Search

This study aimed to investigate the efficacy of the combination of neuron-specific enolase (NSE) measurement and initial neurological examination in predicting the neurological outcomes of patients with cardiac arrest (CA) by retrospectively analyzing data from the Korean Hypothermia Network prospective registry. NSE levels were recorded at 48 and 72 h after CA. The initial Full Outline of UnResponsiveness (FOUR) and Glasgow Coma Scale (GCS) scores were recorded. These variables were categorized using the scorecard method. The primary endpoint was poor neurological outcomes at 6 months. Of the 475 patients, 171 (36%) had good neurological outcomes at 6 months. The areas under the curve (AUCs) of the categorized NSE levels at 72 h, GCS score, and FOUR score were 0.889, 0.722, and 0.779, respectively. The AUCs of the combinations of categorized NSE levels at 72 h with categorized GCS scores and FOUR score were 0.910 and 0.912, respectively. Each combination was significantly higher than the AUC value of the categorized NSE level at 72 h alone (with GCS: p = 0.015; with FOUR: p = 0.026). Combining NSE measurement and initial neurological examination improved the prediction of neurological outcomes., (© 2021. The Author(s).)

Haemophilus influenzae is a common organism of the human upper respiratory tract; this bacterium is responsible of a wide spectrum for respiratory infections and can generate invasive diseases such as meningitis and septicemia. These infections are associated with H. influenzae encapsulated serotype b. However, the incidence of invasive disease caused by nontypeable H. influenzae (NTHi) has increased in the post- H. influenzae serotype b (Hib) vaccine era. Currently, an effective vaccine against NTHi is not available; due to this, it is important to find an antigen capable to confer protection against NTHi infection. In this study, 10 linear B cell epitopes and 13 CTL epitopes and a putative plasminogen-binding motif ( 252 FYNKENGMY 260 ) and the presence of enolase on the surface of different strains of H. influenzae were identified in the enolase sequence of H. influenzae . Both in silico and experimental results showed that recombinant enolase from H. influenzae is immunogenic that could induce a humoral immune response; this was observed mediating the generation of specific polyclonal antibodies anti-rNTHiENO that recognize typeable and nontypeable H. influenzae strains. The immunogenic properties and the superficial localization of enolase in H. influenzae , important characteristics to be considered as a new candidate for the development of a vaccine, were demonstrated., Competing Interests: The authors declared there are no competing financial interests., (Copyright © 2021 Yesenia Osorio-Aguilar et al.)

The method of transferring the immunoreaction from electrode surface to tube can greatly improve the analytical performance of electrochemical immunosensor. In this work, based on PtCu nanoprobe-mediated and iodide-catalyzed cascade reaction, an improved electrochemical immunosensor was elaborately designed for neuron-specific enolase (NSE) detection. PtCu nanoparticles combined with NSE antibody (Ab 2 ) were used as immunoprobes to trigger cascade reaction. With high catalytic activity towards the oxidation of iodide, PtCu nanoprobes catalyzed iodide to iodine in the presence of H 2 O 2 , leading to the decrease of iodide. Subsequently, as a bridge between the tube and iodide-responsive sensing interface, the residual iodide in tube was employed to catalyze the transition from thiol substances (RSH) to disulfide substances (RSSR) on electrode surface. On the basis of this property, thiol-modified DNA (DNA-SH) and 6-mercaptohexanol (MCH) reacted with H 2 O 2 and the residual iodide to form disulfide substances and detach from the electrode surface, causing the decrease of interface resistance in different degrees. Then square wave voltammetry (SWV) was applied for current signal of electrochemical sensing interface to achieve the quantitative detection of NSE. Under optimal conditions, this improved biosensor demonstrated excellent selectivity, stability and reproducibility with wide linear range from 0.0001 to 100 ng mL -1 and ultralow detection limit of 52.14 fg mL -1 for NSE, thus holding great promise for sensitive determination of tumor markers., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The glycolytic enzyme enolase of Staphylococcus aureus is a highly conserved enzyme which binds to human plasminogen thereby aiding the infection process. The cloning, over expression and purification of S. aureus enolase as well as the effect of various metals upon the catalytic activity and structural stability of the enzyme have been reported. The recombinant enzyme (rSaeno) has been purified to homogeneity in abundant amounts (60 mg/L of culture) and the kinetic parameters (K m = 0.23 +/- 0.013 x 10 -3 M; V max = 90.98 +/- 0.00052 U/mg) and the optimum pH were calculated. This communication further reports that increasing concentrations of Na + ions inhibit the enzyme while increasing concentrations of K + ions were stimulatory. In case of divalent cations, it was found that Mg 2+ stimulates the activity of rSaeno while the rest of the divalent cations (Zn 2+ , Mn 2+ , Fe 2+ , Cu 2+ , Ni 2+ and Ca 2+ ) lead to a dose-dependent loss in the activity with a total loss of activity in the presence of Hg 2+ and Cr 2+ . The circular dichroism data indicate that other than Hg 2+ , Ni 2+ and to a certain extent Cu 2+ , none of the other ions destabilized rSaeno. The inhibitory roles of fluorides, as well as neurotoxic compounds upon the catalytic activity of rSaeno, have also been studied. Conformational changes in rSaeno (induced by ions) were studied using partial trypsin digestion.

We propose the first black phosphorus (BP) - fiber optic biosensor for ultrasensitive diagnosis of human neuron-specific enolase (NSE) cancer biomarkers. A novel optical-nano configuration has been exploited by integrating BP nanosheets with a largely tilted fiber grating (BP-TFG), where the BP is bio-functionalized by the poly-L-lysine acting as a critical cross-linker to facilitate bio-nano-photonic interface with extremely enhanced light-matter interaction. BP nanosheets are synthesized by a liquid ultrasonication-based exfoliation and deposited on fiber device by an in-situ layer-by-layer method. The BP-induced optical modulation effects in terms of thickness-tunable feature, polarization-dependence and enhanced light-matter interaction are experimentally investigated. The anti-NSE immobilized BP-TFG biosensor has been implemented to detect NSE biomarkers demonstrating ultrahigh sensitivity with limit of detection down to 1.0 pg/mL, which is 4 orders magnitude lower than NSE cut-off value of small cell lung cancer. The enhanced sensitivity of BP-TFG is 100-fold higher than graphene oxide or AuNPs based biosensors. We believe that BP-fiber optic configuration opens a new bio-nano-photonic platform for the applications in healthcare, biomedical, food safety and environmental monitoring., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Enolase is an evolutionarily conserved enzyme involved in the processes of glycolysis and gluconeogenesis. Mycoplasma hyopneumoniae belongs to Mycoplasma , whose species are wall-less and among the smallest self-replicating bacteria, and is an important colonizing respiratory pathogen in the pig industry worldwide. Mycoplasma hyopneumoniae enolase ( Mhp Eno) expression is significantly increased after infection and was previously found to be a virulence factor candidate. Our studies show that Mhp Eno is a cell surface-localized protein that can adhere to swine tracheal epithelial cells (STECs). Adhesion to STECs can be specifically inhibited by an Mhp Eno antibody. Mhp Eno can recognize and interact with plasminogen with high affinity. Here, the first crystal structure of the mycoplasmal enolase from Mycoplasma hyopneumoniae was determined. The structure showed unique features of Mhp Eno in the S3/H1, H6/S6, H7/H8, and H13 regions. All of these regions were longer than those of other enolases and were exposed on the Mhp Eno surface, making them accessible to host molecules. These results show that Mhp Eno has specific structural characteristics and acts as a multifunctional adhesin on the Mycoplasma hyopneumoniae cell surface.

