Application of the MultiScreen system to cytokine radioreceptor assays

Radioreceptor assays are becoming increasingly valuable in the biotechnology community for a variety of basic and applied research applications. It is clear, for example, that assessing the potential spectrum of biological activities of a novel polypeptide regulatory factor can be greatly simplified by the development of a rapid radioreceptor assay, since a wide variety of cell types can be screened using a single type of assay. By contrast, searching for potentially diverse biological effects can be an extremely time-consuming process. In addition, screening for agonists/antagonists for hormones using radioreceptor assays has a marked advantage compared with biological assays, in that compounds or natural products that are toxic to cells will not read out as false positives in a binding assay. Our laboratory has developed a major program centered on the molecular characterization of receptors for polypeptide hormones involved in immune regulation, including a number of cytokines/interleukins and also several colony stimulating factors. We have developed a variety of radioreceptor- and fluorescence-based assay systems for ligand-receptor interactions, with applications in basic characterization, purification, cDNA cloning, and drug development screens for cytokine receptors. In this report we compare two assay formats, a standard phthalate oil centrifugation method and a novel plate filtration system, using the interaction between interleukin-1 alpha and its receptor as a test system.     

A real-time DNase assay (ReDA) based on PicoGreen fluorescence

DNA nucleases (DNases) perform a wide variety of important cellular functions and are also very useful for research and in biotechnological applications. Due to the biological and technological importance of DNases and their use in a wide range of applications, DNase activity assays are essential. Traditional DNase assays employ radiolabeled DNA substrates and require separation of the products of the reaction from the unreacted substrate before quantification of enzyme activity. As a consequence, these methods are discontinuous. In this report, we describe a continuous DNase assay based on the differential fluorescence output of a DNA dye ligand called PicoGreen. The assay was developed to characterize a processive dsDNA exonuclease, lambda exonuclease. The assay appears to have general utility as it is also suitable for measuring the DNA digestion activities of a processive helicase/nuclease, RecBCD, a distributive exonuclease, T7 gene 6 exonuclease, and an endonuclease, DNaseI. The benefits of, and limitations to, the method are discussed.     

A real-time DNase assay (ReDA) based on PicoGreen® fluorescence

DNA nucleases (DNases) perform a wide variety of important cellular functions and are also very useful for research and in biotechnological applications. Due to the biological and technological importance of DNases and their use in a wide range of applications, DNase activity assays are essential. Traditional DNase assays employ radiolabeled DNA substrates and require separation of the products of the reaction from the unreacted substrate before quantification of enzyme activity. As a consequence, these methods are discontinuous. In this report, we describe a continuous DNase assay based on the differential fluorescence output of a DNA dye ligand called PicoGreen®. The assay was developed to characterize a processive dsDNA exonuclease, lambda exonuclease. The assay appears to have general utility as it is also suitable for measuring the DNA digestion activities of a processive helicase/nuclease, RecBCD, a distributive exonuclease, T7 gene 6 exonuclease, and an endonuclease, DNaseI. The benefits of, and limitations to, the method are discussed.     

Oncology drug discovery applications using the FMAT 8100 HTS system

High-throughput screening (HTS) for potential anticancer agents requires a broad portfolio of assay platforms that may include kinase enzyme assays, protein-protein binding assays, and functional cell-based apoptosis assays. The authors have explored the use of fluorometric microvolume assay technology (the FMAT 8100 HTS System) in three distinct homogeneous HTS assays: (1). a Src tyrosine kinase enzyme assay, (2). a Grb2-SH2 protein-peptide interaction assay, and (3). an annexin V binding apoptosis assay. Data obtained from all three assays suggest that the FMAT system should facilitate the implementation of homogeneous assays for a wide variety of molecular targeted and cell-based screens.     

Microanalytical determination of the activities of phospholipases A, C, and D and of their mixtures

In the course of studies on the catabolite repression of succinate dehydrogenase in yeast (1), the need arose to monitor simultaneously phospholipases A and D activities in broken cell preparations. Although a variety of assay methods are described in the literature for both of these enzymes, none of them appeared to provide a simple and unambiguous measure of both activities simultaneously at the very low enzyme concentrations and with the crude enzyme preparations used. Therefore, a method was developed, based on the use of uniformly labeled ¹⁴C-lecithin, which provides simple and relatively easy assays for phospholipases A, C, and D, as well as for mixtures of the three enzymes, and permits initial-rate measurements. Details are described in this paper. Comparison of the methods described with other assays for phospholipases is presented in the Discussion.     

Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions

We have previously described a strategy for detecting protein protein interactions based on protein interaction assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 beta-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quantitative analysis of a small molecule induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissociation constant and number of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of beta-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array analysis of protein protein or enzyme protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.     

In vitro mammalian mutagenesis as a model for genetic lesions in human cancer

Recently in vitro assays of mutagenesis have been criticized as being poorly predictive of long-term in vivo rodent assays of carcinogenicity. Questions have also been raised concerning the relevance of rodent assays to human risk. In vitro assays using mammalian cells can detect most types of genetic lesions thought to be important in human malignant disease. Molecular and cytogenetic analyses of mutations induced by a variety of genotoxic compounds at the heterozygous thymidine kinase locus in mouse lymphoma cells indicate that this in vitro assay does indeed register the range of genetic lesions recently found in a wide variety of human tumors. The types and complexity of the induced lesions are reflected in mutant colony phenotype in a compound-specific fashion. These studies point to the use of appropriate in vitro mammalian mutagenesis assays as new model systems for dissecting the genetic lesions important in human carcinogenesis, and as a means of determining the potential for compounds to induce such lesions.     

Assay of ribonucleotide reductase activity in intact permeabilized hamster cells: an evaluation

An intact cell assay system, based on Tween-80 permeabilization can be used to investigate ribonucleotide reductase activity in a variety of mammalian cell lines. An important consideration in the use of intact cells is the presence of other nucleotide metabolizing activities. The influence of these activities on estimates of pyrimidine (CDP) and purine (ADP) reductase in permeabilized hamster cells has been examined. Studies on the incorporation of label from CDP and ADP into RNA indicated that a very small proportion of the reductase substrates was eventually incorporated into RNA during routine enzyme assays, and would have no detectable effect on activity estimates. The possibility that the products of the reaction (dCDP and dADP) were eventually phosphorylated and incorporated into DNA was also examined, and it was found that proper permeabilization of the cells eliminated or greatly reduced loss of deoxyribonucleotides to DNA. An analysis by HPLC of nucleotides present during CDP and ADP reductase reactions showed that various kinases and phosphatases were active in permeabilized cells, as all levels of phosphorylation of nucleotide substrates and allosteric effectors were detected. The base composition of the nucleotides added to the assay systems were not altered. Although movement of phosphates occurred during the assay, the concentrations of substrates quickly reached equilibrium (within 1 min) with their respective nucleosides and nucleotides, resulting in a relatively constant although reduced concentration of CDP or ADP substrates during the 20-min assay. Similarly the levels of allosteric effectors, ATP for pyrimidine and dGTP for purine reductase activities, declined within the first minute of the assays and quickly reached an equilibrium with their respective adenine or guanine containing nucleotides during most of the reaction time. Although useful approximations of intracellular reductase activity can be obtained without correcting for modified nucleotide concentrations, precise determinations can be calculated when these alterations are taken into consideration. For example, estimates of intracellular K_m values for CDP closely resembled those reported with highly purified mammalian enzyme preparations in other studies. Clearly, the intact cell assay system provides worthwhile information about mammalian ribonucleotide reductase in its physiologically relevant environment.     

Luminometric quantitation of photinus pyralis firefly luciferase and Escherichia coli beta-galactosidase in blood-contaminated organ lysates

