A sensitive automated method for adenosine triphosphatase kinetics.

An automated method for measuring adenosine triphosphatase (ATPase) activity is described. A modified version of a Technicon Autoanalyzer utilizing a sensitive colourimetric technique for determining inorganic phosphate concentrations (1 nmol/ml) allows investigations on enzymes of low specific activities. Dialysis may be used for measuring tissue homogenate activities or bypassed by examining purified enzyme preparations. When combined with a gradient apparatus, the proposed technique is particularly well suited for the study of enzyme kinetics in relation to cation or anion concentrations.     

A mass spectrometry-based strategy for detecting and characterizing endogenous proteinase activities in complex biological samples

Endogenous proteinases in biological fluids such as human saliva produce a rich peptide repertoire that reflects a unique combination of enzymes, substrates, and inhibitors/activators. Accordingly, this subproteome is an interesting source of biomarkers for disease processes that either directly or indirectly involve proteolysis. However, the relevant proteinases, typically very low abundance molecules, are difficult to classify and identify. We hypothesized that a sensitive technique for monitoring accumulated peptide products in an unbiased, global manner would be very useful for detecting and profiling proteolytic activities in complex biological samples. Building on the longstanding use of 18O isotope-based approaches for the classification of proteolytic and other enzymatic processes we devised a new method for evaluating endogenous proteinases. Specifically, we showed that upon ex vivo incubation endogenous proteinases in human parotid saliva introduced 18O from isotopically enriched water into the C-terminal carboxylic groups of their peptide products. Subsequent peptide sequence determination and inhibitor profiling enabled the detection of discrete subsets of proteolytic products that were generated by different enzymes. As a proof-of-principle we used one of these fingerprints to identify the relevant activity as tissue kallikrein. We termed this technique PALeO. Our results suggest that PALeO is a rapid and highly sensitive method for globally assessing proteinase activities in complex biological samples.     

Farnesyltransferase as target for non-cytotoxic anti-cancer agents: first steps]

Farnesylation is a key maturation step involved in the ras-dependent transformation of cells. This acylation step is catalyzed by protein: farnesyltransferase, a soluble enzyme. The present work describes the use of a new HPLC method of measurement of this enzymatic activity using the K-ras-derived CVIM tetrapeptide as substrate. The method is used to check the activity catalyzed by cytosols issued from various types of cancer cells. J82, a human bladder cancer cell line was retained for measurement of the inhibitory potency of a few peptide sequences and will be used as starting biological material for the purification of the enzyme. This HPLC method presented herein has the main advantages over other published methods of being automatisable and versatile, because it can be used with a wide spectrum of peptide substrates. Results presented herein are only first studies and need some more structural observations. The obtention of the cancer cell line-derived, partially purified farnesyltransferase will hopefully lead us to the discovery of specific inhibitors with potential non-cytotoxic anti-cancer activities.     

A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase.

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5'-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5'-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5'-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes.     

Novel regulatory interactions and activities of mammalian tRNA synthetases

Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to their cognate tRNAs, thereby ensuring the faithful translation of genetic code. In addition to their enzymatic function, these enzymes have been discovered to regulate various cellular functions such as tRNA export, ribosomal RNA synthesis, apoptosis, inflammation and angiogenesis in mammalian. The insights into the noncanonical activities of these enzymes have been obtained from their unique cellular localization, interacting partners, isoform generation and expression control. Mammalian ARSs also form a macromolecular protein complex with a few auxiliary factors. Although the physiological significance of this complex is poorly understood, it also supports the potential of mammalian ARSs as sophisticated multifunctional proteins for regulating various cellular procedures. In this review, the novel regulatory activities of mammalian ARSs will be discussed in different biological processes.     

