Interaction of human leukocyte elastase with soluble and insoluble protein substrates. A practical kinetic approach

A progress-curve kinetic method was developed to investigate the interaction between human leukocyte elastase and macromolecular substrates, such as insoluble elastin and soluble plasma proteins. A fluorogenic, synthetic peptide (reporter substrate) was incubated in the presence of finely powdered elastin and enzyme under continuous stirring. The progress curves, which corresponded to the release of product from the reporter substrate, were very sensitive to the presence of various amounts of the macromolecular substrate. The kinetic parameters for the interaction between elastase and elastin were calculated using a pre-steady-state approach characteristic of slow-binding inhibitors. The interaction of elastase with the soluble protein substrates was studied with similar techniques, but formally treating the substrates as classical, fully competitive inhibitors. The adsorption of elastase on insoluble elastin was a time-dependent process consisting of at least three observable phases: The first step was a rapid formation of an encounter complex followed by a very slow step lasting several minutes, and the third step consisted of a steady-state release of products. On the contrary, elastase very rapidly formed productive complexes with bovine serum albumin and a human monoclonal immunoglobulin G. The progress-curve method was also suitable for analyzing the behavior of inhibitors in the presence of protein substrates. The kinetic parameters which characterize the interaction between elastase and protein substrates represent a practical tool to formulate hypotheses on the efficiency of inhibitors in vivo.     

运用α-氰代酯荧光底物检测抗性棉蚜羧酸酯酶代谢活性

为了研究抗性和敏感棉蚜Aphis gossypii品系对菊酯类药剂代谢的差异, 本实验合成了溴氰菊酯和高效氯氰菊酯报告荧光底物, 应用这两种底物水解后生成具有荧光化合物的特性,测定了不同品系棉蚜羧酸酯酶的代谢活性。结果表明: 氧化乐果棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为10.0和3.4 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为4.0和2.4 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的2.9和1.7倍; 溴氰菊酯棉蚜抗性和敏感品系羧酸酯酶对溴氰菊酯报告荧光底物的代谢活性分别为7.6和6.2 pmol/min·mg; 对高效氯氰菊酯报告荧光底物的代谢活性分别为9.3和5.2 pmol/min·mg, 抗性品系羧酸酯酶对溴氰菊酯和高效氯氰菊酯报告荧光底物的代谢活性分别为敏感品系的1.2和1.8倍。这种衍生的报告荧光底物能够用来检测抗性棉蚜羧酸酯酶的水解活性, 表明羧酸酯酶可能参与棉蚜对溴氰菊酯和氧化乐果抗性的形成。     

Muscle pyruvate kinase: interaction with substrates and analogues studied by difference spectroscopy. Comparative studies of the substrate-binding sites of various ATP phosphotransferases.

The substrate binding sites of pyruvate kinase have been studied by means of spectrophotometric investigations. Two binding sites, one for the nucleotide substrate and one for the acceptor, have been characterized. The interaction of nucleotide substrates with the enzyme, which is metal-dependent, results in a perturbation of the spectrum of the nucleotide chromophore characterized by hypochromic and red shift effects; the hydrophobicity of the nucleotide site was estimated by using a reporter group reagent, 2-(dansylamino)ethyl monophosphate. The comparison between the binding sites of several ATP phosphotransferases is discussed and some common features are reported.     

Real time visualization of protein kinase activity in living cells.

A library of fluorescently labeled protein kinase C (PKC) peptide substrates was prepared to identify a phosphorylation-induced reporter of protein kinase activity. The lead PKC substrate displays a 2.5-fold change in fluorescence intensity upon phosphorylation. PKC activity is readily sampled in cell lysates containing the activated PKCs. Immunodepletion of conventional PKCs from the cell lysate eliminates the fluorescence response, suggesting that this peptide substrate is selectively phosphorylated by PKCalpha, beta, and gamma. Finally, living cells microinjected with the peptide substrate exhibit a 2-fold increase in fluorescence intensity upon exposure to a PKC activator. These results suggest that peptide-based protein kinase biosensors may be useful in monitoring the temporal and spatial dynamics of PKC activity in living cells.     

