scholarly journals The region around residue 115 of human bactericidal/permeability-increasing protein is not involved in lipopolysaccharide binding or bactericidal activity. Chemical synthesis and expression of a gene coding for the active domain and characterization of recombinant proteins

1994 ◽  
Vol 298 (3) ◽  
pp. 711-718 ◽  
Author(s):  
S Y Qi ◽  
Y Li ◽  
C D O'Connor
Keyword(s):  
Bactericidal Activity ◽  
Polymorphonuclear Leucocytes ◽  
Recombinant Polypeptide ◽  
Outer Membranes ◽  
High Affinity ◽  
Gene Coding ◽  
High Concentrations ◽  
Derivatives Of ◽  
Lipopolysaccharide Binding

Bactericidal/permeability-increasing protein (BPI) is a potent antimicrobial agent produced by polymorphonuclear leucocytes that specifically interacts with and kills Gram-negative bacteria. An 825 bp gene determining the bactericidal N-terminal domain of human BPI was chemically synthesized and expressed as inclusion bodies in Escherichia coli. The recombinant polypeptide, BPI', was solubilized and conditions under which it folded to give the active protein were determined. Folding was critically dependent on the urea and salt concentrations as well as the pH. BPI' bound with high affinity to Salmonella typhimurium cells (apparent Kd = 36 nM), permeabilized their outer membranes to actinomycin D, specifically activated a synovial fluid phospholipase A2 and showed potent bactericidal activity. In contrast with the native protein, however, it could not be efficiently released from the cell surface by the addition of high concentrations of Mg2+ ions. Pre-incubation of the protein with lipopolysaccharide or trypsin prevented cytotoxicity. However, boiling BPI' immediately before its addition to cells did not block its bactericidal activity, suggesting that it may be able to function even when presented to cells in an unfolded form. A BPI' derivative, containing a 13-residue foreign antigenic determinant genetically inserted between Ala115 and Asp116, was also produced. The derivative was functional in the above assays and bound with high affinity to S. typhimurium (apparent Kd = 74 nM). These results imply that the region defined by these residues is not involved in the lipopolysaccharide-binding or bactericidal activities of BPI. The availability of functional, nonglycosylated recombinant derivatives of BPI should greatly aid detailed studies on its structure, interactions with lipopolysaccharide and mechanism of action.

scholarly journals Acetylacetone-cleaving enzyme Dke1: a novel C-C-bond-cleaving enzyme from Acinetobacter johnsonii

2003 ◽  
Vol 369 (3) ◽  
pp. 573-581 ◽  
Author(s):  
Grit D. STRAGANZ ◽  
Anton GLIEDER ◽  
Lothar BRECKER ◽  
Douglas W. RIBBONS ◽  
Walter STEINER
Keyword(s):  
Metabolic Pathway ◽  
Metabolic Pathways ◽  
Active Protein ◽  
Acinetobacter Johnsonii ◽  
High Affinity ◽  
Gene Coding ◽  
High Yields ◽  
Relationship Of ◽  
First Time

The toxicity of acetylacetone has been demonstrated in various studies. Little is known, however, about metabolic pathways for its detoxification or mineralization. Data presented here describe for the first time the microbial degradation of acetylacetone and the characterization of a novel enzyme that initiates the metabolic pathway. From an Acinetobacter johnsonii strain that grew with acetylacetone as the sole carbon source, an inducible acetylacetone-cleaving enzyme was purified to homogeneity. The corresponding gene, coding for a 153 amino acid sequence that does not show any significant relationship to other known protein sequences, was cloned and overexpressed in Escherichia coli and gave high yields of active enzyme. The enzyme cleaves acetylacetone to equimolar amounts of methylglyoxal and acetate, consuming one equivalent of molecular oxygen. No exogenous cofactor is required, but Fe2+ is bound to the active protein and essential for its catalytic activity. The enzyme has a high affinity for acetylacetone with a Km of 9.1μM and a kcat of 8.5s-1. A metabolic pathway for acetylacetone degradation and the putative relationship of this novel enzyme to previously described dioxygenases are discussed.