Identification of diadenosine triphosphate in Brugia malayi by reverse phase high performance liquid chromatography and MALDI mass spectrometry

The presence of diadenosine oligophosphates (ApnA) in eukaryotic pathogens has been difficult technically to assess and thus is often overlooked. ApnA are a family of intercellular and intracellular signaling molecules and their biological activities differ relative to the number of phosphate moieties. The application of mass spectrometry to differentiate nucleotide phosphates has been limited by the high salt content in tissue extracts, enzymatic reactions or high performance liquid chromatography (HPLC) buffers, as well as the potential for sample loss when processing and desalting small biological samples. To address this problem a simple reverse phase HPLC (RP-HPLC) method using volatile organic buffers at low pH was developed to create elution profiles of adenosine and diadenosine phosphates. To test this method on a eukaryotic pathogen, small intravascular human filarial parasites (Brugia malayi) were extracted in phosphate buffered saline and a nucleotide phosphate profile was visualized by RP-HPLC. A major peak eluting at 10.4 min was analyzed directly by mass spectrometry and this confirmed the presence of significant quantities of diadenosine triphosphate, Ap3A. Application of this simplified RP-HPLC method will facilitate research on the normal and pathophysiological effects of ApnA particularly in situations when analysis of small biological samples is required.     

Effects of seasonal and decadal warming on soil enzymatic activity in a P‐deficient Mediterranean shrubland

Soil enzymes are central in the response of terrestrial ecosystems to climate change, and their study can be crucial for the models’ implementation. We investigated for 1 year the effects of warming and seasonality on the potential activities of five soil extracellular enzymes and their relationships with soil moisture, phosphorus (P) concentration, and other soil parameters in a P‐limited Mediterranean semiarid shrubland. The site was continuously subjected to warming since 1999, and we compared data from this study to analogous data from 2004. Warming uniformly increased all enzymes activities, but only when a sufficient amount of soil water was available. Seasonality unevenly altered enzyme activities, thus affecting enzymatic stoichiometry. P deficiency affected enzymatic stoichiometry, favoring the activities of the phosphatases. The effect of warming was stronger in 2014 than 2004, excluding the hypothesis of acclimation of rhizospheric responses to higher temperatures and suggesting that further increases in extracellular enzymatic activities are to be expected if sufficient water is available. Climatic warming will likely generally stimulate soil enzymatic activities and accelerate nutrient mineralization and similar ecological processes such as the production and degradation of biomass and changes in community composition, but which will be limited by water availability, especially in Mediterranean soils in summer. Winters in such ecosystems will benefit from a general increase in activity and production, but biological activity could even decrease in summer, potentially leading to a negative overall balance of nutrient mineralization. This study suggests that a general increase in activity due to warming could lead to faster mineralization of soil organic matter and water consumption in colder climates, until one of these factors in turn becomes limiting. Such trade‐offs between water and temperature in relation with enzyme activity should be considered in biogeochemical models.     

Biochemical changes as enzymatic defense system of phenanthrene exposure in olive flounder, Paralichthys olivaceus

The objective of this study was to investigate early biological response in olive flounder exposed to sub-lethal concentrations of waterborne phenanthrene (0.5, 1 or 2 microM). The fish were exposed for 4 weeks and we analyzed their enzymatic defense system, antioxidant and phase II enzyme activities, to evaluate the chronic exposure toxicity of phenanthrene. Waterborne phenanthrene affected antioxidant enzymes and glutathione-mediated detoxification as enzyme defense system. Hepatic, gill and kidney glutathione reductase as well as glutathione S-transferase, and catalase activities were markedly elevated after two or four weeks of exposure. These enzymes activities of olive flounder, Paralichthys olivaceus seem to be a convenient tool for monitoring pollution in coastal areas against PAHs pollution including phenanthrene.     

Activity based subcellular resolution imaging of lipases

Lipases play a key role in whole body energy homeostasis. Dysregulation of lipolytic activities affects lipid absorption, mobilization, and transport, and is causative for lipid-related diseases. Regulation of enzymes involved in lipid metabolism is governed by a complex network of protein-protein and protein-small molecule interactions. Thus these enzymes have to be studied under the physiologically most relevant conditions, that is, in vivo. Our latest generation of activity based probes designed for capturing of lipases employs bioorthogonal chemical linker groups, which are membrane permeable and thus allow studying protein activity in living cells. Another advantage is the virtually unlimited choice of reporter tags. Here we report on a novel method combining in vivo activity based labeling of lipases with in situ detection of lipolytic activities by on slide click chemistry and imaging by fluorescence microscopy. We demonstrate that cytosolic as well as organelle resident lipases are specifically labeled in intact living cells. This method will shed light on the (sub)cellular localization of lipolytic proteomes of cells and tissues in health and disease directly at enzymatic activity level without the need of prior knowledge of the identities of the responsible enzymes or dependence on the availability of specific antibodies.     

