scholarly journals Bacterial attack on phenolic ethers: An enzyme system demethylating vanillic acid

1967 ◽  
Vol 102 (3) ◽  
pp. 826-841 ◽  
Author(s):  
N. J. Cartwright ◽  
A. R. W. Smith
Keyword(s):  
Formate Dehydrogenase ◽  
Soluble Fraction ◽  
Enzyme System ◽  
Reduced Glutathione ◽  
Ring Cleavage ◽  
Formaldehyde Dehydrogenase ◽  
Free System ◽  
Methyl Group ◽  
Oxidative Demethylation ◽  
A Cell

1. A cell-free system from Pseudomonas fluorescens catalysed the oxidative demethylation and subsequent ring-cleavage of vanillate, with uptake of 2.5 moles of oxygen/mole of substrate. 2. Demethylation involved absorption of 0.5 mole of oxygen/mole, and required reduced glutathione (GSH) and nucleotide (probably NADPH) as cofactors, with further possible requirements, the natures of which are discussed. 3. Incomplete evidence suggested that the aromatic ring was opened via protocatechuate and the appropriate oxygenase, with absorption of 1 mole of oxygen/mole of substrate, eventually yielding beta-oxoadipate. 4. The methyl group was removed sequentially as formaldehyde, formate and carbon dioxide, the steps catalysed respectively by formaldehyde dehydrogenase, which required GSH and NAD(+), and formate dehydrogenase. Each enzyme was cytochrome-linked and accounted for absorption of 0.5mole of oxygen/mole of substrate. 5. All enzymes except formate dehydrogenase, which was a cell-wall enzyme, resided in the soluble fraction of the extract. The demethylase could not be resolved because of unknown cofactor requirements.

scholarly journals The separation of vanillate o-demethylase from protocatechuate 3,4-oxygenase by ultracentrifugation

1967 ◽  
Vol 105 (2) ◽  
pp. 767-770 ◽  
Author(s):  
N. J. Cartwright ◽  
J. A. Buswell
Keyword(s):  
Pseudomonas Fluorescens ◽  
Formaldehyde Dehydrogenase ◽  
Cell Free Extract ◽  
Methyl Group ◽  
Oxidative Demethylation ◽  
A Cell ◽  
Trapping Agent ◽  
Soluble Part

1. Protocatechuate 3,4-oxygenase in the soluble part of a cell-free extract of Pseudomonas fluorescens (strain T) sedimented more rapidly than vanillate O-demethylase under specified conditions in a preparative ultracentrifuge. 2. The supernatant from this process contained vanillate O-demethylase and formaldehyde dehydrogenase, and when supplemented with NADH oxidized vanillate with an uptake of 1 mole of oxygen/mole of substrate and accumulation of protocatechuate. 3. This uptake was decreased to 0·5mole/mole of substrate in the presence of semicarbazide as trapping agent for formaldehyde. 4. Reasons are presented for the process of methyl group removal from vanillate being oxidative demethylation.

scholarly journals Alloxan-induced luminol luminescence as a tool for investigating mechanisms of radical-mediated diabetogenicity

1981 ◽  
Vol 200 (3) ◽  
pp. 685-690 ◽  
Author(s):  
K Grankvist
Keyword(s):  
Superoxide Dismutase ◽  
Hydroxyl Radicals ◽  
Metal Ion ◽  
Reduced Glutathione ◽  
Chemical Reactivity ◽  
Free System ◽  
A Cell ◽  
Radical Generation ◽  
Metal Ion Chelators

Chemiluminescence of luminol in a cell-free system was used to investigate the mechanism of alloxan-dependent free-radical generation. In the presence of alloxan and reduced glutathione (GSH), luminescence was greatly stimulated by FeSO4. Replacing GSH by oxidized glutathione or NAD(P)(H), or replacing FeSO4 by CuSO4, ZNSO4 or FeCl3, did not yield chemiluminescence. The chemiluminescence of a mixture of alloxan. GSH, FeSO4 and luminol was inhibited by catalase, superoxide dismutase, scavengers of hydroxyl radicals (sodium benzoate, n-butanol, D-mannitol, dimethyl sulphoxide) or metal-ion chelators (EDTA, diethylenetriaminepenta-acetic acid, diethyldithiocarbamate. desferroxamine), D-glucose, L-glucose, D-mannose, D-fructose, 3-O-methyl-D-glucose, NAD+, NADH, NADP+ or NADPH, but not by urea or enzymically inactive superoxide dismutase. The results support the hypothesis that the diabetogenic action of alloxan is mediated by hydroxyl radicals generated in an iron-catalysed reaction. Protection against alloxan in vivo depends both on the chemical reactivity of protector with radicals or radical-generating systems and on the stereospecific requirement of some strategic site in the B-cell.