Characterization of the high and low affinity components of the renal Ca2+ – Mg2+ ATPase

1990 ◽  
Vol 68 (6) ◽  
pp. 718-726 ◽  
Author(s):  
M. G. Brunette ◽  
J. Mailloux ◽  
M. Chan ◽  
C. Ramachandran
Keyword(s):  
Enzyme Activity ◽  
Atp Hydrolysis ◽  
Free Calcium ◽  
Atp Binding ◽  
High Affinity ◽  
Atp Concentration ◽  
Low Concentrations ◽  
High Affinity Site ◽  
High Concentrations

The purpose of this study was to characterize the interrelationship between free calcium (Ca2+) and magnesium (Mg2+) in the Ca2+ ATPase enzyme cycle of kidney membranes. Experiments were performed with basolateral membranes from rat renal cortex and microdissected proximal and distal tubules from mice. Results were similar in the three types of preparations. We first investigated the effect of ATP concentration on Ca2+- and Mg2+-dependent ATP hydrolysis. With 0.2 μM Ca2+, the enzyme activity, as a function of ATP concentration, showed two saturable components: a high affinity component with a Km of 33 μM ATP and a low affinity component with a Km of 0.63 mM ATP. These components may represent either two distinct sites of ATP binding or two forms of the same site. For the sake of simplicity, it was assumed that the two components correspond to a high affinity and a low affinity substrate site. At the high affinity site (ATP = 50 μM), the Ca2+ dependence of ATP hydrolysis followed a single Michaelis–Menten kinetics with Km for Ca2+ of 0.08 μM. The addition of 1 mM Mg2+ resulted in a relatively constant increase in ATP hydrolysis at all Ca2+ concentrations, indicating that the effects of the two cations were additive. With high ATP concentration (ATP = 3 mM), Ca2+ also induced an ATP hydrolysis according to a saturable process, with a Km for Ca2+ of 0.2 μM. In contrast with what occurred with low concentrations of ATP, addition of millimolar Mg2+ completely curtailed the sensitivity of the enzyme to Ca2+. However, traces of Mg2+ (< 10−6 M), while decreasing the Vmax of the Ca2+ ATPase, increased the affinity of the enzyme for Ca2+. These results indicate that with high concentrations of ATP, Mg2+ not only modifies the enzyme conformation but also displaces Ca2+. The effects of calmodulin, vanadate, and lanthanum were also examined using low and high concentrations of ATP. With 50 μM ATP, neither 0.5 μM calmodulin nor 100 μM lanthanum had any influence on the Ca2+-dependent ATP hydrolysis; in contrast, 100 μM vanadate significantly diminished the enzyme activity. With 3 mM ATP, 0.5 μM calmodulin increased the affinity of the enzyme for Ca2+, whereas 100 μM vanadate decreased it. These results suggest that the effect of calmodulin occurs only when the two sites are occupied, whereas vanadate interferes in presence of both low and high ATP concentrations. Based on the analogy with the sequential elementary steps described for other membrane Ca2+ ATPases and for Na+–K+ ATPase, and on the similarities between these characteristics of ATP hydrolysis and those of the enzyme phosphorylation on one hand and ATP-dependent Ca2+ transport on the other hand, we propose that the phosphoenzyme formation occurs at the high affinity site and that Ca2+ transport is directly related to ATP binding at the low affinity site, which might be considered as a regulatory site.Key words: kidney Ca2+ ATPase, ATP magnesium and calcium on Ca2+ ATPase, ATP-dependent calcium transport in kidney.

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

Synthesis and Spectral Characterization of Derivatives of Benzotriazole and Piperidine

2018 ◽  
Vol 8 (2) ◽  
pp. 278-287
Author(s):  
Selvarathy Grace P ◽  
Ravindran Durainayagam B ◽  
Pon Matheswari P.
Keyword(s):  
Spectral Characterization ◽  
Derivatives Of

Potential Contribution of Nutrients and Polycyclic Aromatic Hydrocarbons from the Creeks of Cootes Paradise Marsh

1996 ◽  
Vol 31 (3) ◽  
pp. 485-504 ◽  
Author(s):  
Patricia Chow-Fraser ◽  
Barb Crosbie ◽  
Douglas Bryant ◽  
Brian McCarry
Keyword(s):  
Aromatic Hydrocarbons ◽  
Laboratory Experiments ◽  
Dry Weight ◽  
Common Source ◽  
Potential Contribution ◽  
Nitrogen And Phosphorus ◽  
Engine Combustion ◽  
Polycyclic Aromatic ◽  
High Concentrations ◽  
Derivatives Of