Firefly luciferase and Escherichia coli beta-galactosidase chemiluminescent reporter gene assays are rapid and sensitive means of detecting reporter enzyme activities in cell lysates of both eukaryotic and prokaryotic systems. In these assays, expression vectors containing the luciferase or beta-galactosidase genes are transferred to cells in culture or animal tissues in vivo. Crude cell or organ lysates are then prepared and submitted to enzyme assays. The level of enzyme activity is proportional to the efficiency of gene delivery and expression. When used with modified substrates that emit light when cleaved by the appropriate enzyme, luciferase and beta-galactosidase activity can be detected luminometrically. Attempts to apply these assays to cell lysates contaminated with blood, as from any whole organ lysate, have had questionable results thus far because of light absorption by hemoglobin in the ranges of light emission by both of these assays. We have made several adjustments to standard chemiluminescent reporter gene assay protocols to minimize errors in quantitation contributed by hemoglobin. To this end, we have developed a method for quantitating the protein due to blood and due to the organ itself in a blood-contaminated organ lysate. We have also found that the use of a colorimetric protein assay that is unaffected by hemoglobin absorbance is preferred for protein quantitation. In conclusion, luciferase and beta-galactosidase assays can be applied to blood-contaminated organ lysates; however, the luciferase assay proved to be superior due to minimal endogenous activity and lower absorption by hemoglobin of light emitted by the enzyme product.     

Angiotensin I-converting enzyme localization on cultured fibroblasts by immunofluorescence

Angiotensin I-converting enzyme is responsible for the activation of angiotensin I and the inactivation of bradykinin. It has been localized by immunofluorescence on the endothelium of a variety of tissues and has been considered to be a specific marker for endothelial cells in culture. The present paper demonstrates, by immunofluorescence, the presence of angiotensin I-converting enzyme in monolayer cultures of fibroblasts derived from adult rat lung, bovine calf pulmonary artery, and human foreskin (CF-3 cells). Fluorescent localization of angiotensin I-converting enzyme was observed over the cytoplasm of adult rat lung and bovine calf pulmonary artery fibroblasts and as distinct areas overlying the nuclei of human foreskin fibroblasts. Determination of angiotensin I-converting enzyme activity by fluorimetric assay in parallel studies confirmed the presence of angiotensin I-converting enzyme activity in cultured fibroblasts. Immunofluorescent studies with antibody to Factor VIII demonstrated the presence of Factor VIII on cultured endothelial cells but not on fibroblasts. These results indicate that angiotensin I-converting enzyme is not confined to endothelial cells, and thus may not serve as a specific marker for endothelial cells in culture. Factor VIII may be a more specific marker for these cells.     

Measurement of key metabolic enzyme activities in mammalian cells using rapid and sensitive microplate‐based assays

Sensitive microplate‐based assays to determine low levels of key enzyme activities in mammalian cells are presented. The enzyme platform consists of four cycling assays to measure the activity of 28 enzymes involved in central carbon and glutamine metabolism. The sensitivity limit of all cycling assays was between 0.025 and 0.4 nmol product. For the detection of glutaminase activity, a new glutamate cycle system involving the enzymes glutamate dehydrogenase and aspartate transaminase was established. The relative standard deviation of the method was found to be 1.7% with a limit of detection of 8.2 pmol and a limit of quantitation of 24.8 pmol. Hence, cell extracts could be highly diluted to reduce interferences caused by other components in the extract, which in addition minimized underestimates or overestimates of actual enzyme activities. Since substrate concentrations could be maintained at a nearly constant level throughout the assay product accumulation during the reaction was low, which minimized product inhibition. As an example, the enzyme platform was used to investigate maximum enzyme activities of stationary‐phase MDCK cells grown in serum‐containing GMEM medium as typically used in influenza vaccine production. Biotechnol. Bioeng. 2010;107: 566–581. © 2010 Wiley Periodicals, Inc.     

Quantitative detection of blood-brain barrier-associated enzymes in cultured endothelial cells of procine brain microvessels

Summary The present study deals with a rapid and convenient assay for blood-brain barrier (BBB)-associated enzymes, γ-glutamyl transpeptidase (γ-GTP) and alkaline phosphatase (ALP), in cultured endothelial cells and other cells. These enzyme activities in cultured cells could be efficiently measured by direct incubation of each substrate in the culture plates without pretreatment of the cells. This new direct in situ-in plate assay was more rapid and convenient than conventional ex-plate assays, and these assays gave similar values for specific enzyme activities. γ-GTP and ALP activities could be detected by this in situ method in primary-cultured endothelial cells of porcine brain microvessels, but their levels were lower than those before culture. The degree of loss due to culture differed, between γ-GTP and ALP; a relatively large amount of ALP remained but the γ-GTP level decreased greatly In this direct in situ-in plate assay, cultured porcine aortic endothelial cells exhibited negligibly small activities for both enzymes, whereas cultured astroglial cells of neonatal porcine brain showed moderate γ-GTP activity and a trace of ALP activity. This direct in situ-in plate assay can be used for microculture and automatic measurement and offers a convenient means for studying the possible regulatory mechanisms of the expression of the BBB-associated enzymes.     