This research aims to engineer molecularly imprinted polymer (MIP)-based synthetic receptors for the molecular recognition of neuron specific enolase (NSE) biomarker. The synthetic peptide derived from the NSE was synthesized along with its cysteine and histidine modified versions. The modified peptides were utilized as templates for molecular imprinting, which was achieved by combination of epitope- and electrochemical surface imprinting strategy. The subsequently generated imprinted cavities were used for the detection of the NSE derived peptide and NSE. The imprints created with cysteine (CME) and histidine modified epitopes (HME) could detect the peptide in a concentration range of 2-128 µM and 15.6 nM to 128 µM, respectively. The recognition of NSE was achieved by the same imprints in a linear range of 1-64 ng mL -1 (CME) and 0.25-64 ng mL -1 (HME), respectively. The target molecules bound to the control polymer very weakly, confirming the high selectivity of the MIP cavities. Selectivity studies resulted in imprinting factors of 8.8 and 11 for the CME and HME imprints, respectively. The affinity analyses provided dissociation constants of 2.3 × 10 -10 M and 3 × 10 -11 M for NSE recognition using the corresponding epitope imprints. Cross-reactivity studies with non-specific molecules proved high specificity of the artificial receptors for the targets., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A molecularly imprinted electrochemical sensor for neuron specific enolase (NSE) was developed by electrochemical polymerizing ionic liquid, which was functionalized with pyrrole moiety, in between gold nanoarrays. A well-defined 3D structured gold nanoarray was fabricated on a glassy carbon electrode (GCE) surface by using template-assisted electrochemical deposition technique. 1-(3-mercaptopropyl)-3-vinyl-imidazolium tetrafluoroborate was self-assembled onto the surface of gold nanoarrays to produce active sites for anchoring the molecularly imprinted film. Subsequently, an electrochemical polymerization procedure was carried out in an aqueous solution containing 1,3-di(3-N-pyrrolpropyl)imidazolium bromine ionic liquid and neuron specific enolase (NSE). After removing NSE templates, a molecularly imprinted sensor was successfully fabricated. The sensor showed high selectivity and sensitivity towards NSE, produced a linear response in the concentration range from 0.01 to 1.0ngmL -1 and had a detection limit of 2.6pgmL -1 with an incubation time of 15min. The developed sensor was demonstrated successful in determining NSE in clinical serum samples., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Neuron-specific enolase (NSE) had clinical significance on diagnosis, staging, monitoring effect and judging prognosis of small cell lung cancer. Thus, there had a growing demand for the on-site testing of NSE. Here, a wireless point-of-care testing (POCT) system with electrochemical measurement for NSE detection was developed and verified. The wireless POCT system consisted of microfluidic paper-based analytical devices (μPADs), electrochemical detector and Android's smartphone. Differential pulse voltammetry (DPV) measurement was adopted by means of electrochemical detector which including a potentiostat and current-to-voltage converter. μPADs were modified with nanocomposites synthesized by Amino functional graphene, thionine and gold nanoparticles (NH 2 -G/Thi/AuNPs) as immunosensors for NSE detection. Combined with μPADs, the performance of the wireless POCT system was evaluated. The peak currents showed good linear relationship of the logarithm of NSE concentration ranging from 1 to 500ngmL -1 with the limit of detection (LOD) of 10pgmL -1 . The detection results were automatically stored in EEPROM memory and could be displayed on Android's smartphone through Bluetooth in real time. The detection results were comparable to those measured by a commercial electrochemical workstation. The wireless POCT system had the potential for on-site testing of other tumor markers., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Brucella abortus , the causative agent of bovine brucellosis, invades and replicates within cells inside a membrane-bound compartment known as the Brucella containing vacuole (BCV). After trafficking along the endocytic and secretory pathways, BCVs mature into endoplasmic reticulum-derived compartments permissive for bacterial replication. Brucella Type IV Secretion System (VirB) is a major virulence factor essential for the biogenesis of the replicative organelle. Upon infection, Brucella uses the VirB system to translocate effector proteins from the BCV into the host cell cytoplasm. Although the functions of many translocated proteins remain unknown, some of them have been demonstrated to modulate host cell signaling pathways to favor intracellular survival and replication. BPE123 (BAB2_0123) is a B. abortus VirB-translocated effector protein recently identified by our group whose function is yet unknown. In an attempt to identify host cell proteins interacting with BPE123, a pull-down assay was performed and human alpha-enolase (ENO-1) was identified by LC/MS-MS as a potential interaction partner of BPE123. These results were confirmed by immunoprecipitation assays. In bone-marrow derived macrophages infected with B. abortus , ENO-1 associates to BCVs in a BPE123-dependent manner, indicating that interaction with translocated BPE123 is also occurring during the intracellular phase of the bacterium. Furthermore, ENO-1 depletion by siRNA impaired B. abortus intracellular replication in HeLa cells, confirming a role for α-enolase during the infection process. Indeed, ENO-1 activity levels were enhanced upon B. abortus infection of THP-1 macrophagic cells, and this activation is highly dependent on BPE123. Taken together, these results suggest that interaction between BPE123 and host cell ENO-1 contributes to the intracellular lifestyle of B. abortus .

Renal targets of autoimmunity in human lupus nephritis (LN) are unknown. We sought to identify autoantibodies and glomerular target antigens in renal biopsy samples from patients with LN and determine whether the same autoantibodies can be detected in circulation. Glomeruli were microdissected from biopsy samples of 20 patients with LN and characterized by proteomic techniques. Serum samples from large cohorts of patients with systemic lupus erythematosus (SLE) with and without LN and other glomerulonephritides were tested. Glomerular IgGs recognized 11 podocyte antigens, with reactivity varying by LN pathology. Notably, IgG2 autoantibodies against α-enolase and annexin AI were detected in 11 and 10 of the biopsy samples, respectively, and predominated over other autoantibodies. Immunohistochemistry revealed colocalization of α-enolase or annexin AI with IgG2 in glomeruli. High levels of serum anti-α-enolase (>15 mg/L) IgG2 and/or anti-annexin AI (>2.7 mg/L) IgG2 were detected in most patients with LN but not patients with other glomerulonephritides, and they identified two cohorts: patients with high anti-α-enolase/low anti-annexin AI IgG2 and patients with low anti-α-enolase/high anti-annexin AI IgG2. Serum levels of both autoantibodies decreased significantly after 12 months of therapy for LN. Anti-α-enolase IgG2 recognized specific epitopes of α-enolase and did not cross-react with dsDNA. Furthermore, nephritogenic monoclonal IgG2 (clone H147) derived from lupus-prone MRL-lpr/lpr mice recognized human α-enolase, suggesting homology between animal models and human LN. These data show a multiantibody composition in LN, where IgG2 autoantibodies against α-enolase and annexin AI predominate in the glomerulus and can be detected in serum., (Copyright © 2014 by the American Society of Nephrology.)

