Mass-tag technology for monitoring of protein kinase activity using mass spectrometry

Monitoring of intracellular protein kinase activity is very important for fields involving diagnosis and drug screening. However, current methods, such as radiometry using (32)P, or ELISA, are laborious and time-consuming. We have developed high-throughput assay system of protein kinase activity using mass-tagged substrate peptide probes and mass spectrometry. This assay system can easily evaluate target kinase activity and will potentially be able to simultaneously profile many protein kinase activities.     

A quantitative continuous enzyme assay of intramolecularly quenched fluorogenic phospholipase substrates for molecular imaging

There has been recent growth in the development of activatable near-infrared (NIR) fluorescent probes for molecular imaging, generally designed by placing fluorochromes on a cleavable substrate in close proximity to one another, such that they self-quench, but fluoresce on separation via enzymatic cleavage of the substrate. Although these probes offer excellent contrast, the detection of enzyme activity has largely only been described qualitatively. In order to assess the effectiveness of a probe, it is useful to have a quantitative measure, such as the enzyme-substrate kinetic parameters. We have developed an assay to determine kinetic parameters and applied it to an intramolecularly quenched molecule, Pyro-PtdEtn-BHQ, a NIR fluorescent probe specific to phosphatidylcholine-specific phospholipase C. The development of this assay includes corrections for intermolecular quenching, calibration, optimization of reaction mixtures, and determination of kinetic and inhibition parameters. This assay can easily be extended to analyze and compare the efficiency of other fluorescent activatable phospholipase probes as suitable molecular imaging agents.     

Synthesis of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and its use as a substrate to assess feruloyl esterase activity

A synthetic scheme was developed for the production of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside (FA-Ara) in gram quantities. This molecule accurately models the chemical attachment of ferulic acid to polysaccharides found in cell walls of plants in the Gramineae family. It is therefore a realistic substrate that can be used to monitor feruloyl esterase activity. Ultraviolet spectral analysis indicated that FA-Ara has an absorption maximum distinct from the hydrolytic product, ferulic acid (FA), over a wide range of solution pH values. The log molar extinction coefficient ranges from 4.16 to 4.36 for FA-Ara and 4.16 to 4.33 for FA depending upon the pH of the buffered solution. Consequently a convenient spectrophotometric assay can be utilized to monitor esterase activity. Three different methods were developed for using this model substrate to assess esterase activity, including thin-layer chromatography, a spectrophotometric assay, and the use of high-performance liquid chromatography.     

A spectrophotometric assay for the transphosphatidylation activity of phospholipase D enzyme

We developed a specific spectrophotometric assay for the quantitative determination of phospholipase D-catalyzed transphosphatidylation activity. The assay measures p-nitrophenol liberated by phospholipase D-catalyzed reaction of phosphatidyl-p-nitrophenol and ethanol in an aqueous-organic emulsion system. The release of p-nitrophenol was linear to reaction time at an early stage of the reaction with phospholipase D from Streptomyces sp. In the spectrophotometric assay for the reaction with phospholipase D from Streptomyces chromofuscus, which has higher hydrolytic activity than transphosphatidylation activity, p-nitrophenol was not found. The advantages of this novel method for measuring the transphosphatidylation activity of phospholipase D are that (i) it does not use radioactive compounds, (ii) it can measure the initial velocity of the reaction, and (iii) it is rapid, easy, and accurate to perform.     

Guanine Nucleotide-Binding Protein Regulation of Microsomal Phospholipase D Activity of Canine Cerebral Cortex

The hydrolytic activity of microsomal phospholipase D from canine cerebral cortex was measured by a radiochemical assay using 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl[3H]choline and 1-palmitoyl-2-[9,10(n)-3H]palmitoyl-sn-glycerol-3-phosphorylcholine as the exogenous substrates. Of several detergents tested, Triton X-100 was found to be the most effective in allowing expression of phospholipase D hydrolytic activity. The microsomal phospholipase D does not require any metal ion for its hydrolytic activity. Calcium and magnesium were slightly inhibitory between concentrations of 1 and 4 mM, but zinc was greatly inhibitory, causing a loss of greater than 90% activity at the 4 mM concentration. Non-hydrolyzable guanine nucleotide analogues such as guanosine 5'-(3-O-thio)triphosphate and guanyl-5'-yl-(beta, gamma-methylene)diphosphonate but not guanosine 5'-(2-thio)diphosphate were able persistently to stimulate phospholipase D hydrolytic activity at micromolar concentrations. Guanosine 5'-(2-thio)diphosphate was capable of partially blocking guanosine 5'-(3-O-thio)triphosphate stimulation of phospholipase D. Aluminum fluoride was able to cause a two- to threefold increase in hydrolytic activity of the phospholipase D. Cholera toxin had a stimulatory effect on the hydrolytic activity of phospholipase D, whereas islet-activating protein pertussis toxin had no effect. These results indicate that regulation of microsomal phosphatidylcholine phospholipase D activity by the guanine nucleotide-binding protein(s) in canine cerebral cortex may play an important role in signal transduction processes as well as in brain choline metabolism.     