Mass spectrometry analysis of oxidized phosphatidylcholine and phosphatidylethanolamine

Oxidized phospholipids (OxPLs) are rapidly becoming recognized as important mediators of cellular and immune signaling. They are generated either enzymatically or non-enzymatically and 100s of structures exist of which only a small fraction have been analyzed to date. Pleiotropic activities, including regulation of adhesion molecule expression, pro-coagulant activity and inhibition of Toll-like receptor signaling have been observed and some are detected in models of human and animal disease, including atherosclerosis and infection. More recently, the acute generation of specific oxidized phospholipids by cellular enzymes in immune cells was reported. Assays for analysis and quantification of OxPLs were first developed approx 15years ago, primarily for hydro(pero)xy-species. Many were based on monitoring a single precursor ion with/without LC separation, based on the PL headgroup. Others combined LC with monitoring precursor to product transitions, but were unable to provide information regarding position of oxidation on unsaturated sn-2 fatty acid due to sensitivity issues. More recently, LC/MS/MS methods for specific OxPLs have been reported that enable high sensitivity quantitation in biological samples. In this review, widely used methods for detecting and quantifying various classes of OxPL will be summarized, along with practical advice for their use. In particular, the focus will be on LC/MS/MS, which today is almost universally the method of choice.     

Tobacco,a highly efficient green bioreactor for production of therapeutic proteins

Molecular farming of pharmaceuticals in plants has the potential to provide almost unlimited amounts of recombinant proteins for use in disease diagnosis, prevention or treatment. Tobacco has been and will continue to be a major crop for molecular farming and offers several practical advantages over other crops. It produces significant leaf biomass, has high soluble protein content and is a non-food crop, minimizing the risk of food-chain contamination. This, combined with its flexibility and highly-efficient genetic transformation/regeneration, has made tobacco particularly well suited for plant-based production of biopharmaceutical products. The goal of this review is to provide an update on the use of tobacco for molecular farming of biopharmaceuticals as well the technologies developed to enhance protein production/purification/efficacy. We show that tobacco is a robust biological reactor with a multitude of applications and may hold the key to success in plant molecular farming.     

Advances in using MRI probes and sensors for in vivo cell tracking as applied to regenerative medicine

The field of molecular and cellular imaging allows molecules and cells to be visualized in vivo non-invasively. It has uses not only as a research tool but in clinical settings as well, for example in monitoring cell-based regenerative therapies, in which cells are transplanted to replace degenerating or damaged tissues, or to restore a physiological function. The success of such cell-based therapies depends on several critical issues, including the route and accuracy of cell transplantation, the fate of cells after transplantation, and the interaction of engrafted cells with the host microenvironment. To assess these issues, it is necessary to monitor transplanted cells non-invasively in real-time. Magnetic resonance imaging (MRI) is a tool uniquely suited to this task, given its ability to image deep inside tissue with high temporal resolution and sensitivity. Extraordinary efforts have recently been made to improve cellular MRI as applied to regenerative medicine, by developing more advanced contrast agents for use as probes and sensors. These advances enable the non-invasive monitoring of cell fate and, more recently, that of the different cellular functions of living cells, such as their enzymatic activity and gene expression, as well as their time point of cell death. We present here a review of recent advancements in the development of these probes and sensors, and of their functioning, applications and limitations.KEY WORDS: Regenerative medicine, Stem cells, Magnetic resonance imaging, Paramagnetic contrast agents, CEST, Perfluorocarbon particles, Biosensor, Cell labeling, Cellular function     

Finding Sequences for over 270 Orphan Enzymes

Despite advances in sequencing technology, there are still significant numbers of well-characterized enzymatic activities for which there are no known associated sequences. These ‘orphan enzymes’ represent glaring holes in our biological understanding, and it is a top priority to reunite them with their coding sequences. Here we report a methodology for resolving orphan enzymes through a combination of database search and literature review. Using this method we were able to reconnect over 270 orphan enzymes with their corresponding sequence. This success points toward how we can systematically eliminate the remaining orphan enzymes and prevent the introduction of future orphan enzymes.     

