Inorganic pyrophosphatase and photosynthesis by isolated chloroplasts. I. Characterisation of chloroplast pyrophosphatase and its relation to the response to exogenous pyrophosphate

1. 1. A soluble, alkaline, Mg²⁺-dependent inorganic pyrophosphatase (EC 3.6.1.1) has been isolated from the stroma of intact spinach and pea chloroplasts and purified some 100-fold. The enzyme has a high specificity for inorganic pyrophosphate and Mg²⁺, and exhibits maximal activity at pH 8.2–8.6. The enzyme shows allosteric characteristics with Mg²⁺ as activator and optimal rates are obtained with a ratio of Mg²⁺ to PP_i of approximately 4 to 1. The enzyme is inhibited by anionic PP_i and by its own reaction product, orthophosphate.

2. 2. If Mg²⁺ is excluded from the medium in which isolated chloroplasts are assayed, active photosynthetic oxygen evolution can still be observed. The addition of P_i, but not PP_i, will then offset a phosphate deficiency. If external Mg²⁺ is present PP_i will also offset a phosphate deficiency and in these circumstances the rapidity and nature of the response is related to the external pyrophosphatase activity.

3. 3. Evidence is presented that the chloroplast envelope is relatively impermeable to PP_i and that the response to added PP_i is due to external hydrolysis followed by entry of P_i to the chloroplast. These results have significance concerning proposed mechanisms for control of photosynthesis.

A novel high-throughput screening assay for discovery of molecules that increase cellular tetrahydrobiopterin

Tetrahydrobiopterin (BH(4)) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH(4) has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH(4). The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH(4) levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH(4) levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z' factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein-protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH(4) levels.     

Evaluation of alkoxyresorufins as fluorescent substrates for cytochrome P450 BM3 and site-directed mutants

In this study, the first fluorescent assay for bacterial cytochrome P450 BM3 (BM3) and mutants is described. BM3 mutants are potentially very versatile biocatalysts for the production of fine chemicals. A fluorescent assay would be very useful for the identification of nonnatural ligands in high-throughput inhibition assays. Because of the ease and sensitivity of alkoxyresorufin O-dealkylation assays, four different alkoxyresorufins were evaluated as substrates. Wild-type BM3 showed extremely low activity toward all four alkoxyresorufins tested. Five different BM3 mutants were constructed, carrying different combinations of mutations R47L, F87V, and L188Q, which were previously shown to increase activity toward nonnatural substrates. For all mutants, a high benzyloxyresorufin O-dealkylation (BROD) activity was found. The triple mutant of BM3, R47L/F87V/L188Q, showed the highest activity, increasing 900-fold compared to wild-type BM3. The BROD assay could also be applied in whole Escherichia coli cells; permeabilization by lipopolysaccharide deficiency strongly increased activity. To demonstrate the applicability of the BROD assay to screening for novel ligands of BM3 R47L/F87V/L188Q, a library of 45 drug-like compounds was tested for inhibition. Of these compounds, 8 showed strong inhibition of the BROD activity, demonstrating for the first time that drug-like molecules also can bind with high affinity to BM3 mutants.     

真核细胞顺乌头酸酶活性检测新技术——胶内酶活性分析法

顺乌头酸酶（aconitase,Aco）是细胞内重要的铁硫蛋白酶,它催化细胞内柠檬酸经中间产物顺乌头酸生成异柠檬酸. 真核细胞中顺乌头酸酶有两种,分别定位在细胞质的顺乌头酸酶1（c-Aco）和定位在线粒体的顺乌头酸酶2（m-Aco）.检测它们活性的变化能敏感地反映出细胞中能量代谢、自由基产生、铁硫簇组装及铁代谢水平的改变. 顺乌头酸酶活性的传统检测方法通常是测定细胞中总的顺乌头酸酶活性,该方法难以准确区分出c-Aco和m-Aco各自的活性变化.因此我们建立一种胶内酶活性分析法检测顺乌头酸酶活性. 该方法利用非变性电泳技术将c-Aco和m-Aco浓缩分离,通过泡染底物显色,条带颜色深浅反映了酶活性的强弱. 同时,比较了胶内酶活性分析法和分光光度法检测细胞内c-Aco和m-Aco的活性,并对比检测了过氧化氢处理细胞前后Aco活性的变化.结果显示,这两种方法均可敏感地检测出Aco的活性改变,并有广泛的细胞系实用性,但胶内酶活性分析法可区别测定c-Aco和m-Aco活性,不需繁琐的细胞质和线粒体分离,简便易行.文中介绍的线粒体分离纯化技术也为线粒体功能深入研究提供了一个快速、高效的分离纯化方法.     

