuromuscular blocking agents. The assay plays an important role in clinical chemistry as a good diagnostic marker for intoxication with pesticides and nerve agents. Furthermore, the assay is also commonly used for in vitro characterization of cholinesterases, their toxins and drugs. There is still lack of standardized procedure for measurement of BChE activity and many laboratories use different substrates at various concentrations. The purpose of this study was to validate the BChE activity assay to determine the best dilution of human serum and the most optimal concentration of substrates and inhibitors. Serum BChE activity was measured using modified Ellman’s method applicable for a microplate reader. We present our experience and new insights into the protocol for high-throughput routine assays of human plasma cholinesterase activities adapted to a microplate reader. During our routine assays used for the determination of BChE activity, we have observed that serum dilution factor influences the results obtained. We show that a 400-fold dilution of serum and 5mM S-butyrylthiocholine iodide can be successfully used for the accurate measurement of BChE activity in human serum. We also discuss usage of various concentrations of dibucaine and fluoride in BChE phenotyping. This study indicates that some factors of such a multicomponent clinical material like serum can influence kinetic parameters of the BChE. The observed inhibitory effect is dependent on serum dilution factor used in the assay.     

A high-throughput colorimetric assay to measure the activity of glutamate decarboxylase

A pH-sensitive colorimetric assay has been established to quantitatively measure glutamate decarboxylase (GAD) activity in bacterial cell extracts using a microplate format. GAD catalyzes the irreversible α-decarboxylation of l-glutamate to γ-aminobutyrate. The assay is based on the color change of bromocresol green due to an increase in pH as protons are consumed during the enzyme-catalyzed reaction. Bromocresol green was chosen as the indicator because it has a similar pK_a to the acetate buffer used. The corresponding absorbance change at 620 nm was recorded with a microplate reader as the reaction proceeded. A difference in the enzyme preparation pH and optimal pH for GAD activity of 2.5 did not prevent this method from successfully allowing the determination of reaction kinetic parameters and the detection of improvements in enzymatic activity with a low coefficient of variance. Our assay is simple, rapid, requires minimal sample concentration and can be carried out in robotic high-throughput devices used as standard in directed evolution experiments. In addition, it is also applicable to other reactions that involve a change in pH.