Neuraminidase-triggered activation of prodrug-type substrate of 4-nitroaniline

Artificial substrates for probing neuraminidase activity are powerful tools for studying the physiological and pathological roles of neuraminidases. Most of the substrates are α-O-linked sialosides involving hydroxyl-containing reporters for visualization, and neuraminidase-catalyzed cleavage of the sialic acid residues directly activates the reporters. However, the use of amine-containing reporters has been avoided because α-N-linked sialosides are marginal substrates for neuraminidases. To expand the applicability of reporters to amine-containing compounds, we have focused on prodrug design. Herein we describe the synthesis and enzymatic study of a model substrate involving 4-nitroaniline as an amine-containing chromogenic reporter. The substrate can respond to neuraminidase from Clostridium perfringens. Neuraminidase-mediated hydrolysis of the sialic acid moiety of the substrate initiates self-immolative elimination of the linker moiety, leading the liberation of yellow-colored reporter 4-nitroaniline. The elimination process involves generation of quinone methide intermediate, which causes to neutralize neuraminidase. The substrate, thus, works as not only a chromogenic substrate but also a suicide inactivator.     

A survey of the kinetic parameters of class C beta-lactamases. Cephalosporins and other beta-lactam compounds.

Various cephalosporins, cefoxitin, moxalactam, imipenem and aztreonam were studied as substrates of six class C beta-lactamases. Nitrocefin, cephaloridine, cefazolin, cephalothin and cephalexin were good substrates, with kcat. values ranging from 27 to 5000 s-1. Cefuroxime, cefotaxime and cefoxitin exhibited low kcat. values (0.010-1.7 s-1) and low Km values, which suggested a rate-limiting deacylation. Imipenem and aztreonam were even poorer substrates (kcat. 2 x 10(-4)-3 x 10(-2) s-1) and, in the presence of a reporter substrate, behaved as transient inactivators. With moxalactam, biphasic kinetics were observed, indicating a possible rearrangement of the acyl-enzyme.     

Sensitive analysis of recombination activity using integrated cell surface reporter substrates.

BACKGROUND:Recombination processes play a crucial role in the functioning of the immune system and are also involved in mutation events that result in various malignancies. So far the study of recombination activity has frequently relied on the use of reporter substrates that are limited by low sensitivity as well as tedious and distorting readout procedures. METHODS:Immunoglobulin class switch recombination substrates were generated which, upon recombination, resulted in the surface expression of human CD4 or murine MHC class I H-2K(k) and thus allowed for cytometric evaluation. RESULTS:Recombining cells harboring integrated reporter substrates were analyzed by immunofluorescence and flow cytometry and could easily be isolated by high-gradient magnetic cell sorting (MACS). The analysis was not influenced by cloning efficiencies, as would be the case after drug selection, or prokaryotic recombination that might occur after analysis of recovered substrates in bacteria. In addition, cytometric readout is much faster, as it can be performed immediately after recombination. The substrate exhibited properties compatible with the detection of immunoglobulin class switch recombination and permitted the detection of recombination events down to 10(-5) per cell and generation. CONCLUSIONS:The high sensitivity of this system allows precise detection of very rare recombination events and thus permits the study of cell types with extremely low recombination activities.     

A novel fluorogenic substrate for ribonucleases. Synthesis and enzymatic characterization.

The synthesis and enzymatic characterization of DUPAAA, a novel fluorogenic substrate for RNases of the pancreatic type is described. It consists of the dinucleotide uridylyl-3',5'-deoxyadenosine to which a fluorophore, o-aminobenzoic acid, and a quencher, 2,4-dinitroaniline, have been attached by means of phosphodiester linkages. Due to intramolecular quenching the intact substrate displayed very little fluorescence. Cleavage of the phosphodiester bond at the 3'-side of the uridylyl residue by RNase caused a 60-fold increase in fluorescence. This allowed the continuous and highly sensitive monitoring of enzyme activity. The substrate was turned over efficiently by RNases of the pancreatic type, but no cleavage was observed with the microbial RNase T1. Compared to the dinucleotide substrate UpA, the specificity constant with RNase A, RNase PL3 and RNase U(s) increased 6-, 18-, and 29-fold, respectively. These differences in increased catalytic efficiency most likely reflect differences in the importance of subsites on the enzyme in the binding of elongated substrates. Studies on the interactions of RNase inhibitor with RNase A using DUPAAA as a reporter substrate showed that it was well suited for monitoring this very tight protein-protein interaction using pre-steady-state kinetic methods.     