Development of a novel assay for human tyrosyl DNA phosphodiesterase 2

Tyrosyl DNA phosphodiesterase 2 (TDP2), a newly discovered enzyme that cleaves 5′-phosphotyrosyl bonds, is a potential target for chemotherapy. TDP2 possesses both 3′- and 5′-tyrosyl-DNA phosphodiesterase activity, which is generally measured in a gel-based assay using 3′- and 5′-phosphotyrosyl linkage at the 3′ and 5′ ends of an oligonucleotide. To understand the enzymatic mechanism of this novel enzyme, the gel-based assay is useful, but this technique is cumbersome for TDP2 inhibitor screening. For this reason, we have designed a novel assay using p-nitrophenyl-thymidine-5′-phosphate (T5PNP) as a substrate. This assay can be used in continuous colorimetric assays in a 96-well format. We compared the salt and pH effect on product formation with the colorimetric and gel-based assays and showed that they behave similarly. Steady-state kinetic studies showed that the 5′ activity of TDP2 is 1000-fold more efficient than T5PNP. Tyrosyl DNA phosphodiesterase 1 (TDP1) and human AP-endonuclease 1 (APE1) could not hydrolyze T5PNP. Sodium orthovanadate, a known inhibitor of TDP2, inhibits product formation from T5PNP by TDP2 (IC₅₀ = 40 mM). Our results suggest that this novel assay system with this new TDP2 substrate can be used for inhibitor screening in a high-throughput manner.     

A sensitive and quantitative assay for measuring cleavage of presenilin substrates.

The presenilin (PS) proteins are components of the gamma-secretase activity, which is central in the pathogenesis of Alzheimer's disease. Here we present a novel cell-based reporter gene assay for the quantification of PS-controlled gamma-secretase cleavage of the Alzheimer amyloid precursor protein (APP). We show that this assay offers several advantages, including increased sensitivity and specificity, improved quantification of cleavage, and simultaneous detection of all gamma-secretase cleavages in APP. Furthermore, the APP assay can be used in parallel with a similar assay that records gamma-secretase cleavage of a Notch receptor. The use of these assays to analyze the effects of two known gamma-secretase inhibitors and postulated PS active site mutants on APP and Notch processing demonstrated that inhibitors and mutants that differently affect Notch and APP cleavage can be identified rapidly. The possibility in using these assays for high throughput screening of candidate gamma-secretase inhibitors for APP and Notch in parallel opens up new vistas to systematically search for novel inhibitors that selectively block APP cleavage while not affecting Notch signaling.     

An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases

Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone, and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays either use radiolabeled substrates and are discontinuous or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format, and that it can reproduce IC(50) values for several previously reported FDPS inhibitors. This new method offers a simple, safe, and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target.     

Molecular and biochemical markers for monitoring dynamic shifts of cellulolytic protozoa in Reticulitermes flavipes

Lower termites rely on cellulolytic protozoa to aid in the digestion of their wood-based diet. However, despite the major contribution of protozoa to the lower termite digestive system, few techniques have been developed to monitor shifts in protozoan populations. This study investigated whether quantitative real-time PCR (qRT-PCR) and/or cellulase enzyme assays can be used to monitor changes of cellulolytic protozoan populations in the lower termite, Reticulitermes flavipes (Kollar). Previously developed cellulase primer sets were used to test for changes in cellulase gene expression, while three different cellulase enzyme assays were used to assess changes in cellulase enzyme activity. The results from this study indicate that qRT-PCR is a reliable method to monitor shifts in cellulolytic protozoan populations. Specifically, qRT-PCR can serve as a useful monitoring technique during high-throughput screening of novel termite control agents such as cellulase inhibitors, and help to answer questions relating to whether or not such control agents impact cellulolytic protozoan populations.     

An enzyme-linked immunosorbent assay for the Raf/MEK1/MAPK signaling cascade.