Aim: A study was performed on the articular disk and periarticular tissues of the temporo-mandibular joint (TMJ) with immunohistochemical techniques to give evidence to the presence of neuroreceptors (NRec) in these sites., Methods: The study was carried out on tissue samples obtained from 10 subjects without TMJ disease and from 7 patients with severe TMJ arthritis and arthrosis. We use antibodies directed against following antigens: Gliofibrillary Acidic Protein (GFAP), Leu-7, Myelin Basic Protein (MBP), Neurofilaments 68 kD (NF), Neuron Specific Enolase (NSE), S-100 protein (S-100) and Synaptophysin (SYN)., Results: This study revealed that Ruffini's-like, Pacini's-like and Golgi's-like receptors can be demonstrated in TMJ periarticular tissues and that free nervous endings are present in the subsynovial tissues but not within the articular disk. We observed elongated cytoplamic processes of chondrocytes that demonstrated strong S-100 immunoreactivity but they were unreactive with all other antibodies. These cytoplamic processes were more abundant and thicker in the samples obtained from patients with disease TMJ., Conclusion: The results of this study confirm that different Nrec are detectable in TMJ periarticular tissues but they are absent within the articular disk. In the latter site, only condrocytic processes are evident, especially in diseased TMJ, and they might have been confused with nervous endings in previous morphological studies. Nevertheless the absence of immunoreactivity for NF, NSE and SYN proves that they are not of neural origin.

Novel synthetic peptides represent smart molecules for antigen-antibody interactions in several bioanalytics applications, from purification to serum screening. Their immobilization onto a solid phase is considered a key point for sensitivity increasing. In this view, we exploited Quartz Crystal Microbalance with simultaneous frequency and dissipation monitoring (QCM-D) with a double aim, specifically, as investigative tool for spacers monolayer assembling and its functional evaluation, as well as high sensitive method for specific immunosorbent assays. The method was applied to pancreatic ductal adenocarcinoma (PDAC) detection by studying the interactions between synthetic phosphorylated and un-phosphorylated α-enolase peptides with sera of healthy and PDAC patients. The synthetic peptides were immobilized on the gold surface of the QCM-D sensor via a self-assembled alkanethiol monolayer. The presented experimental results can be applied to the development of surfaces less sensitive to non-specific interactions with the final target to suggest specific protocols for detecting PDAC markers with un-labeled biosensors., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The enolase EnoA1 of Lactobacillus plantarum is here shown to interact with human plasminogen (Plg). By sequence alignment of EnoA1 with Streptococcus pneumoniae and Bifidobacterium lactis enolases, we identified BS1 and BS2 Plg-binding sites. A structure prediction of EnoA1 showed lysine residues in position 255 (BS2), and 422 (BS1) exposed on protein surface. A lysine residue in position 259 was as well identified as surface-exposed amino acid. The enoA1 gene was site directed-mutagenized to generate four mutated proteins, carrying K255A, K259A, K422A and K259A/K422A substitutions. The functional role of these lysine residues was assessed evaluating specific Plg-binding activity of the mutated proteins. While the binding activity of the mutated proteins was drastically reduced, the residual enzymatic activity was more than 50% of EnoA1. Our results show that L. plantarum EnoA1 exhibits the Plg-BS1, and the Plg-BS2 extending up to the lysine residue in position 259, therefore consisting of 12-aa residues instead of 9-aa residues described in S. pneumoniae. A test performed on whole cells of L. plantarum, demonstrated that after inducing conversion of the cell-bound plasminogen to plasmin, this was released into the medium, unlike the mechanism reported for most pathogens, that retained plasmin bound to the cell surface., (Copyright © 2012. Published by Elsevier GmbH.)

We performed proteomics analysis of the cerebrospinal fluid (CSF) of healthy dogs and dogs with meningoencephalitis of unknown etiology (MUE). By comparing two-dimensional electrophoreses (2DE), an upregulated spot was found in MUE dogs. This protein was identified as a neuron-specific enolase (NSE) by analysis with MALDI-TOF mass spectrometry. In comparing dot blots using an antibody against NSE, the NSE levels in the CSF of MUE dogs was significantly higher than that of the controls. NSE is a diagnostic marker of neuroendocrine tumors, brain injury and spinal cord trauma in humans. It seems that the NSE concentration in the CSF is increased by cellular destruction in canine encephalitis. Though elevation of NSE may not be specific in canine encephalitis because the NSE level was increased in other CNS diseases, further study including measurement with serum is necessary.

Recombinant Lactobacillus jensenii enolase fused to a C-terminal noncleavable His tag was expressed in Escherichia coli, purified and crystallized by sitting-drop vapor diffusion. A complete data set was collected to 3.25 A resolution. The crystals belonged to space group I4, with unit-cell parameters a=b=145.31, c=99.79 A. There were two protein subunits in the asymmetric unit, which gave a Matthews coefficient VM of 2.8 A3 Da(-1), corresponding to 55.2% solvent content.

During oilseed embryo development, carbon from sucrose is utilized for fatty acid synthesis in the plastid. The role of plastidial glycolysis in Arabidopsis embryo oil accumulation was investigated. Genes encoding enolases (ENO) and phosphoglyceromutases (PGlyM) were identified, and activities and subcellular locations were established by expression of recombinant and green fluorescent protein (GFP)-fusion proteins. Mutant Arabidopsis plants lacking putative plastidial isoforms were characterized with respect to isoform composition and embryo oil content. In the developing embryo, ENO1 and ENO2 account for most or all of the plastidial and cytosolic ENO activity, respectively, and PGLYM1 accounts for most or all of the plastidial PGlyM activity. The eno1 and pglym1 mutants, in which plastidic ENO and PGlyM activities were undetectable, had wild-type amounts of seed oil at maturity. It is concluded that although plastids of developing Arabidopsis embryos have the capacity to carry out the lower part of the glycolytic pathway, the cytosolic glycolytic pathway alone is sufficient to support the flux from 3-phosphoglycerate to phosphoenolpyruvate required for oil production. The results highlight the importance for oil production of translocators that facilitate interchange of glycolytic intermediates between the cytosol and the plastid stroma.

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pI 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.

The Arabidopsis thaliana gene At1g74030 codes for a putative plastid phosphoenolpyruvate (PEP) enolase (ENO1). The recombinant ENO1 protein exhibited enolase activity and its kinetic properties were determined. ENO1 is localized to plastids and expressed in most heterotrophic tissues including trichomes and non-root-hair cells, but not in the mesophyll of leaves. Two T-DNA insertion eno1 mutants exhibited distorted trichomes and reduced numbers of root hairs as the only visible phenotype. The essential role of ENO1 in PEP provision for anabolic processes within plastids, such as the shikimate pathway, is discussed with respect to plastid transporters, such as the PEP/phosphate translocator.

Multiple reaction monitoring (MRM) mass spectrometry identifies and quantifies specific peptides in a complex mixture with very high sensitivity and speed and thus has promise for the high throughput screening of clinical samples for candidate biomarkers. We have developed an interactive software platform, called MRMer, for managing highly complex MRM-MS experiments, including quantitative analyses using heavy/light isotopic peptide pairs. MRMer parses and extracts information from MS files encoded in the platform-independent mzXML data format. It extracts and infers precursor-product ion transition pairings, computes integrated ion intensities, and permits rapid visual curation for analyses exceeding 1000 precursor-product pairs. Results can be easily output for quantitative comparison of consecutive runs. Additionally MRMer incorporates features that permit the quantitative analysis experiments including heavy and light isotopic peptide pairs. MRMer is open source and provided under the Apache 2.0 license.