Expression, purification, and initial characterization of human alanine aminotransferase (ALT) isoenzyme 1 and 2 in High-five insect cells

Alanine aminotransferase (ALT) is a key enzyme for gluconeogenesis as well as a widely used serum marker for liver injury. We have identified two ALT isoenzymes, ALT1 and ALT2, which are encoded by separate genes. In this study, we described the expression, purification and initial characterization of human ALT1 and ALT2 proteins in High-five insect cells. Human ALT1 and ALT2 were expressed as His-tagged fusion proteins by recombinant baculovirus in insect cells and purified into homogeneity in one step by using immobilized Ni²⁺-affinity chromatography. Tag-free ALT1 and ALT2 were obtained by cleavage of enterokinase digestion and used for initial characterization of the enzymes. The specific ALT activity of purified fusion or His-tag-removed ALT1 was about 15-fold higher than that of ALT2 and their enzymatic activities decreased quickly at 37 °C and −20 °C, but were well preserved at −80 °C. Nevertheless, the ALT1 and ALT2 activities remained stable in a buffer containing 25% glycerol. The pH profile was similar between hALT1 and hALT2 in that both enzymes remained fully active between pH 6.5 and 8.0. The purified ALT recombinant proteins can not only be used as a reference protein standard for the ALT assay in clinical chemistry, but also will be useful for understanding the biochemical and biological significance of the isoenzymes and for developing ALT isoform-specific assays for clinical or preclinical diagnostic use.     

Adapting capillary gel electrophoresis as a sensitive,high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner.     

InXy and SeXy, compact heterologous reporter proteins for mammalian cells

Mammalian reporter proteins are essential for gene-function analysis, drugscreening initiatives and as model product proteins for biopharmaceutical manufacturing. Bacillus subtilis can maintain its metabolism by secreting Xylanase A (XynA), which converts xylan into shorter xylose oligosaccharides. XynA is a family 11 xylanase monospecific for D-xylose containing substrates. Mammalian cells transgenic for constitutive expression of wild-type xynA showed substantial secretion of this prokaryotic enzyme. Deletion analysis confirmed that a prokaryotic signal sequence encoded within the first 81 nucleotides was compatible with the secretory pathway of mammalian cells. Codon optimization combined with elimination of the prokaryotic signal sequence resulted in an exclusively intracellular mammalian Xylanase A variant (InXy) while replacement by an immunoglobulin-derived secretion signal created an optimal secreted Xylanase A derivative (SeXy). A variety of chromogenic and fluorescence-based assays adapted for use with mammalian cells detected InXy and SeXy with high sensitivity and showed that both reporter proteins resisted repeated freeze/thaw cycles, remained active over wide temperature and pH ranges, were extremely stable in human serum stored at room temperature and could independently be quantified in samples also containing other prominent reporter proteins such as the human placental alkaline phosphatase (SEAP) and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Glycoprofiling revealed that SeXy produced in mammalian cells was N- glycosylated at four different sites, mutation of which resulted in impaired secretion. SeXy was successfully expressed in a variety of mammalian cell lines and primary cells following transient transfection and transduction with adeno-associated virus particles (AAV) engineered for constitutive SeXy expression. Intramuscular injection of transgenic AAVs into mice showed significant SeXy levels in the bloodstream. InXy and SeXy are highly sensitive, compact and robust reporter proteins, fully compatible with pre-existing marker genes and can be assayed in high-throughput formats using very small sample volumes.     