The Ras-MAPK signaling cascade transmits mitogenic stimuli from growth factor receptors and activated Ras to the cell nucleus. Inappropriate Ras activation is associated with approximately 30% of all human cancers. The kinase components of the Ras-MAPK signaling cascade are attractive targets for pharmaceutical intervention. Therefore, we have developed a high-throughput, nonradioactive ELISA method to monitor Raf and MEK1 kinase activity. In this assay system activated Raf phosphorylates and activates MEK1, which in turn phosphorylates MAPK. Antibodies that specifically detect phosphorylated MAPK (vs. nonphosphorylated MAPK) made enzyme-linked immunosorbent assay (ELISA) development possible. This assay detects inhibitors of Raf and/or MEK1 and has been used to screen large numbers of random compounds. The specific target of inhibition in the Raf/MEK1/MAPK ELISA can be subsequently identified by secondary assays which directly measure Raf phosphorylation of MEK1 or MEK1 phosphorylation of MAPK.     

A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate

Tyrosyl-tRNA synthetase catalyzes the attachment of tyrosine to the 3′ end of tRNA^Tyr, releasing AMP, pyrophosphate, and l-tyrosyl-tRNA as products. Because this enzyme plays a central role in protein synthesis, it has garnered attention as a potential target for the development of novel antimicrobial agents. Although high-throughput assays that monitor tyrosyl-tRNA synthetase activity have been described, these assays generally use stoichiometric amounts of tRNA, limiting their sensitivity and increasing their cost. Here, we describe an alternate approach in which the Tyr-tRNA product is cleaved, regenerating the free tRNA substrate. We show that cyclodityrosine synthase from Mycobacterium tuberculosis can be used to cleave the l-Tyr-tRNA product, regenerating the tRNA^Tyr substrate. Because tyrosyl-tRNA synthetase can use both l- and d-tyrosine as substrates, we replaced the cyclodityrosine synthase in the assay with d-tyrosyl-tRNA deacylase, which cleaves d-Tyr-tRNA. This substitution allowed us to use the tyrosyl-tRNA synthetase assay to monitor the aminoacylation of tRNA^Tyr by d-tyrosine. Furthermore, by making Tyr-tRNA cleavage the rate-limiting step, we are able to use the assay to monitor the activities of cyclodityrosine synthetase and d-tyrosyl-tRNA deacylase. Specific methods to extend the tyrosyl-tRNA synthetase assay to monitor both the aminoacylation and post-transfer editing activities in other aminoacyl-tRNA synthetases are discussed.     

A simple whole cell based high throughput screening protocol using Mycobacterium bovis BCG for inhibitors against dormant and active tubercle bacilli

This study aimed at developing a whole cell based high throughput screening protocol to identify inhibitors against both active and dormant tubercle bacilli. A respiratory type of nitrate reductase (NarGHJI), which was induced during dormancy, could reflect the viability of dormant bacilli of Mycobacterium bovis BCG in microplate adopted model of in vitro dormancy. Correlation between reduction in viability and nitrate reductase activity was seen clearly when dormant stage inhibitor metronidazole and itaconic anhydride were applied in this in vitro microplate model. Active replicating stage could also be monitored in the same assay by measuring the A(620) of the culture. MIC values of 0.08, 0.075, 0.3 and 3.0 microg/ml, determined through monitoring A(620) in this assay for rifampin, isoniazid, streptomycin and ethambutol respectively, were well in agreement with previously reported by BACTEC and Bio-Siv assays. S/N ratio and Z' factor for the assay were 8.5 and 0.81 respectively which indicated the robustness of the protocol. Altogether the assay provides an easy, inexpensive, rapid, robust and high content screening tool to search novel antitubercular molecules against both active and dormant bacilli.     

Oxalate oxidase: a novel reporter gene for monocot and dicot transformations

A wheat germin gene, with oxalate oxidase (OxO) activity, can be used as a sensitive reporter gene in both monocot and dicot transformations. Detection of H₂O₂ generated from OxO oxidation of oxalate provides simple, rapid detection of gene expression. Inexpensive substrates are required for both assays. OxO activity, could be detected histochemically in minutes, without chlorophyll clearing procedures. This assay was used to optimize transformation procedures and to track stable transgene expression in breeding populations over many generations. A simple spectrophotometric quantitative enzyme activity assay was used to select lines with various levels of transgene expression and to monitor transgene silencing phenomena. The quantitative OxO assay can also be used as an internal DNA delivery standard with a second reporter gene used in gene expression studies. The simplicity of the assay is ideal for screening large populations to identify primary transgenics, for monitoring transgene segregation in large populations in field studies and for assessing stability of transgene expression over numerous generations.     