Introduction of a novel HRP substrate-Nanogold probe for signal amplification in immunocytochemistry.

Amplification of immunological signals with catalyzed reporter deposition (CARD) allows improved detection of scarce tissue antigens in light and electron microscopy. The technique takes advantage of the oxidation ability of horseradish peroxidase (HRP), in the presence of hydrogen peroxide, to yield the accumulation of one of its specific reporter-tagged substrates. This immunocytochemical approach continues to be improved by the introduction of new reporter molecules tagged to tyramine or to other HRP substrates. In this study we introduced a novel HRP substrate tagged to Nanogold particles. The amplification protocol is based on the application of a specific primary antibody, a biotinylated secondary antibody, streptavidin-HRP, and an HRP substrate coupled to Nanogold, followed by silver intensification. In addition to amplification of immunological signals of high resolution, direct accumulation of Nanogold particles at target sites by enzymatic activity of HRP improves the efficiency of the technique compared to other amplification protocols. Moreover, this approach combines the CARD amplification potentials with the ultrasmall gold probe and the silver intensification method. Immunolabeling obtained by light and electron microscopy, as well as immunodot assay using this new amplification strategy, appear to be highly sensitive, specific, and of enhanced intensity.     

Steady-state kinetic analysis of human cholinesterases over wide concentration ranges of competing substrates

Substrate competition for human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) was studies under steady-state conditions using wide range of substrate concentrations. Competing couples of substates were acetyl-(thio)esters. Phenyl acetate (PhA) was the reporter substrate and competitor were either acetylcholine (ACh) or acetylthiocholine (ATC). The common point between investigated substrates is that the acyl moiety is acetate, i.e. same deacylation rate constant for reporter and competitor substrate.Steady-state kinetics of cholinesterase-catalyzed hydrolysis of PhA in the presence of ACh or ATC revealed 3 phases of inhibition as concentration of competitor increased: a) competitive inhibition, b) partially mixed inhibition, c) partially uncompetitive inhibition for AChE and partially uncompetitive activation for BChE. This sequence reflects binding of competitor in the active centrer at low concentration and on the peripheral anionic site (PAS) at high concentration. In particular, it showed that binding of a competing ligand on PAS may affect the catalytic behavior of AChE and BChE in an opposite way, i.e. inhibition of AChE and activation of BChE, regardless the nature of the reporter substrate.For both enzymes, progress curves for hydrolysis of PhA at very low concentration (?K_m) in the presence of increasing concentration of ATC showed that: a) the competing substrate and the reporter substrate are hydrolyzed at the same time, b) complete hydrolysis of PhA cannot be reached above 1 mM competing substrate. This likely results from accumulation of hydrolysis products (P) of competing substrate and/or accumulation of acetylated enzyme·P complex that inhibit hydrolysis of the reporter substrate.     

Photometric and electrochemical enzyme-multiplied assay techniques using beta-galactosidase as reporter enzyme

Beta-galactosidase (beta-gal) is shown to be a versatile new reporter enzyme in both photometric and electrochemical enzyme-multiplied assay techniques (EMATs). The well-known beta-gal substrate analog, o-nitrophenyl beta-d-galactopyranoside, yields the visibly colored, o-nitrophenol product upon hydrolysis, whereas the substrate, p-aminophenyl beta-D-galactopyranoside, gives rise to an electrooxidizable product, p-aminophenol. These beta-gal substrates made possible the demonstration of both photometric and electrochemical signal transduction schemes for beta-gal-based EMAT detection of estradiol (as the estradiol-bovine serum albumin (E-BSA) conjugate). The EMAT system is composed of the reporter enzyme, beta-gal, with covalently attached estradiol, and estrogen antibody, which inhibits enzyme activity of the beta-gal-estradiol conjugate up to approximately 75%. Reporter enzyme inhibition is relieved significantly by addition of < or =2 ng/mL of estradiol (as E-BSA), which competes for binding with the antibody. Thus, the presence of analyte (E-BSA) is reported by the enzyme (beta-gal), which amplifies the ligand-protein dissociation event by turning over its substrate repeatedly. The electrochemical version of EMAT, based on amperometric detection of p-aminophenol, is responsive to added estradiol within minutes. These results show that beta-gal may serve as a useful alternative to glucose-6-phosphate dehydrogenase, which currently is used as reporter enzyme in commercially available EMAT systems.     