Streptococcus suis is an important swine pathogen that causes meningitis, endocarditis, arthritis and septicaemia. As a zoonotic agent, S. suis also causes similar diseases in humans. Binding of pathogenic bacteria to extracellular matrix components enhances their adhesion to and invasion of host cells. In the present study we isolated and identified a novel fibronectin-binding protein from S. suis. The native protein (designated SsEno) possessed not only high homology with other bacterial enolases but also enolase activity. We cloned, expressed and purified SsEno and showed that it is ubiquitously expressed by all S. suis serotypes and we identified its surface localization using immunoelectron microscopy. ELISA demonstrated that SsEno binds specifically to fibronectin and plasminogen in a lysine-dependent manner. Additional surface plasmon resonance assays demonstrated that SsEno binds to fibronectin or plasminogen with low nanomolar affinity. Inhibition experiments with anti-SsEno antibodies also showed that bacterial SsEno is important for the adhesion to and invasion of brain microvascular endothelial cells by S. suis. Overall, the present work is the first study, to our knowledge, to demonstrate a fibronectin-binding activity of a bacterial enolase, and shows that, similar to other bacterial fibronectin-binding proteins, SsEno may contribute to the virulence of S. suis.

Trichomonas vaginalis is a protist that causes the most common human sexually transmitted infection. A T. vaginalis cDNA expression library was screened with pooled sera from patients with trichomoniasis. A highly reactive cDNA clone of 1,428 bp encoded a trichomonad protein of 472 amino acids with sequence identity to alpha-enolase (tv-eno1). The sequence alignment confirmed the highly conserved nature of the enzyme with 65% to 84% identity among organisms. The expression of tv-eno1 was up-regulated by contact of parasites with vaginal epithelial cells, and this is the first report demonstrating up-regulation by cytoadherence of a plasminogen-binding alpha-enolase in T. vaginalis. Immunofluorescence with monoclonal antibody of nonpermeabilized trichomonads showed tv-ENO1 on the surface. The recombinant tv-ENO1 was expressed in Escherichia coli as a glutathione S-transferase (GST)::tv-ENO1 fusion protein, which was cleaved using thrombin to obtain affinity-purified recombinant tv-ENO1 protein (tv-rENO1) detectable in immunoblots by sera of patients. Immobilized tv-rENO1 bound human plasminogen in a dose-dependent manner, and plasminogen binding by tv-rENO1 was confirmed in a ligand blot assay. The plasminogen-specific inhibitor epsilon-aminocaproic acid blocked the tv-rENO1-plasminogen association, indicating that lysines play a role in binding to tv-rENO1. Further, both parasites and tv-rENO1 activate plasminogen to plasmin that is mediated by tissue plasminogen activator. These data indicate that as with other bacterial pathogens, tv-ENO1 is an anchorless, surface-associated glycolytic enzyme of T. vaginalis.

Plasminogen (Plg) binding to the cell surface of Mycoplasma fermentans results in a marked increase in the maximal adherence of the organism to HeLa cells, enhanced Plg activation by the urokinase-type Plg activator, and the induction of the internalization of M. fermentans by eukaryotic host cells (A. Yavlovich, A. Katzenell, M. Tarshis, A. A. Higazi, and S. Rottem, Infect. Immun. 72:5004-5011, 2004). In this study, the M. fermentans Plg binding protein was isolated by affinity chromatography of Triton X-100-solubilized M. fermentans membranes by utilizing a column of a Plg-biotin complex attached to avidin that was eluted with epsilon-aminocaproic acid. The eluted approximately 50-kDa protein was identified by mass spectrometric techniques as alpha-enolase. The possibility that alpha-enolase, a key cytoplasmatic glycolytic enzyme, resides also on the cell surface of M. fermentans was supported by an immunoblot analysis using polyclonal anti-alpha-enolase antiserum, which showed that alpha-enolase was present in a purified M. fermentans membrane preparation, as well as by immunochemical criteria and by immunoelectron microscopy analysis. Our observation that Plg blocked the binding of anti-alpha-enolase antibodies to a 50-kDa polypeptide band resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of M. fermentans membrane or soluble preparations further supports our notion that mycoplasmal surface alpha-enolase is a major Plg binding protein of M. fermentans.

We have cloned, over-expressed and purified enolase from Plasmodium falciparum strain NF54 in Escherichia coli in active form, as an N-terminal His6-tagged protein. The sequence of the cloned enolase from the NF54 strain is identical to that of strain 3D7 used in full genome sequencing. The recombinant enolase (r-Pfen) could be obtained in large quantities (approximately 50 mg per litre of culture) in a highly purified form (> 95%). The purified protein gave a single band at approximately 50 kDa on SDS/PAGE. MALDI-TOF analysis gave a mean +/- SD mass of 51396 +/- 16 Da, which is in good agreement with the mass calculated from the sequence. The molecular mass of r-Pfen determined in gel-filtration experiments was approximately 100 kDa, indicating that P. falciparum enolase is a homodimer. Kinetic measurements using 2-phosphoglycerate as substrate gave a specific activity of approximately 30 U.mg(-1) and K(m2PGA) = 0.041 +/- 0.004 mm. The Michaelis constant for the reverse reaction (K(mPEP)) is 0.25 +/- 0.03 mm. pH-dependent activity measurements gave a maximum at pH 7.4-7.6 irrespective of the direction of catalysis. The activity of this enzyme is inhibited by Na+, whereas K+ has a slight activating effect. The cofactor Mg2+ has an apparent activation constant of 0.18 +/-0.02 mm. However, at higher concentrations, it has an inhibitory effect. Polyclonal antibody raised against pure recombinant P. falciparum enolase in rabbit showed high specificity towards recombinant protein and is also able to recognize enolase from the murine malarial parasite, Plasmodium yoelii, which shares 90% identity with the P. falciparum protein.

A protein kinase of 57 kDa, able to phosphorylate tyrosine in synthetic substrates pol(Glu4,Tyr1) and a fragment of Src tyrosine kinase, was isolated and partly purified from maize seedlings (Zea mays). The protein kinase was able to phosphorylate exogenous proteins: enolase, caseins, histones and myelin basic protein. Amino acid analysis of phosphorylated casein and enolase, as well as of phosphorylated endogenous proteins, showed that both Tyr and Ser residues were phosphorylated. Phosphotyrosine was also immunodetected in the 57 kDa protein fraction. In the protein fraction there are present 57 kDa protein kinase and enolase. This co-purification suggests that enolase can be an endogenous substrate of the kinase. The two proteins could be resolved by two-dimensional electrophoresis. Specific inhibitors of typical protein-tyrosine kinases had essentially no effect on the activity of the maize enzyme. Staurosporine, a nonspecific inhibitor of protein kinases, effectively inhibited the 57 kDa protein kinase. Also, poly L-lysine and heparin inhibited tyrosine phosphorylation by 57 kDa maize protein kinase. The substrate and inhibitor specificities of the 57 kDa maize protein kinase phosphorylating tyrosine indicate that it is a novel plant dual-specificity protein kinase.