Novel and highly sensitive fluorescent assay for leucine aminopeptidases

l-Leucine aminopeptidases (LAPs) are implicated in the progress of many pathological disorders and play some regulatory roles in tumor cell proliferation, invasion, and/or angiogenesis. Thus, LAPs not only could become new diagnostic or prognostic biomarkers but also may have potential as novel molecular targets for the treatment of several cancers. Highly sensitive assays are critical for early detection of changes in LAP activity and for screening potent LAP inhibitors. In this study, we developed a novel and highly sensitive fluorescent assay for LAPs based on substituted aminopyridines as fluorescent reporters. This assay was at least 100- and 20-fold more sensitive than commercial colorimetric and fluorescent LAP substrates, respectively. We also showed that this assay was a useful tool for monitoring LAP activities in extracts from cancer cell lines, as well as for the high-throughput screening of inhibitors, which could lead to new cancer treatments.     

D-myo-inositol 1-phosphate as a surrogate of D-myo-inositol 1,4,5-tris phosphate to monitor G protein-coupled receptor activation

Phospholipase C beta (PLC-beta)-coupled G protein-coupled receptor (GPCR) activities traditionally are assessed by measuring Ca2+ triggered by D-myo-inositol 1,4,5-trisphosphate (IP3), a PLC-beta hydrolysis product, or by measuring the production of inositol phosphate using cumbersome radioactive assays. A specific detection of IP3 production was also established using IP3 binding proteins. The short lifetime of IP3 makes this detection very challenging in measuring GPCR responses. Indeed, this IP3 rapidly enters the metabolic inositol phosphate cascade. It has been known for decades that lithium chloride (LiCl) leads to D-myo-inositol 1-phosphate accumulation on GPCR activation by inhibiting inositol monophosphatase, the final enzyme of the IP3 metabolic cascade. We show here that IP1 can be used as a surrogate of IP3 to monitor GPCR activation. We developed a novel homogeneous time-resolved fluorescence (HTRF) assay that correlates perfectly with existing methods and is easily amenable to high-throughput screening. The IP-One assay was validated on various GPCR models. It has the advantage over the traditional Ca2+ assay of allowing the measurement of inverse agonist activity as well as the analysis of PLC-beta activity in any nontransfected primary cultures. Finally, the high assay specificity for D-myo-inositol 1 monophosphate (IP1(1)) opens new possibilities in developing selective assays to study the functional roles of the various isoforms of inositol phosphates.     

'One bead two compound libraries' for detecting chemical and biochemical conversions

When combinatorial chemistry was introduced 13 years ago, the expectations were high for the delivery of results, particularly in the pharmaceutical industry. However, combinatorial chemistry was implemented independently of the application for which the products were going to be used. Resins developed only for efficient solid-phase synthesis were used and products were employed in existing assays developed for traditional solution studies. There was almost no assay or technology development and the use of real combinatorial methods soon had to give way to high-throughput synthesis and traditional screening. However, during recent years more sophisticated resins and assay techniques have been developed that may result in a second and more successful implementation of real integrated combinatorial chemistry. The first in this line of new developments is the 'one bead two compound' assay, in which the resin bead in addition to a combinatorial library member contains a reporter compound that can act as a beacon to monitor the activity of the library member. This powerful concept can be generally applied in all fields of combinatorial chemistry including drug, catalysts and material development.     

Development of a highly sensitive, high-throughput, mass spectrometry-based assay for rat procollagen type-I N-terminal propeptide (PINP) to measure bone formation activity

Type-I procollagen aminoterminal propeptide (PINP) is a useful biomarker for bone formation activity that is used to monitor treatment of bone disorders including osteoporosis. Studies with human patients under long-term therapy for osteoporosis by daily injection of parathyroid hormone (PTH) demonstrated that the circulating level of PINP at 3 months of treatment, measured by radioimmunoassay, was a good predictor for bone mineral density (BMD) at 18 months. It is important to have PINP assays for other species to elucidate processes of bone formation and for the development of new therapeutic options that can enhance bone formation activity. Currently, only a human PINP radioimmunoassay is commercially available for clinical use, which may not be cross reactive with PINP from other species. For example, rat PINP has little amino acid sequence homology to human PINP. Therefore, we developed a new, highly sensitive, high-throughput mass spectrometry-based assay for PINP from rat plasma or serum that does not rely on antibody reagents. Circulating levels of PINP showed age-dependent changes in rats. Prednisolone treatment, which is known to retard bone formation activity, led to a significant decrease in PINP, whereas PTH treatment dose-dependently increased PINP. The throughput of the assay parallels that of most antibody-based assays so that it can handle a large number of samples that are generated from preclinical animal studies. PINP in rats may serve as a biomarker for bone formation activity, and this assay could be instrumental in studying bone physiology in rat experimental models.