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.     

Development of a differential medium for bile salt hydrolase-active Lactobacillus spp.

An agar plate assay was developed to detect bile salt hydrolase activity in lactobacilli. On Lactobacillus-selective MRS or Rogosa SL medium supplemented with taurodeoxycholic, taurocholic, or taurochenodeoxycholic acids, bile salt hydrolysis was manifested at two intensities: (i) the formation of precipitate halos around colonies or (ii) the formation of opaque granular white colonies. Sixty-six lactobacilli were tested for bile salt hydrolase activity by both the plate assay and a sensitive radiochemical assay. No false-positive or false-negative results were detected by the plate assay. Based on results of experiments with Eubacterium lentum and Bacteroides species, the plate assay was dependent on two factors: (i) the presence of bile salt hydrolytic activity and (ii) the ability of the organism to sufficiently acidify the medium to protonate free bile acids. The availability of a differential medium for determination of bile salt hydrolase activity will provide a rapid method for determining shifts in a specific functional activity of intestinal Lactobacillus species and provide a rapid screening capability for identifying bile salt hydrolase-deficient mutants. The latter application should allow bile salt hydrolase activity to be used as a marker enzyme in genetic experiments.     

Development of a differential medium for bile salt hydrolase-active Lactobacillus spp

An agar plate assay was developed to detect bile salt hydrolase activity in lactobacilli. On Lactobacillus-selective MRS or Rogosa SL medium supplemented with taurodeoxycholic, taurocholic, or taurochenodeoxycholic acids, bile salt hydrolysis was manifested at two intensities: (i) the formation of precipitate halos around colonies or (ii) the formation of opaque granular white colonies. Sixty-six lactobacilli were tested for bile salt hydrolase activity by both the plate assay and a sensitive radiochemical assay. No false-positive or false-negative results were detected by the plate assay. Based on results of experiments with Eubacterium lentum and Bacteroides species, the plate assay was dependent on two factors: (i) the presence of bile salt hydrolytic activity and (ii) the ability of the organism to sufficiently acidify the medium to protonate free bile acids. The availability of a differential medium for determination of bile salt hydrolase activity will provide a rapid method for determining shifts in a specific functional activity of intestinal Lactobacillus species and provide a rapid screening capability for identifying bile salt hydrolase-deficient mutants. The latter application should allow bile salt hydrolase activity to be used as a marker enzyme in genetic experiments.     

A novel assay for monoacylglycerol hydrolysis suitable for high-throughput screening

A simple assay for monoacylglycerol hydrolysis suitable for high-throughput screening is described. The assay uses [(3)H]2-oleoylglycerol as substrate, with the tritium label in the glycerol part of the molecule and the use of phenyl sepharose gel to separate the hydrolyzed product ([(3)H]glycerol) from substrate. Using cytosolic fractions derived from rat cerebella as a source of hydrolytic activity, the assay gives the appropriate pH profile and sensitivity to inhibition with compounds known to inhibit hydrolysis of this substrate. The assay could also be adapted to a 96-well plate format, using C6 cells as the source of hydrolytic activity. Thus the assay is simple and appropriate for high-throughput screening of inhibitors of monoacylglycerol hydrolysis.     

Screening for the rice blast resistance gene Pi-ta using LNA displacement probes and real-time PCR

Single nucleotide polymorphisms (SNPs) will become a molecular breeding tool of increasing significance as a growing range of SNP data becomes available. In order for these markers to be incorporated into breeding programs, simple, high throughput and low cost detection methods need to be available. We have developed such an assay using LNA containing displacement probes for the Pi-ta gene, an important blast resistance gene in rice. This assay gave superior performance in comparison with TaqMan MGB probes and was able to accurately identify the presence of low frequency genotypes in artificially created mixed samples. The ability to pool samples for screening purposes offers the potential to significantly increase throughput and reduce per sample detection cost, particularly for low frequency alleles.     