Principle and application of a multicuvette for determination of enzyme activities and substrate concentrations in microsamples.

A method of photometric analysis is described that allows to determine enzyme activities or substrate concentrations of 50 samples simultaneously in a multicuvette. Test volume is in the range of 30-85 micronl, light path between 0.5 and 2.0 mm. The testing principle can be used for colorimetric measurements and for NADH dependent reactions as well. The procedure is particularly suited for kinetic determinations. Variation coefficients for determinations of substrate concentrations and enzyme activities are in the range of conventional enzymatic tests.     

Immobilized enzymes – valuable tools for the indication of temperature events

Abstract

The enzymes trypsin and urease were covalently tethered to cellulose to utilize their ability to produce colored products as a consequence of enzymatic activity. Therefore, cellulose had to be chemically modified first in order to generate appropriate chemical functionalities. Different approaches including periodate supported oxidation followed by immobilization via reductive amination, insertion of a reactive polymer interface, and cross-linking inside the cellulose matrix were utilized for the immobilization. The success of immobilization was assessed by the quantification of surface-bound protein as well as by recording of enzymatic activities under different conditions. The enzymatic activity of trypsin and urease was maintained best when a hydrophilic intermediate polymer layer was used for immobilization. The applicability of immobilized enzymes as temperature indicators was demonstrated using cross-linked urease.     

In situ staining of activities of enzymes involved in carbohydrate metabolism in plant tissues.

A powerful technique is described to localize the activities of a range of enzymes in a wide variety of plant tissues. The method is based on the coupling of the enzymatic reaction to the reduction of NAD and subsequent reduction and precipitation of nitroblue tetrazolium. Enzymes that did not reduce NAD could be visualized by coupling their activities to glucose-6-phosphate dehydrogenase activity via one or more intermediary 'coupling' enzymes. The method is shown to be applicable for the detection of the activities of hexokinase, fructokinase, sucrose synthase, uridine 5'-diphospho-glucose pyrophosphorylase, ADP-glucose pyrophosphorylase, phosphoglucomutase, and phosphoglucose isomerase. It could be used for all tissues tested, including green leaves, stems, roots, fruits, and seeds. The method is specific, very sensitive, and has a high spatial resolution, giving information at the cellular and the subcellular level. The localization of sucrose synthase, invertase, and uridine 5'-diphospho-glucose pyrophosphorylase in transgenic potato plants, carrying a cytokinin biosynthesis gene, is studied and compared with wild-type plants.     

Selective permeabilization for the high-throughput measurement of compartmented enzyme activities in mammalian cells

Permeabilization was evaluated as a rapid method to prepare mammalian cells for subcellular enzyme activity measurement. It was observed that enzymes can be measured directly in cell suspensions permeabilized by Triton X-100 and digitonin with various concentrations. Total enzyme activities measured in permeabilized cells were identical to those measured in sonicated cells showing that permeabilization can replace the more complicated sonication method. Tuning of digitonin concentration allowed selective permeabilization of plasma and mitochondrial membranes. This was studied by analyzing the release of extramitochondrial and mitochondrial marker enzymes on treatment with different concentrations of the agent. Solely the plasma membrane was permeabilized by using 0.01–0.02% (w/v) digitonin. Access to all cellular enzymes was achieved by using 0.05% (v/v) Triton X-100. This selective permeabilization was further evaluated in a 96-well plate format by testing additional marker enzymes and additional cell lines, Hep G2 and CHO-K1, applying the developed protocol. The presented method is well suited for the high-throughput analysis of subcellular localization and activity of enzymes. The method is simple and enables one to distinguish between mitochondrial and extramitochondrial activities, which is usually achieved only by much more complicated and time-consuming cell preparation.     