Mitochondria fulfill a wide range of metabolic functions in addition to the synthesis of ATP and contain a diverse array of proteins to perform these functions. Here, we present the unexpected discovery of the presence of the enzymes of glycolysis in a mitochondrial fraction of Arabidopsis cells. Proteomic analyses of this mitochondrial fraction revealed the presence of 7 of the 10 enzymes that constitute the glycolytic pathway. Four of these enzymes (glyceraldehyde-3-P dehydrogenase, aldolase, phosphoglycerate mutase, and enolase) were also identified in an intermembrane space/outer mitochondrial membrane fraction. Enzyme activity assays confirmed that the entire glycolytic pathway was present in preparations of isolated Arabidopsis mitochondria, and the sensitivity of these activities to protease treatments indicated that the glycolytic enzymes are present on the outside of the mitochondrion. The association of glycolytic enzymes with mitochondria was confirmed in vivo by the expression of enolase- and aldolase-yellow fluorescent protein fusions in Arabidopsis protoplasts. The yellow fluorescent protein fluorescence signal showed that these two fusion proteins are present throughout the cytosol but are also concentrated in punctate regions that colocalized with the mitochondrion-specific probe Mitotracker Red. Furthermore, when supplied with appropriate cofactors, isolated, intact mitochondria were capable of the metabolism of (13)C-glucose to (13)C-labeled intermediates of the trichloroacetic acid cycle, suggesting that the complete glycolytic sequence is present and active in this subcellular fraction. On the basis of these data, we propose that the entire glycolytic pathway is associated with plant mitochondria by attachment to the cytosolic face of the outer mitochondrial membrane and that this microcompartmentation of glycolysis allows pyruvate to be provided directly to the mitochondrion, where it is used as a respiratory substrate.

Infection by the human opportunistic fungal pathogen Candida albicans has been increasing over recent years. In an attempt to understand the molecular mechanism of Candida invasion across host tissues, the relationship of C. albicans enolase to human plasminogen/plasmin was investigated. C. albicans enolase is a cell-surface protein and an immunodominant antigen in infected patients' sera. Plasminogen is an abundant plasma protein. Several lines of evidence support the binding of C. albicans enolase to human plasminogen. Firstly, it was found that various Candida strains were able to bind to plasminogen and its active form, plasmin. Secondly, recombinant Candida enolase was retained in a nickel-chelating affinity column matrix that can bind (125)I-labelled plasminogen or plasmin in a dose-dependent manner. Plasmin(ogen)-specific inhibitors, such as epsilon -aminocaproic acid and aprotinin, can effectively block plasmin-binding activity. Thirdly, as with many plasminogen receptors, binding of Candida enolase to plasmin(ogen) is lysine-dependent, whereas little inhibition occurred with arginine, aspartate and glutamate. Fourthly, immobilized enolase enhanced plasminogen's affinity for streptokinase at least tenfold, as demonstrated by its activation of plasmin activity. To elucidate the biological significance of this result, it was demonstrated that the plasmin(ogen)-bound Candida cells were able to induce fibrinolysis activity in a matrix-gel assay. Furthermore, plasmin-bound Candida cells had an increased ability to cross an in vitro blood-brain barrier system. The results given here indicate that Candida enolase is a plasminogen- and plasmin-binding protein and that the interaction of C. albicans enolase with the plasminogen system may contribute to invasion of the tissue barrier.

Rhodotorula mucilaginosa (also known as R. rubra) is among the most commonly found yeast strains in our environment. However, allergens from R. mucilaginosa have not yet been characterized at the molecular level. The purpose of this study was to characterize the enolase allergen from R. mucilaginosa and examine the allergenic/antigenic cross-reactivity among fungal enolases. The full-length cDNA encoding the R. mucilaginosa enolase was isolated through the reverse transcriptase-polymerase chain reaction in conjunction with the 5'-end and 3'-end rapid amplification cDNA end reactions. The corresponding natural enolase from R. mucilaginosa was identified using two-dimensional gel electrophoresis and N-terminal amino acid sequence analysis. The results showed that the enolase from R. mucilaginosa is a protein of 439 residues and is encoded by a cDNA of 1497 bp. It shares high sequence identity with enolase allergens from Candida albicans (85%), Saccharomyces cerevisiae (76%), Penicillium citrinum (76%), Aspergillus fumigatus (76%), Cladosporium herbarum (76.5%), and Alternaria alternata (74%). A 47-kD component in the R. mucilaginosa extracts was found to react with IgE or rabbit anti-enolase antiserum and has an N-terminal amino acid sequence identical to that deduced from the isolated enolase cDNA. Sera from three (21%) of 14 allergic patients sensitized to R. mucilaginosa showed IgE binding to this 47-kD R. mucilaginosa component and the His-tagged recombinant enolase. A rabbit antiserum against the P. citrinum enolase and a monoclonal antibody (MoAb; Afueno 8) against the A. fumigatus enolase reacted with all 5 fungal enolases tested. However, an MoAb (E2a) generated by using the Saccharomyces enolase as antigen could only recognize the immunizing enolase. In addition, heterogeneity in immunoblot profiles of IgE antibodies in serum samples from 9 allergic patients against 5 different fungal enolases tested was also observed. The presence of IgE cross-reactivity among enolase allergens from R. mucilaginosa, C. albicans and P. citrinum was detected by immunoblot inhibition. In conclusion, a new and cross-reactive enolase allergen from R. mucilaginosa (Rho m 1) was identified. Although enolases are highly conserved allergens among different fungal species, most of the allergic patients examined in this study differed in their IgE reactivity to the 5 different fungal enolases tested. The results obtained will be of value in understanding the role of enolase allergen in clinical mould allergy., (Copyright 2002 National Science Council, ROC and S. Karger AG, Basel)

Many pathogenic bacteria express plasminogen receptors on their surface, which may play a role in the dissemination of organisms by binding plasminogen that, when converted to plasmin, can digest extracellular matrix proteins. A 45-kDa protein was purified from Streptococcus pneumoniae and confirmed as an alpha-enolase by its ability to catalyse the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate and by N-terminal sequencing. The activity of alpha-enolase was found in the cytoplasm and in whole cells. Activity was also demonstrated in cell wall fractions, which confirmed that alpha-enolase is a cytoplasmic antigen also expressed on the surface of S. pneumoniae. The plasminogen-binding activity of alpha-enolase was examined by Western blot, which showed that purified alpha-enolase was able to bind human plasminogen. Immunoblots of the purified 45-kDa alpha-enolase with 22 sera from patients with pneumococcal disease showed binding in 15 cases, indicating that pneumococcal enolase is immunogenic.

Natural rubber latex allergy is an IgE-mediated disease that is caused by proteins that elute from commercial latex products. A complementary DNA (cDNA) coding for Hev b 9, an enolase (2-phospho-D-glycerate hydrolyase) and allergen from latex of the rubber tree Hevea brasiliensis, was amplified by PCR. The PCR primers were designed according to conserved regions of enolases from plants. The obtained cDNA amplification product consisted of 1651 bp and encoded a protein of 445 amino-acid residues with a calculated molecular mass of 47.6 kDa. Sequence comparisons revealed high similarities of the Hevea latex enolase to mold enolases that have been identified as important allergens. In addition, the crucial amino-acid residues that participate in the formation of the catalytic site and the Mg2+ binding site of enolases were also conserved. Hevea latex enolase was produced as a recombinant protein in Escherichia coli with an N-terminal hexahistidyl tag, and purified by affinity chromatography. The yield amounted to 110 mg of purified Hev b 9 per litre of bacterial culture. The recombinant allergen bound IgE from latex, as well as mold-allergic patients, in immunoblot and ELISA experiments. The natural enolase was isolated from Hevea latex by (NH4)2SO4 precipitation and ion exchange chromatography. The natural and the recombinant (r)Hev b 9 showed equivalent enzymatic activity. Patients' IgE-antibodies preincubated with rHev b 9 lost their ability to bind to natural (n) Hev b 9, indicating the identity of the B-cell epitopes on both molecules. Cross-reactivity with two enolases from Cladosporium herbarum and Alternaria alternata was determined by inhibition of IgE-binding to these enolases by rHev b 9. Therefore, enolases may represent another class of highly conserved enzymes with allergenic potentials.