scholarly journals The transformation of the cytoplasmic oestradiol–receptor complex into the nuclear complex in a uterine cell-free system

1972 ◽  
Vol 128 (3) ◽  
pp. 611-616 ◽  
Author(s):  
Michael Gschwendt ◽  
Terrell H. Hamilton
Keyword(s):  
High Speed ◽  
Soluble Fraction ◽  
Supernatant Fraction ◽  
Receptor Complex ◽  
Free System ◽  
Cell Free System ◽  
Nuclear Complex ◽  
Nuclear Particle ◽  
A Cell ◽  
A Subunit

Experiments performed with a cell-free system in tris–EDTA buffer, pH 7.4, indicate that the high-speed supernatant fraction of the rat uterus contains all the factors necessary to transform the 8S cytoplasmic oestradiol–receptor complex to the nuclear complex. The transformation is temperature-dependent. This nuclear complex was extracted in the form of a 5S particle with 0.4m-KCl from sediments of either uterine or heart nuclei that had been incubated together with the cytoplasmic soluble fraction of the uterus at 2°C for 30min. This complex can also be obtained similarly from the soluble fraction of the uterus, incubated in the absence of nuclei. Previous warming of the soluble fraction to 37°C for 7min was necessary for the successful extraction of the nuclear particle under these conditions of incubation. After an incubation of the transformed complex with the nuclear sediment at 37°C for 7min, the 5S complex was extractable from the uterine nuclear sediment but not from the heart nuclear sediment, which may indicate the tissue specificity of the nuclear acceptor sites for the transformed complex. The extracted uterine nuclear complex sediments in the 5S region, but whether it is the native complex or a subunit or other part of the native complex resulting from the extraction with salt is unknown.

scholarly journals Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs.

1988 ◽  
Vol 106 (1) ◽  
pp. 1-12 ◽  
Author(s):  
M A Sheehan ◽  
A D Mills ◽  
A M Sleeman ◽  
R A Laskey ◽  
J J Blow
Keyword(s):  
Dna Replication ◽  
Soluble Fraction ◽  
Replication Initiation ◽  
Sperm Chromatin ◽  
Nuclear Pore ◽  
Free System ◽  
Cell Free System ◽  
Nuclear Assembly ◽  
Single Membrane ◽  
A Cell

We have studied the pathway of nuclear assembly from demembranated sperm chromatin by fractionating a cell-free system from Xenopus eggs (Lohka, M. J., and Y. Masui. 1983. Science (Wash. DC). 220:719-721). Both the soluble fraction and a washed vesicular fraction are required for formation of normal nuclei that initiate replication in vitro. The soluble fraction alone decondenses chromatin and the vesicular fraction alone surrounds chromatin with membranes. Both fractions are required for formation of nuclear pore complexes. Recombining these two fractions recovers approximately 100% of the nuclear assembly and DNA replication activities. Restricting the proportion of the vesicular fraction slows acquisition of the nuclear membrane and allows observation of immature nuclear pores ("prepores"). These form as arrays around and within the chromatin mass before membranes form. Subsequently membrane vesicles bind to these prepores, linking them by a single membrane throughout the chromatin mass. At the periphery this single membrane is surrounded by an outer membrane. In mature nuclei all membranes are at the periphery, the two membranes are linked by pores, and no prepores are seen. Nuclear assembly and replication are inhibited by preincubating the chromatin with the vesicular fraction. However nuclear assembly is accelerated by preincubating the condensed chromatin with the soluble fraction. This also decreases the lag before DNA replication. Initiation of DNA replication is only observed after normal nuclei have fully reassembled, increasing the evidence that replication depends on nuclear structure. The pathway of nuclear assembly and its relationship to DNA replication are discussed.

scholarly journals Metabolism of 4-chloro-2-methylphenoxyacetate by a soil pseudomonad. Ring-fission, lactonizing and delactonizing enzymes

1971 ◽  
Vol 122 (4) ◽  
pp. 533-542 ◽  
Author(s):  
J. K. Gaunt ◽  
W. C. Evans
Keyword(s):  
Metabolic Pathway ◽  
Reduced Glutathione ◽  
Single Step ◽  
Reaction Sequence ◽  
Free System ◽  
Cell Free System ◽  
Ring Fission ◽  
A Cell