Quantitative, standardized assays for determining the concentrations of bovine lactoperoxidase, human salivary peroxidase, and human myeloperoxidase

Because of the important biological functions of peroxidases, there is growing interest in the measurement of their concentrations in various secretions. At present, there is no standard method which allows for comparisons in reported activities. This report describes procedures which can be used to measure peroxidase enzyme concentrations by commonly employed assays. Regression equations have been determined which can be used to calculate concentrations of bovine lactoperoxidase (LPO), human salivary peroxidase (SPO), and human myeloperoxidase (MPO) from activities measured with the following donors: pyrogallol, guaiacol, 2,2'-azinobis(3-ethylbenzylthiazoline-6-sulfonic acid), and thiocyanate (SCN-). The peroxidation rates of these donors depend upon the concentrations of hydrogen peroxide (H2O2) used in the individual assays and thus, for accurate, reproducible results, these concentrations must be carefully controlled. The SCN- normally present in human saliva will reduce observed reaction rates by simple competition kinetics in the ABTS, guaiacol and pyrogallol assays and will increase the rates observed when Cl- is used as a donor in NBS assay for MPO. Therefore, SCN- must be removed from saliva samples prior to peroxidase activity determination by all assays except the thionitrobenzoic acid (NBS) assay. LPO cannot be used as a standard for either SPO or MPO because the specific activities of LPO, SPO, and MPO are significantly different.     

A kinetic epoxidation assay for chloroperoxidase

Chloroperoxidase exhibits a wide variety of enantioselective epoxidation reactions. Until now, the epoxidation activities have been mainly evaluated using elaborate gas chromatographic methods. This paper reports a rapid and convenient spectrophotometric assay for CPO. The disappearance of indene by catalytic epoxidation is monitored at 250 nm and this is used as an index of enzyme activity. This method will prove to be highly useful in large-scale screening of mutants.     

Determination of myrosinase (thioglucoside glucohydrolase) activity by a spectrophotometric coupled enzyme assay

The hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme system was used to assay for plant thioglucoside glucohydrolase (myrosinase, EC 3.2.3.1) by measuring the rate of glucose released during hydrolysis of glucosinolates. This coupled assay was compared with two other assays for myrosinase: a pH-stat assay that measures the rate of acid released during glucosinolate hydrolysis, and a spectrophotometric assay in which the decrease in the absorbance at 227.5 nm is used to measure the disappearance of the substrate, 2-propenylglucosinolate (DSA assay). The coupled and pH-stat assays were found to give comparable activities and were linear with enzyme concentration over the range 0 to 30 micrograms. The DSA assay gave lower myrosinase activity in comparison to the coupled and pH-stat assays. This is due to the lower concentrations of substrate and activator (ascorbate) which must be used in the assay. The DSA assay was found to give a nonlinear relationship with enzyme concentration over the range 2 to 30 micrograms. For these reasons this assay was found to be unsatisfactory. The coupled assay was found to be more sensitive and more widely applicable than the pH-stat assay as a routine continuous assay for myrosinase activity.     

Quantification of uridine 5'-diphosphate (UDP)-glucose by high-performance liquid chromatography and its application to a nonradioactive assay for nucleoside diphosphate kinase using UDP-glucose pyrophosphorylase as a coupling enzyme

We describe a method for the detection and quantification of nucleoside diphosphate kinase (NDPK). NDPK catalyzes the transfer of the gamma-phosphate of cytidine 5'-triphosphate on uridine 5'-diphosphate (UDP) to produce uridine 5'-triphosphate (UTP). The method uses a nonradioactive coupled enzyme assay in which UTP produced by NDPK is utilized by UDP-glucose pyrophosphorylase. This latter enzyme synthesizes UDP-glucose and inorganic phosphate in the presence of glucose 1-phosphate. UDP-glucose is detected at 260 nm after separation of the reaction mixture by high-performance liquid chromatography (HPLC) on a strong anion-exchange column. The assay is reliable, specific, and linear with respect to time and enzyme amount. Using 15 min incubation time, the method allows detection of NDPK activity below 10 pmol/min. It can be used to analyze kinetic behavior and to quantify NDPK from a wide variety of animal, microbial, and plant sources. It also provides an alternative to radiometric assays and an improvement on pyruvate kinase-linked spectrophotometric assays, which can be hampered by pigments present in crude extracts. Furthermore, we show that the HPLC method developed here can be directly used to assay enzymes for which UDP-glucose is a product.