Genomic or cDNA clones for the glycolytic enzyme enolase were isolated from the amitochondriate pelobiont Mastigamoeba balamuthi, from the kinetoplastid Trypanosoma brucei, and from the euglenid Euglena gracilis. Clones for the cytosolic enzyme were found in all three organisms, whereas Euglena was found to also express mRNA for a second isoenzyme that possesses a putative N-terminal plastid-targeting peptide and is probably targeted to the chloroplast. Database searching revealed that Arabidopsis also possesses a second enolase gene that encodes an N-terminal extension and is likely targeted to the chloroplast. A phylogeny of enolase amino acid sequences from 6 archaebacteria, 24 eubacteria, and 32 eukaryotes showed that the Mastigamoeba enolase tended to branch with its homologs from Trypanosoma and from the amitochondriate protist Entamoeba histolytica. The compartment-specific isoenzymes in Euglena arose through a gene duplication independent of that which gave rise to the compartment-specific isoenzymes in Arabidopsis, as evidenced by the finding that the Euglena enolases are more similar to the homolog from the eubacterium Treponema pallidum than they are to homologs from any other organism sampled. In marked contrast to all other glycolytic enzymes studied to date, enolases from all eukaryotes surveyed here (except Euglena) are not markedly more similar to eubacterial than to archaebacterial homologs. An intriguing indel shared by enolase from eukaryotes, from the archaebacterium Methanococcus jannaschii, and from the eubacterium Campylobacter jejuni maps to the surface of the three-dimensional structure of the enzyme and appears to have occurred at the same position in parallel in independent lineages.

Cell surface-associated materials of Actinobacillus actinomycetemcomitans were extracted by a short incubation of the cell suspension in a Tris-buffered saline in the presence and absence of a restriction enzyme, EcoRI. The supernatants (which we termed EcoRI extract and surface extract, respectively) contained a number of extracellularly released proteins. Of these proteins, four major proteins were identified by N-terminal sequencing to be the 34 and 39 kDa outer membrane proteins, the GroEL-like protein, and a 47 kDa protein homologous to Haemophilus influenzae enolase. Enolase activity was found in the extracts and its relative amount of activity in the EcoRI extract from a culture of the mid-exponential growth phase was estimated as 5.7% of total enzyme activity. In contrast, the relative amount of activity of another cytosolic enzyme, lactate dehydrogenase, was extremely low in the extracts and also in the culture supernatant. These results suggest the external localization of enolase in this bacterium.

It was reported previously that enolase enzyme activity and ENO1 transcript levels are induced by anaerobic stress in maize (Zea mays). Here we show that not all isoforms of maize enolase are anaerobically induced. We cloned and sequenced a second enolase cDNA clone (pENO2) from maize. Sequence analysis showed that pENO2 shares 75.6% nucleotide and 89.5% deduced amino acid sequence identity with pENO1 and is encoded by a distinct gene. Expression of ENO2 is constitutive under aerobic conditions, whereas ENO1 levels are induced 10-fold in maize roots after 24 h of anaerobic treatment. Western-blot analysis and N-terminal sequencing of in vivo-labeled maize roots identified two major proteins selectively synthesized upon anaerobic stress as isozymes of enolase. We describe the expression of enolase in maize roots under anaerobic stress.

NADH-dichlorophenol-indophenol oxidoreductases (PMOs) were purified from synaptic plasma membranes or synaptic vesicles (small recycling vesicles) from both bovine and rat brains and from a neuroblastoma cell line, NB41A3. Several isoforms could be identified in purified plasma membranes and vesicles. Purification of the enzyme activity involved protein extraction with detergents, (NH4)2SO4 precipitation, chromatography under stringent conditions and native PAGE. PMO activity could be attributed to a very tight complex of several proteins that could not be separated except by SDS/PAGE. SDS/PAGE resolved the purified complex into at least five proteins, which could be micro-sequenced and identified unambiguously as hsc70, TOAD64 and glyceraldehyde-3-phosphate dehydrogenase tightly associated with the brain-specific proteins aldolase C and enolase-gamma. Enzyme activity could be purified from both synaptic plasma membranes and recycling vesicles, yields being much greater from the latter source. Highly purified plasma membranes (prepared from a neuroblastoma cell line NB41A3 by iminobiotinylation of intact cells and affinity purification with avidin and anti-avidin antibodies under very stringent conditions) also displayed PMO activity tightly associated with TOAD64. The association of PMO in a tight complex was confirmed by its immunoprecipitation from cellular and membrane extracts of NB41A3 using antibodies directed against any component protein of the complex followed by immunodetection with antibodies directed against the other members. Antibodies also inhibited the enzyme activity synergistically. In addition, induction of the different components of the complex during dichlorophenol-indophenol stress was demonstrated by the S1 RNase-protection assay in synchronized NB41A3 cells. The role of the complex in membrane fusion and cellular response to extracellular oxidative stress during growth and development is discussed.

The glycolytic enzyme enolase (EC 4.2.1.11) is active as dimers formed from three subunits encoded by different genes. The embryonic alphaalpha isoform remains distributed in many adult cell types, whereas a transition towards betabeta and gammagamma isoforms occurs in striated muscle cells and neurons respectively. It is not understood why enolase exhibits tissue-specific isoforms with very close functional properties. We approached this problem by the purification of native betabeta-enolase from mouse hindlimb muscles and by raising specific antibodies of high titre against this protein. These reagents have been useful in revealing a heterogeneity of the beta-enolase subunit that changes with in vivo and in vitro maturation. A basic carboxypeptidase appears to be involved in generating an acidic beta-enolase variant, and may regulate plasminogen binding by this subunit. We show for the first time that pure betabeta-enolase binds with high affinity the adjacent enzymes in the glycolytic pathway (pyruvate kinase and phosphoglycerate mutase), favouring the hypothesis that these three enzymes form a functional glycolytic segment. betabeta-Enolase binds with high affinity sarcomeric troponin but not actin and tropomyosin. Some of these binding properties are shared by the alphaalpha-isoenolase, which is also expressed in striated muscle, but not by the neuron-specific gammagamma-enolase. These results support the idea that specific interactions with macromolecules will address muscle enolase isoforms at the subcellular site where ATP, produced through glycolysis, is most needed for contraction. Such a specific targeting could be modulated by post-translational modifications.

125I-Labeled recombinant human neuron-specific enolase (R-NSE) was inadequate for RIA as a labeled antigen. The binding activity of labeled R-NSE to the antibody was markedly decreased. To supplement this defect and facilitate purification, we constructed two R-NSE derivatives, Y-NSE (one tyrosine residue was added at the N-terminal of R-NSE) and Y-NSE.H6 (six histidine residues were further added at the C-terminal of Y-NSE). The biochemical and immunochemical characteristics of these R-NSE derivatives were essentially the same to those of R-NSE. These derivatives were useful not only as standards for enzyme immunoassay (EIA), but also as labeled antigens for RIA. These results clearly indicate that the reactivity of these modified NSEs to anti-NSE antibody is almost equivalent to that of human brain gammagamma-enolase (B-NSE), and that even if the modified NSEs are labeled, they retain their binding affinities to antibodies in contrast to R-NSE.

Enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) has been identified as an anaerobic stress protein in Echinochloa oryzoides based on the homology of its internal amino acid sequence with those of enolases from other organisms, by immunological reactivity, and induction of catalytic activity during anaerobic stress. Enolase activity was induced 5-fold in anoxically treated seedlings of three flood-tolerant species (E. oryzoides, Echinochloa phyllopogon, and rice [Oryza sativa L.]) but not in the flood-intolerant species (Echinochloa crus-pavonis). A 540-bp fragment of the enolase gene was amplified by polymerase chain reaction from cDNAs of E. phyllopogon and maize (Zea mays L.) and used to estimate the number of enolase genes and to study the expression of enolase transcripts in E. phyllopogon, E. crus-pavonis, and maize. Southern blot analysis indicated that only one enolase gene is present in either E. phyllopogon or E. crus-pavonis. Three patterns of enolase gene expression were observed in the three species studied. In E. phyllopogon, enolase induction at both the mRNA and enzyme activity levels was sustained at all times with a further induction after 48 h of anoxia. In contrast, enolase was induced in hypoxically treated maize root tips only at the mRNA level. In E. crus-pavonis, enolase mRNA and enzyme activity were induced during hypoxia, but activity was only transiently elevated. These results suggest that enolase expression in maize and E. crus-pavonis during anoxia are similarly regulated at the transcriptional level but differ in posttranslational regulation, whereas enolase is fully induced in E. phyllopogon during anaerobiosis.

Enolase (2-phospho-D-glycerate hydrolase; EC 4.2.1.11) from the hyperthermophilic bacterium Thermotoga maritima was purified to homogeneity. The N-terminal 25 amino acids of the enzyme reveal a high degree of similarity to enolases from other sources. As shown by sedimentation analysis and gel-permeation chromatography, the enzyme is a 345-kDa homoctamer with a subunit molecular mass of 48 +/- 5 kDa. Electron microscopy and image processing yield ring-shaped particles with a diameter of 17 nm and fourfold symmetry. Averaging of the aligned particles proves the enzyme to be a tetramer of dimers. The enzyme requires divalent cations in the activity assay, Mg2+ being most effective. The optimum temperature for catalysis is 90 degrees C, the temperature dependence yields a nonlinear Arrhenius profile with limiting activation energies of 75 kJ mol-1 and 43 kJ mol-1 at temperatures below and above 45 degrees C. The pH optimum of the enzyme lies between 7 and 8. The apparent Km values for 2-phospho-D-glycerate and Mg2+ at 75 degrees C are 0.07 mM and 0.03 mM; with increasing temperature, they are decreased by factors 2 and 30, respectively. Fluoride and phosphate cause competitive inhibition with a Ki of 0.14 mM. The enzyme shows high intrinsic thermal stability, with a thermal transition at 90 and 94 degrees C in the absence and in the presence of Mg2+.

An immunodominant antigen with enolase enzyme activity was purified and used for the development of an assay to detect antibodies directed against this antigen in sera from patients with either invasive candidiasis or Candida colonization. The Au enzyme-linked immunosorbent assay established with the Candida enolase antigen was able to discriminate significantly between invasive candidiasis and colonization in both immunocompetent and immunodeficient groups of patients. The test had a sensitivity of 50% and a specificity of 86% in the immunocompetent patient group. In the immunodeficient patient group, a sensitivity of 53% and a specificity of 78% were established. Antibody levels determined by a counterimmunoelectrophoresis assay with the same set of sera resulted in a better sensitivity for sera from the immunocompetent patient group but a lower specificity, i.e., 80 and 29%, respectively. The counterimmunoelectrophoresis assay of sera from the immunodeficient patient group was not able to discriminate significantly between invasive candidiasis and colonization. With the use of more serum from each patient, the sensitivity of the antibody detection assays increased, while the specificity was maintained. The increase, however, was not statistically significant. Combining the results of the antibody assays with antigen titers obtained by the Cand-Tec assay did not improve the predictive value with respect to invasive candidiasis, as determined by multivariance regression analysis. Furthermore, it was demonstrated by performance of Western blots (immunoblots) that sera from patients as well as a rabbit antiserum cross-reacted with the Candida enolase and baker's yeast enolase enzyme. However, by tandem crossed immunoelectrophoresis it was demonstrated that the antibodies were directed toward different epitopes of the antigen.

The methionine salvage pathway converts the methylthioribose moiety of 5'-(methylthio)-adenosine to methionine via a series of biochemical steps. One enzyme active in this pathway, a bifunctional enolase-phosphatase called E-1 that promotes oxidative cleavage of the synthetic substrate 2,3-diketo-1-phosphohexane to 2-keto-pentanoate, has been purified from Klebsiella pneumoniae and is characterized in the preceding paper (Myers, R., Wray, J., Fish, S., and Abeles, R. H. (1993) J. Biol. Chem. 268, 24785-24791). We synthesized degenerate oligonucleotides corresponding to portions of the amino terminus of E-1. These oligonucleotides were used as polymerase chain reaction primers on whole genomic DNA from Klebsiella oxytoca. This resulted in an 82-base pair DNA fragment that was used as a hybridization probe to obtain a clone of the E-1 gene from a K. oxytoca gene library. The DNA sequence of the E-1 coding region was determined, and the amino acid sequence of E-1 was deduced. E-1 appears to represent a novel class of enzymes since no homology to known enzymes was found. Cloning the gene from K. oxytoca on a multicopy plasmid leads to overproduction of E-1 enzyme that has properties indistinguishable from those of the enzyme from K. pneumoniae.

The 5-methylthio-D-ribose moiety of 5'-(methylthio)-adenosine is converted to methionine in a wide variety of organisms. 2,3-Diketo-5-methylthio-1-phosphopentane is an advanced intermediate in the methionine recycling pathway present in the Gram-negative bacterium Klebsiella pneumoniae. This unusual metabolite is oxidatively cleaved to yield formate (from C-1), 2-keto-4-methylthiobutyrate (the transamination product of methionine), and 3-methylthiopropionate. To further characterize this oxidative conversion, the desthio analog of the naturally occurring diketone, namely 2,3-diketo-1-phosphohexane I, was synthesized. If the metabolism of I is analogous to that of 2,3-diketo-5-methylthio-1-phosphopentane it should be converted to formate, 2-ketopentanoate, and butyrate. An enzyme (E-1), which mediates the oxidative conversion of I to formate and 2-ketopentanoate, was isolated from extracts of K. pneumoniae. E-1 was purified 100-fold to homogeneity in 10% yield. The native enzyme is a monomeric protein of M(r) 27,000. The activity of E-1 requires magnesium ion as a cofactor. No other prosthetic groups were detected. Incubation of the enzyme with I, under anaerobic conditions, led to the discovery of two intermediates. These species have been identified by 1H and 13C NMR, UV-visible spectroscopy, and model chemistry studies as 2-hydroxy-3-keto-1-phospho-1-hexene II, generated by enolization of I; and 1,2-dihydroxy-3-keto-1-hexene III, generated by enzymatic dephosphorylation of II. Intermediates II and III are released from the active site of the enzyme; III accumulates under anaerobic conditions. Under aerobic conditions, III is non-enzymically oxidized to 2-ketopentanoate, formate, and other products. Compound II was also generated by heating I at pH 7.5 for 7 min. Action of alkaline phosphatase on II produces III.