1. A cell-free system, prepared from Pseudomonas N.C.I.B. 9340 grown on 4-chloro-2-methylphenoxyacetate (MCPA) was shown to catalyse the reaction sequence: 5-chloro-3-methylcatechol → cis–cis-γ-chloro-α-methylmuconate → γ-carboxymethylene-α-methyl-Δαβ-butenolide → γ-hydroxy-α-methylmuconate. 2. The activity of the three enzymes involved in these reactions was completely resolved and the lactonizing and delactonizing enzymes were separated. 3. This part of the metabolic pathway of 4-chloro-2-methylphenoxyacetate is thus confirmed for this bacterium. 4. The ring-fission oxygenase required Fe2+ or Fe3+ and reduced glutathione for activity; the lactonizing enzyme is stimulated by Mn2+, Mg2+, Co2+ and Fe2+; no cofactor requirement could be demonstrated for the delactonizing enzyme. 5. cis–cis-γ-Chloro-α-methylmuconic acid was isolated and found to be somewhat unstable, readily lactonizing to γ-carboxymethylene-α-methyl-Δαβ-butenolide. 6. Enzymically the lactonization appears to be a single-step dehydrochlorinase reaction.

Combination of ribosome and phage display for fast selection of high affinity VHH antibody fragments

2019 ◽  
pp. 27-33
Author(s):  
YuE Kravchenko ◽  
SV Ivanov ◽  
DS Kravchenko ◽  
EI Frolova ◽  
SP Chumakov
Keyword(s):  
Phage Display ◽  
Selection Procedure ◽  
Free System ◽  
Phage Displays ◽  
High Affinity ◽  
Binding Domains ◽  
A Cell ◽  
Vhh Antibodies ◽  
Efficiency Of Selection ◽  
Selection Of

Selection of antibodies using phage display involves the preliminary cloning of the repertoire of sequences encoding antigen-binding domains into phagemid, which is considered the bottleneck of the method, limiting the resulting diversity of libraries and leading to the loss of poorly represented variants before the start of the selection procedure. Selection in cell-free conditions using a ribosomal display is devoid from this drawback, however is highly sensitive to PCR artifacts and the RNase contamination. The aim of the study was to test the efficiency of a combination of both methods, including pre-selection in a cell-free system to enrich the source library, followed by cloning and final selection using phage display. This approach may eliminate the shortcomings of each method and increase the efficiency of selection. For selection, alpaca VHH antibody sequences suitable for building an immune library were used due to the lack of VL domains. Analysis of immune libraries from the genes of the VH3, VHH3 and VH4 families showed that the VHH antibodies share in the VH3 and VH4 gene groups is insignificant, and selection from the combined library is less effective than from the VHH3 family of sequences. We found that the combination of ribosomal and phage displays leads to a higher enrichment of high-affinity fragments and avoids the loss of the original diversity during cloning. The combined method allowed us to obtain a greater number of different high-affinity sequences, and all the tested VHH fragments were able to specifically recognize the target, including the total protein extracts of cell cultures.

scholarly journals Effect of detergents on sterol synthesis in a cell-free system of yeast

1982 ◽  
Vol 23 (6) ◽  
pp. 803-810
Author(s):  
S Hata ◽  
T Nishino ◽  
N Ariga ◽  
H Katsuki
Keyword(s):  
Sterol Synthesis ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Reconstitution of Endosomal Proteolysis in a Cell-free System

1989 ◽  
Vol 264 (10) ◽  
pp. 5392-5399
Author(s):  
L S Mayorga ◽  
R Diaz ◽  
P D Stahl
Keyword(s):  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Novel Method for Quantifying AhR-Ligand Binding Affinities Using Microscale Thermophoresis

Biosensors ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 60
Author(s):  
Anne Stinn ◽  
Jens Furkert ◽  
Stefan H. E. Kaufmann ◽  
Pedro Moura-Alves ◽  
Michael Kolbe
Keyword(s):  
Ligand Binding ◽  
Environmental Pollutants ◽  
Screening Strategy ◽  
Pharmacological Intervention ◽  
Binding Affinities ◽  
Free System ◽  
Microscale Thermophoresis ◽  
Aryl Hydrocarbon ◽  
Promising Target ◽  
A Cell

The aryl hydrocarbon receptor (AhR) is a highly conserved cellular sensor of a variety of environmental pollutants and dietary-, cell- and microbiota-derived metabolites with important roles in fundamental biological processes. Deregulation of the AhR pathway is implicated in several diseases, including autoimmune diseases and cancer, rendering AhR a promising target for drug development and host-directed therapy. The pharmacological intervention of AhR processes requires detailed information about the ligand binding properties to allow specific targeting of a particular signaling process without affecting the remaining. Here, we present a novel microscale thermophoresis-based approach to monitoring the binding of purified recombinant human AhR to its natural ligands in a cell-free system. This approach facilitates a precise identification and characterization of unknown AhR ligands and represents a screening strategy for the discovery of potential selective AhR modulators.

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