Functional analysis of a Golgi-localized Kex2p-like protease in tobacco suspension culture cells

Kex2p is the prototype of a Golgi-resident protease responsible for the processing of prohormones in yeast and mammalian cells. A Kex2p-like pathway was shown to be responsible for processing the fungal KP6 protoxin in transgenic tobacco plants. We previously described a chimeric integral membrane reporter protein that traffics through Golgi to the lytic prevacuole where it was proteolytically processed. As a first step to isolate and clone the Kex2p-like protease in plant cells, we designed and used a similar chimeric reporter protein containing Kex2 cleavage sites to assay the Kex2p-like activity and to determine its substrate specificity in tobacco cells. Here we demonstrate that the Kex2 cleavage sites of the reporter were specifically processed by a protease activity with a substrate specificity characteristic of yeast Kex2p. This Kex2p-like protease in tobacco cells is also a Golgi-resident enzyme. Thus, the reporter protein provides a biochemical marker for studying protein traffic through the Golgi in plant cells. These results additionally should allow the design of synthetic substrates for use in biochemical purification of the plant enzyme.     

Factors influencing expression of luxCDABE and nah genes in Pseudomonas putida RB1353(NAH7, pUTK9) in dynamic systems.

Bioluminescent reporter organisms have been successfully exploited as analytical tools for in situ determination of bioavailable levels of contaminants in static environmental samples. Continued characterization and development of such reporter systems is needed to extend the application of these bioreporters to in situ monitoring of degradation in dynamic environmental systems. In this study, the naphthalene-degrading, lux bioreporter bacterium Pseudomonas putida RB1353 was used to evaluate the relative influences of cell growth stage, cell density, substrate concentration, oxygen tension, and background carbon substrates on both the magnitude of the light response and the rate of salicylate disappearance. The effect of these variables on the lag time required to obtain maximum luminescence and degradation was also monitored. Strong correlations were observed between the first three factors and both the magnitude and induction time of luminescence and degradation rate. The maximum luminescence response to nonspecific background carbon substrates (soil extract broth or Luria broth) was 50% lower than that generated in response to 1 mg of sodium salicylate liter(-1). Oxygen tension was evaluated over the range of 0.5 to 40 mg liter(-1), with parallel inhibition to luminescence and degradation rate (20 mg of sodium salicylate liter(-1)) observed at 1.5 mg liter(-1) and below and no effect observed above 5 mg liter(-1). Oxygen tensions from 2 to 4 mg liter(-1) influenced the magnitude of luminescence but not the salicylate degradation rate. The results suggest that factors causing parallel shifts in the magnitude of both luminescence and degradation rate were influencing regulation of the nah operon promoters. For factors that cause nonparallel shifts, other regulatory mechanisms are explored. This study demonstrates that lux reporter bacteria can be used to monitor both substrate concentration and metabolic response in dynamic systems. However, each lux reporter system and application will require characterization and calibration.     

Monitoring the reaction of carbachol with acetylcholinesterase by thioflavin T fluorescence and acetylthiocholine hydrolysis

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex and proceed to an acylated enzyme intermediate which is then hydrolyzed. However, the hydrolysis of the carbamoylated enzyme is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here we show that the reaction of carbachol (carbamoylcholine) with AChE can be monitored both with acetylthiocholine as a reporter substrate and with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. These fluorescence changes allow not only the monitoring of the course of the carbamoylation reaction but also the determination of carbachol affinities for the A- and P-sites.     

Recent advances in catalytic peroxidase histochemistry.