We isolated and sequenced a clone for Candida albicans enolase from a C. albicans cDNA library by using molecular genetic techniques. The 1.4-kbp cDNA encoded one long open reading frame of 440 amino acids which was 87 and 75% similar to predicted enolases of Saccharomyces cerevisiae and enolases from other organisms, respectively. The cDNA included the entire coding region and predicted a protein of molecular weight 47,178. The codon usage was highly biased and similar to that found for the highly expressed EF-1 alpha proteins of C. albicans. Northern (RNA) blot analysis showed that the enolase cDNA hybridized to an abundant C. albicans mRNA of 1.5 kb present in both yeast and hyphal growth forms. The polypeptide product of the cloned cDNA, which was purified as a recombinant protein fused to glutathione S-transferase, had enolase enzymatic activity and inhibited radioimmunoprecipitation of a single C. albicans protein of molecular weight 47,000. Analysis of the predicted C. albicans enolase showed strong conservation in regions of alpha helices, beta sheets, and beta turns, as determined by comparison with the crystal structure of apo-enolase A of S. cerevisiae. The lack of cysteine residues and a two-amino-acid insertion in the main domain differentiated C. albicans enolase from S. cerevisiae enolase. Immunofluorescence of whole C. albicans cells by using a mouse antiserum generated against the purified fusion protein showed that enolase is not located on the surface of C. albicans. Recombinant C. albicans enolase will be useful in understanding the pathogenesis and host immune response in disseminated candidiasis, since enolase is an immunodominant antigen which circulates during disseminated infections.

The gamma subunit of enolase (gamma-enolase) was purified from the brain tissues of cow, dog, goat, pig, rabbit, and rat. The purification was achieved in only three steps: ammonium sulfate-precipitation, DE 53 cellulose ion-exchange chromatography, and polyacrylamide gel electrophoresis (PAGE) in a preparative mode. The purification procedure was comparatively more simple than previously reported methods, and the yield of gamma-enolase was sufficient for subsequent structural and immunological analyses. In all mammals, the purified gamma-enolase migrated in sodium dodecyl sulfate-PAGE (SDS-PAGE) with a molecular mass of 46 kilodaltons (kDa), and the immunological cross-reactivity between those gamma-enolases was very strong. The structural homology of these gamma-enolases was examined by peptide mapping using cyanogen bromide cleavage and subsequent two-dimensional electrophoresis. The resulting peptide patterns were highly similar and in cow, dog, and goat, the patterns were almost identical. These results indicate that structural homology, that is, the species non-specificity of gamma-enolase, appears to be very high.

Transcription of the yeast enolase gene ENO2 is reduced 20- to 50-fold in strains carrying a null mutation in the positive regulatory gene GCR1. A small deletion mutation within one of two upstream activation sites (UAS elements) in the 5'-flanking region of ENO2 permitted wild-type levels of ENO2 gene expression in a strain carrying the gcr1 null mutation. These data show that sequences required for UAS element activity in GCR1 strains were required to repress ENO2 expression in a gcr1 strain. Protein factors that specifically bound to this UAS/repression site were identified. We show that the DNA-binding protein ABFI (autonomously replicating sequence-binding factor) is the major protein which binds the UAS/repression site. Minor DNA-binding activities that interact specifically with the UAS/repression site were also identified and may correspond to proteolytic breakdown products of ABFI. None of the observed binding activities were encoded by the GCR1 structural gene. A double-stranded oligonucleotide that included the UAS/repression site activated transcription of UAS-less ENO1 and ENO2 gene cassettes in vivo to wild-type levels in strains carrying the GCR1 allele as well as the gcr1 null mutation. These latter data show that the UAS/repression site is sufficient for transcriptional activation but is not sufficient to repress transcription of the enolase genes in a gcr1 genetic background.

One aspect in a broad spectrum of possible mechanisms of cariostatic reactions of fluoride is its interaction with the metabolism of oral bacteria. Information on the mechanisms and kinetics of fluoride inhibition of essential enzymes of the glycolytic pathway of the relevant bacteria is lacking. In this work, the isolation and purification of enolase from Streptococcus rattus and its characterization are described. The enzyme has been isolated in a monomeric (22 kilodaltons) and dimeric (49 kilodaltons) form. The Km for 2-phosphoglycerate is 4.35 mM. Fluoride inhibition kinetics have competitive character, while phosphate in concentrations above 2 mM and in the presence of 0.5 mM fluoride alters the inhibition kinetics from competitive to noncompetitive. Without fluoride, 2 mM phosphate has a slight stimulatory effect on the enzyme. Monofluorophosphate has a noncompetitive inhibiting effect on the enzyme. This finding suggests that the effect of phosphate may be due to an additional binding of fluoride to the enolase, resulting in a conformational change of the enzyme.

The complete amino acid sequence of chicken skeletal-muscle enolase, comprising 433 residues, was determined. The sequence was deduced by automated sequencing of hydroxylamine-cleavage, CNBr-cleavage, o-iodosobenzoic acid-cleavage, clostripain-digest and staphylococcal-proteinase-digest fragments. The presence of several acid-labile peptide bonds and the tenacious aggregation of most CNBr-cleavage fragments meant that a commonly used sequencing strategy involving initial CNBr cleavage was unproductive. Cleavage at the single Asn-Gly peptide bond with hydroxylamine proved to be particularly useful. Comparison of the sequence of chicken enolase with the two yeast enolase isoenzyme sequences shows that the enzyme is strongly conserved, with 60% of the residues identical. The histidine and arginine residues implicated as being important for the activity of yeast enolase are conserved in the chicken enzyme. Secondary-structure predictions are analysed in an accompanying paper [Sawyer, Fothergill-Gilmore & Russell (1986) Biochem. J. 236, 127-130].

Considerable amounts of five glycolytic enzymes glucosephosphate isomerase, glyceraldehyde-phosphate dehydrogenase, aldolase, pyruvate kinase, and lactate dehydrogenase, became fixed when intact synaptosomes were incubated with glutaraldehyde. Other glycolytic enzymes were immobilized much less by this procedure. The lactate dehydrogenase isoenzymes showed a variable response to glutaraldehyde fixation. The isoenzymes enriched in muscle subunits were rapidly immobilized by glutaraldehyde, while the isoenzymes enriched in heart subunits, especially H4, were not. It is suggested that the enzymes which were immobilized are located near the synaptosomal membrane, perhaps in association with actin, which is found at this site. The enzymes that showed a much smaller degree of fixation were either randomly distributed in the synaptoplasm or less susceptible to fixation.

The enolase from a highly fluoride-sensitive strain of Streptococcus salivarius and its fluoride-resistant mutant, as well as those from strains of Streptococcus sanguis and Streptococcus mutans with intermediate and low sensitivities to fluoride have been shown to be inhibited by fluoride. Comparisons of the purified, strain-specific enzymes showed a high degree of similarity for all preparations. The Michaelis constants for the substrate 2-phosphoglycerate were 1.3 x 10(-4) to 2.4 x 10(-4) M, pH optima were 7.3 to 7.7, and Mg2+ optima were 2 mEq/liter for all. Inhibition by fluoride required the presence of inorganic phosphate and was competitive in nature, and the calculated modified inhibition indices were found to be in the range from 3.3 x 10(-14) to 5.8 x 10(-14) M4. Percent inhibitions were determined under standardized conditions (0.16 mM NaF, 2 mM MgSO4, 0.5 mM Pi, and 0.5 mM 2-phosphoglycerate) and were found to range from 53.3 to 65.9% for all of the purified enzymes. The differences do not appear to be meaningful metabolically. Inhibition was reduced to about 14% at pH 6.0. From the similarities in the behavior of the strain-specific enzymes it is concluded that the differences in the glycolytic sensitivities of the different strains of streptococci to fluoride are not the consequence of any kinetic differences between the respective enolases.