Characterization of oxidized nicotinamide adenine dinucleotide (NAD(+)) analogues using a high-pressure-liquid-chromatography-based NAD(+)-glycohydrolase assay and comparison with fluorescence-based measurements

A high-pressure-liquid-chromatography (HPLC)-based technique was developed to assess the oxidized nicotinamide adenine dinucleotide (NAD(+))-glycohydrolase activity of the catalytic domain of Pseudomonas exotoxin A containing a hexa-His tag. The assay employs reverse-phase chromatography to separate the substrate (NAD(+)) and products (adenosine 5'-diphosphate-ribose and nicotinamide) produced over the reaction time course, whereby the peak area of nicotinamide is correlated using a standard curve. This technique was used to determine whether the NAD(+) analogue, 2'-F-ribo-NAD(+), was a competing substrate or a competitive inhibitor for this toxin. This NAD(+) analogue was hydrolyzed at a rate of 0.2% that of NAD(+) yet retained the same binding affinity for the toxin as the parent compound. Finally, the rate that a fluorescent NAD(+) analogue, epsilon-NAD(+), is hydrolyzed by the toxin was also investigated. This analogue was hydrolyzed six times slower than NAD(+) as determined using HPLC. The rate of hydrolysis of epsilon-NAD(+) calculated using the fluorometric version of the assay shows a sixfold increase in reaction rate compared to that determined by HPLC. This HPLC-based assay is adaptable to any affinity-tagged enzyme that possesses NAD(+)-glycohydrolase activity and offers the advantage of directly measuring the enzyme-catalyzed hydrolytic rate of NAD(+) and its analogues.     

Protein phosphatase inhibitory activity of tautomycin photoaffinity probes evaluated at femto-molar level

Herein we describe the further improvement of our in-house developed firefly bioluminescence assay system for the determination of inhibition of protein phosphatase (PP). The advantage with the new system is higher sensitivity as well as being time and sample efficient. The inhibition activity of tautomycin with PP1gamma was determined using the upgraded test system and Ki was found to be 4.5 nM, which compare favorably with the activity reported previously by others using different methods. The test system was then used in order to determine the activity of nine tautomycin (TTM) photoaffinity probes. One of the TTM photoaffinity probes (anti-10) was found to possess higher activity than the natural product itself with a Ki of 3.4 nM, while the remaining photoaffinity probes were found to possess Ki in the range of 8.0-213 nM.     

Application of a unique server-based oligonucleotide probe selection tool toward a novel biosensor for the detection of Streptococcus pyogenes

We developed a software program for the rapid selection of detection probes to be used in nucleic acid-based assays. In comparison to commercially available software packages, our program allows the addition of oligotags as required by nucleic acid sequence-based amplification (NASBA) as well as automatic BLAST searches for all probe/primer pairs. We then demonstrated the usefulness of the program by designing a novel lateral flow biosensor for Streptococcus pyogenes that does not rely on amplification methods such as the polymerase chain reaction (PCR) or NASBA to obtain low limits of detection, but instead uses multiple reporter and capture probes per target sequence and an instantaneous amplification via dye-encapsulating liposomes. These assays will decrease the detection time to just a 20 min hybridization reaction and avoid costly enzymatic gene amplification reactions. The lateral flow assay was developed quantifying the 16S rRNA from S. pyogenes by designing reporter and capture probes that specifically hybridize with the RNA and form a sandwich. DNA reporter probes were tagged with dye-encapsulating liposomes, biotinylated DNA oligonucleotides were used as capture probes. From the initial number of capture and reporter probes chosen, a combination of two capture and three reporter probes were found to provide optimal signal generation and significant enhancement over single capture/reporter probe combinations. The selectivity of the biosensor was proven by analyzing organisms closely related to S. pyogenes, such as other Streptococcus and Enterococcus species. All probes had been selected by the software program within minutes and no iterative optimization and re-design of the oligonucleotides was required which enabled a very rapid biosensor prototyping. While the sensitivity obtained with the biosensor was only 135 ng, future experiments will decrease this significantly by the addition of more reporter and capture probes for either the same rRNA or a different nucleic acid target molecule. This will lead to the possibility of detecting S. pyogenes with a rugged assay that does not require a cell culturing or gene amplification step and will therefore enable rapid, specific and sensitive onsite testing.     