Immunoassays have developed to become an important analytical tool in life sciences for detection of endogenous and exogenous targets. Among the most important enzyme labels horseradish peroxidase (HRP), alkaline phosphatase (AP), and beta-D-galactosidase (GAL) is HRP the smallest enzyme and plays nowadays an outstanding role. The oldest substrates are chromogens widely applied for localization of sites of peroxidase (PO) activity in histochemistry as well as for colorimetric applications. They are represented by a diversity of aromatic amines and phenols. Encouraged by development of light excitation and measuring techniques and the commercial availability of highly sensitive equipment, luminescent labels represent the most sensitive and worthwhile detection tools to date. In contrast to chromogens fluorescent labels for detection PO activity are confined only to a few substrates developed more recently. These substrates are mostly applied in histochemistry at a short time scale due to their frequently high solubility. At the long time scale sole exception is so far the tyramine based fluorochome deposition technique (more general: catalytic reporter deposition, CARD). Despite quite different staining behavior both fluorometric and product deposition related principles are based on 4-hydroxy phenylalkyl substrates. The following article reviews basic principles of peroxidatic substrate degradation processes including chromogenic and fluorescent approaches with emphasis on recent advances in development of chromogens and fluorogens for application in histology. As a result of systematic efforts towards the design of substrates, the range of classical precipitating chromogens as well as fluorescent techniques could be complimented by novel highly sensitive substrates with superior staining capabilities: a) Metal chelating 2-hydroxy benzylamines are derived from classical aniline substrates (two steps) and utilize metal catalytic effects in an efficient intramolecular way. The enzymatically yielded dark colored polycondensation products are applicable in histochemistry, in colorimetry and especially as precipitating electron opaque labels with enhanced osmiophilic properties for light and electron microscopy. b) Fluorescent 4-hydroxy-styryl derivatives are capable of oxidative selfanchoring reactions at the cellular level close to sites of PO activity. In contrast to deposition of tyramine conjugated fluorochromes an altered fluorochrome with improved fluorescence properties is furnished during oxidative crosslinking of the substrate. This results in a highly specific and photostable fluorescence response and an outstanding low background staining. Histochemical and immunohistochemical applications are presented.     

Enhanced detection of beta-galactosidase reporter activation is achieved by a reduction of hemoglobin content in tissue lysates

beta-galactosidase (beta-gal), the product of the E. coli LacZ gene, has been used extensively as a reporter in numerous systems. Until recently, the most commonly used method of detecting beta-gal reporter enzymatic activity was a colormetric assay based on the cleavage of the beta-gal substrate 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) to form a blue precipitate. However, when increased sensitivity is needed, many investigators now turn to alternate substrates that produce fluorescent or luminescent products upon cleavage by beta-gal. These products are much more easily quantified than X-gal. The luminescent and fluorometric assays work very well in cultured cells but are often less sensitive in whole tissue lysates. In this study, we have evaluated the sensitivity of a fluorescent and a luminescent substrate in whole tissue lysates cleared of red blood cells or washed with PBS only. We have found that both assays show increased low-end sensitivity in tissues with reduced levels of hemoglobin (Hb). Hb is apparently able to quench luminescent and, to a lesser degree, fluorescent reporter light emission. Therefore, steps should be taken to reduce Hb levels either by lysis, perfusion, or both to enhance the sensitivity of these assays.     

Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay

A radiometric procedure to detect the presence of proteolytic enzymes and analyze their substrate specificity is described. The enzymatic activity is first measured by the release into solution of a radiolabeled reporter group from an immobilized peptidyl substrate. Two peptidyl substrates encompassing multiple cleavage sites, a derivative of Leu-enkephalin and a peptide related to the bait region of human alpha 2-macroglobulin, are prepared and linked via a spacer molecule to an insoluble support. The labeled peptides released are then separated by high-performance liquid chromatography. The position of the released peptides upon chromatography allows direct identification of the sites of cleavage. The assay, using a radioactive iodinated tyrosine residue as reporter group, is extremely sensitive (less than 0.02 pg/ml of trypsin), reproducible, and easy to perform while yielding unambiguous identification of the sites of cleavage. This assay can be used to detect the presence of enzymatic activities and/or of enzyme inhibitors. Furthermore, it can be easily adapted to detect from a variety of sources all four classes of enzymes known by using appropriate peptidyl substrate sequences, buffer, pH, and incubation conditions.