Abstract During the summer of 1994, we compared the physical and nutrient characteristics of the three main tributaries of Cootes Paradise: Spencer, Chedoke and Borer’s creeks. On all sampling occasions, concentrations of CHL α and nutrients were always lowest in Borer’s Creek and highest in Chedoke Creek. There were generally 10-fold higher CHL α concentrations and 2 to 10 times higher levels of nitrogen and phosphorus in Chedoke Creek compared with Spencer Creek. Despite this, the light environment did not differ significantly between Spencer and Chedoke creeks because the low algal biomass in Spencer Creek was balanced by a relatively high loading of inorganic sediments from the watershed. Laboratory experiments indicated that sediments from Chedoke Creek released up to 10 µg/g of soluble phosphorus per gram (dry weight) of sediment, compared with only 2 µg/g from Spencer Creek. By contrast, sediment samples from Spencer Creek contained levels of polycyclic aromatic hydrocarbon that were as high as or higher than those from Chedoke Creek, and much higher than those found in Borer’s Creek. The distribution of normalized PAH concentrations suggests a common source of PAHs in all three tributaries, most likely automobile exhaust, since there were high concentrations of fluoranthene and pyrene, both of which are derivatives of engine combustion.

Acid-Sensing Ion Channels

2020 ◽  
Author(s):  
Stefan Gründer
Keyword(s):  
Nervous System ◽  
Ion Channels ◽  
Excitatory Synapses ◽  
Detailed Characterization ◽  
High Affinity ◽  
Acid Sensing Ion Channels ◽  
Long Time ◽  
Pathological Pain

Acid-sensing ion channels (ASICs) are proton-gated Na+ channels. Being almost ubiquitously present in neurons of the vertebrate nervous system, their precise function remained obscure for a long time. Various animal toxins that bind to ASICs with high affinity and specificity have been tremendously helpful in uncovering the role of ASICs. We now know that they contribute to synaptic transmission at excitatory synapses as well as to sensing metabolic acidosis and nociception. Moreover, detailed characterization of mouse models uncovered an unanticipated role of ASICs in disorders of the nervous system like stroke, multiple sclerosis, and pathological pain. This review provides an overview on the expression, structure, and pharmacology of ASICs plus a summary of what is known and what is still unknown about their physiological functions and their roles in diseases.

scholarly journals Adsorption Studies on Magnetic Nanoparticles Functionalized with Silver to Remove Nitrates from Waters

Water ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 1757
Author(s):  
Yesica Vicente-Martínez ◽  
Manuel Caravaca ◽  
Antonio Soto-Meca ◽  
Miguel Ángel Martín-Pereira ◽  
María del Carmen García-Onsurbe
Keyword(s):  
Magnetic Nanoparticles ◽  
Nitrate Content ◽  
Adsorption Efficiency ◽  
Experimental Conditions ◽  
Dependent Behavior ◽  
Before And After ◽  
Naoh Solution ◽  
High Concentrations ◽  
Interference Studies

This paper presents a novel procedure for the treatment of contaminated water with high concentrations of nitrates, which are considered as one of the main causes of the eutrophication phenomena. For this purpose, magnetic nanoparticles functionalized with silver (Fe3O4@AgNPs) were synthesized and used as an adsorbent of nitrates. Experimental conditions, including the pH, adsorbent and adsorbate dose, temperature and contact time, were analyzed to obtain the highest adsorption efficiency for different concentration of nitrates in water. A maximum removal efficiency of 100% was reached for 2, 5, 10 and 50 mg/L of nitrate at pH = 5, room temperature, and 50, 100, 250 and 500 µL of Fe3O4@AgNPs, respectively. The characterization of the adsorbent, before and after adsorption, was performed by energy dispersive X-ray spectroscopy, scanning electron microscopy, Brunauer-Emmett-Teller analysis and Fourier-transform infrared spectroscopy. Nitrates can be desorbed, and the adsorbent can be reused using 500 µL of NaOH solution 0.01 M, remaining unchanged for the first three cycles, and exhibiting 90% adsorption efficiency after three regenerations. A deep study on equilibrium isotherms reveals a pH-dependent behavior, characterized by Langmuir and Freundlich models at pH = 5 and pH = 1, respectively. Thermodynamic studies were consistent with physicochemical adsorption for all experiments but showed a change from endothermic to exothermic behavior as the temperature increases. Interference studies of other ions commonly present in water were carried out, enabling this procedure as very selective for nitrate ions. In addition, the method was applied to real samples of seawater, showing its ability to eliminate the total nitrate content in eutrophized waters.

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