The use of biological activities to monitor the removal of fuel contaminants—perspective for monitoring hydrocarbon contamination: a review

Soil biological activities are vital for the restoration of soil contaminated with hydrocarbons. Their role includes the biotransformation of petroleum compounds into harmless compounds. In this paper, the use of biological activities as potential monitoring tools or bioindicators during bioremediation of hydrocarbon-contaminated soil are reviewed. The use of biological activities as bioindicators of hydrocarbon removal in soil has been reported with variable success. This variability can be attributed partially to the spatial variability of soil properties, which undoubtedly plays a role in the exposure of organisms to contaminants. Widely used bioindicators have been enzyme activities, seed germination, earthworm survival and microorganisms or microbial bioluminescence. A mixture of some successful utilization of biological activities and several failures, and inconsistencies reported, show that at this stage there is no general guarantee of successful utilization of biological activities as monitoring tools. Wherever possible, the use of biological activities as bioindicators of hydrocarbon removal must be used to complement existing traditional monitoring tools.     

Screening of enzymatic activities for the depolymerisation of the marine bacterial exopolysaccharide HE800

The exopolysaccharide (EPS) HE800 is a marine-derived polysaccharide (from 8 × 10(5) to 1.5 × 10(6) g mol(-1)) produced by Vibrio diabolicus and displaying original structural features close to those of glycosaminoglycans. In order to confer new biological activities to the EPS HE800 or to improve them, structural modifications need to be performed. In particular, depolymerisation is required to generate low-molecular-weight derivatives. To circumvent the use of chemical methods that lack specificity and reproducibility, enzymes able to perform such reaction are sought. This study reports the screening for enzymes capable of depolymerising the EPS HE800. A large diversity of enzyme sources has been studied: commercially available glycoside hydrolases with broad substrate specificity, lyases, and proteases as well as growing microorganisms. Interestingly, we found that the genus Enterococcus and, more particularly, the strain Enterococcus faecalis were able to depolymerise the EPS HE800. Partial characterization of the enzymatic activity gives evidence for a random and incomplete depolymerisation pattern that yields low-molecular-weight products of 40,000 g mol(-1). Genomic analysis and activity assays allowed the identification of a relevant open reading frame (ORF) which encodes an endo-N-acetyl-galactosaminidase. This study establishes the foundation for the development of an enzymatic depolymerisation process.     

Ribosome-inactivating proteins from plants: more than RNA N-glycosidases?

Many plants contain proteins that are capable of inactivating ribosomes and accordingly are called ribosome-inactivating proteins or RIPs. These typical plant proteins receive a lot of attention in biological and biomedical research because of their unique biological activities toward animal and human cells. In addition, evidence is accumulating that some RIPs play a role in plant defense and hence can be exploited in plant protection. To understand the mode of action of RIPs and to optimize their medical and therapeutical applications and their use as antiviral compounds in plant protection, intensive efforts have been made to unravel the enzymatic activities of RIPs and provide a structural basis for these activities. Though marked progress has been made during the last decade, the enzymatic activity of RIPs has become a controversial issue because of the concept that RIPs possess, in addition to their classical RNA N-glycosidase and polynucleotide:adenosine glycosidase activity, other unrelated enzymatic activities. Moreover, the presumed novel enzymatic activities, especially those related to diverse nuclease activities, are believed to play an important role in various biological activities of RIPs. However, both the novel enzymatic activities and their presumed involvement in the biological activities of RIPs have been questioned because there is evidence that the activities observed are due to contaminating enzymes. We offer a critical review of the pros and cons of the putative novel enzymatic activities of RIPs. Based on the available data, it is suggested that there is little conclusive evidence in support of the presumed activities and that in the past too little attention has been given to the purity of the RIP preparation. The antiviral activity and mode of action of RIPs in plants are discussed in view of their classical and presumed novel enzymatic activities.