Role of phosphorolytic cleavage in cellobiose and cellodextrin metabolism by the ruminal bacterium Prevotella ruminicola.

In bacteria, cellobiose and cellodextrins are usually degraded by either hydrolytic or phosphorolytic cleavage. Prevotella ruminicola B(1)4 is a noncellulolytic ruminal bacterium which has the ability to utilize the products of cellulose degradation. In this organism, cellobiose hydrolytic cleavage activity was threefold greater than phosphorolytic cleavage activity (113 versus 34 nmol/min/mg of protein), as measured by an enzymatic assay. Cellobiose phosphorylase activity (measured as the release of P(i)) was found in cellobiose-, mannose-, xylose-, lactose-, and cellodextrin-grown cells (> 92 nmol of P(i)/min/mg of protein), but the activity was reduced by more than 74% for cells grown on fructose, L-arabinose, sucrose, maltose, or glucose. A small amount of cellodextrin phosphorylase activity (19 nmol/min/mg of protein) was also detected, and both phosphorylase activities were located in the cytoplasm. Degradation involving phosphorolytic cleavage conserves more metabolic energy than simple hydrolysis, and such degradation is consistent with substrate-limiting conditions such as those often found in the rumen.     

Tandem optimization of target activity and elimination of mutagenic potential in a potent series of N-aryl bicyclic hydantoin-based selective androgen receptor modulators

Pharmacokinetic studies in cynomolgus monkeys with a novel prototype selective androgen receptor modulator revealed trace amounts of an aniline fragment released through hydrolytic metabolism. This aniline fragment was determined to be mutagenic in an Ames assay. Subsequent concurrent optimization for target activity and avoidance of mutagenicity led to the identification of a pharmacologically superior clinical candidate without mutagenic potential.     

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．     

Simultaneous determination of gene expression and bacterial identity in single cells in defined mixtures of pure cultures.

A protocol was developed to achieve the simultaneous determination of gene expression and bacterial identity at the level of single cells; a chromogenic beta-galactosidase activity assay was combined with in situ hybridization of fluorescently labelled oligonucleotide probes to rRNA. The method allows monitoring of gene expression and quantification of beta-galactosidase activity in single cells.     

Heat stable lipase activity of thermotolerant bacteria from hot springs at Orissa, India

Thermotolerant bacteria (35 in toto) isolated from three hot springs (Atri, Taptapani and Deuljhari, Orissa), were screened for lipase activities. Of these, nine strains of Bacillus spp. and three strains of Pseudomonas spp. showed heat stable lipase activity at 60 degrees C. The hydrolytic activity of these bacteria was tested using Tween-20 and Tween-80 as substrates at different temperatures using plate assay and titration techniques. The hydrolytic activity at different pH values and salt concentrations was investigated.     

High-throughput fluorogenic assay for determination of botulinum type B neurotoxin protease activity

Botulinum neurotoxins are responsible for botulism, a flaccid muscular paralysis caused by inhibition of acetylcholine release at the neuromuscular junction. This occurs by cleavage of conserved proteins involved in exocytosis such as synaptobrevin by the zinc metallopeptidase activity of the light chain of some botulinum neurotoxins. Botulism, for which there is presently no therapy available, is a relatively widespread disease that may result in death. Consequently, the development of drugs able to inhibit the hydrolytic activity of these neurotoxins is of great interest. Design and screening of such inhibitors could be largely facilitated by using high-throughput assays. With this aim, a novel in vitro test for quantifying the proteolytic activity of botulinum type B neurotoxin was developed. The substrate is the 60--94 fragment of human synaptobrevin-1 which was modified by introduction of the fluorescent amino acid l-pyrenylalanine in position 74 and a p-nitrophenylalanyl residue as quenching group in position 77. The cleavage of Syb 60-94 [Pya(74), Nop(77)] by the toxin active chain occurs selectively between residues 76 and 77 as in the case of the unmodified synaptobrevin and is directly quantified by measuring the strong fluorescence of the formed metabolite Syb 60-76 [Pya(74)]. This is the easiest, quickest, and cheapest assay described to date for measuring the proteolytic activity of botulinum type B neurotoxin. It can be easily automated for high-throughput screening. Moreover, amounts of about 3.5 pg/ml of botulinum type B neurotoxin could be detected by this method.