scholarly journals Chemically defined inducers of alkylsulphatases present in Pseudomonas C12B

1974 ◽  
Vol 138 (1) ◽  
pp. 53-62 ◽  
Author(s):  
Kenneth S. Dodgson ◽  
John W. Fitzgerald ◽  
William J. Payne
Keyword(s):  
Secondary Alcohol ◽  
Sodium Dodecyl ◽  
Nutrient Broth ◽  
Phase Cell ◽  
Alkyl Sulphate ◽  
Cell Extracts ◽  
Complex Process ◽  
Commercial Detergent ◽  
Process Studies ◽  
Long Chain

When Pseudomonas C12B is grown on nutrient broth to the stationary phase, cell extracts contain two secondary alkylsulphatases (S1 and S2) active towards potassium decan-5-yl sulphate but not towards potassium pentan-3-yl sulphate and one primary alkylsulphatase (P1) active towards sodium dodecan-1-yl sulphate (sodium dodecyl sulphate). When 10mm-sodium hexan-1-yl sulphate is included in the nutrient broth an additional primary alkylsulphatase (P2) is produced. The S1, S2, P1 and P2 enzymes are also present in extracts of cells grown on broth containing the commercial detergent Oronite, together with an additional secondary alkylsulphatase (S3) active towards pentan-3-yl sulphate as well as decan-5-yl sulphate. The P2 primary alkylsulphatase can be induced by a number of primary and secondary alkyl sulphate esters but the induction of the S3 enzyme appears to be a more specific and complex process. Studies on the ability of different fractions separated from Oronite to act as inducers suggest that the combination of a long-chain secondary alkyl sulphate(s) and a long-chain secondary alcohol(s) is responsible for the appearance of the S3 enzyme. Potassium hexadecan-2-yl sulphate or potassium tetradecan-2-yl sulphate, in combination with either hexadecan-2-ol or tetradecan-2-ol, can serve as inducers for the enzyme. Some characteristics of these specific inducer systems have been elucidated.

scholarly journals Novel NADP-linked alcohol–aldehyde/ketone oxidoreductase in thermophilic ethanologenic bacteria

1981 ◽  
Vol 195 (1) ◽  
pp. 183-190 ◽  
Author(s):  
R J Lamed ◽  
J G Zeikus
Keyword(s):  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Secondary Alcohol ◽  
Sodium Dodecyl ◽  
Secondary Alcohols ◽  
Metal Ion Binding ◽  
Primary Alcohols ◽  
Cell Extracts ◽  
Alcohol Dehydrogenase Activity ◽  
Clostridium Thermohydrosulfuricum

An NADP-specific alcohol--aldehyde/ketone oxidoreductase was detected in cell extracts of Thermoanaerobium brockii and Clostridium thermohydrosulfuricum, but not in Thermobacteroides acetoethylicus or Clostridium thermocellum. The enzyme was purified from Ta. brockii by differential procedures that included heat treatment and an affinity-chromatography step on Blue Dextran--Sepharose. The 44-fold-purified enzyme displayed one band (mol.wt. approx. 40000) after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme had a broad substrate specificity that included linear and branched primary alcohols, linear and cyclic secondary alcohols, linear and cyclic ketones, and acetaldehyde. The NADP-specific alcohol--aldehyde/ketone oxidoreductase was considerably more active towards secondary alcohols than towards other substrates. The enzyme had remarkable stability to heating at 86 degrees C for 70 min, but was rapidly denatured on boiling. Secondary-alcohol dehydrogenase activity displayed a noticeable inflexion point at 50 degrees C in Arrhenius plots and a high Q10 value (greater than 2.0). The enzyme was inactivated by the thiol-blocking reagent p-chloromercuribenzoate, but was not significantly inhibited by common metal-ion-binding agents. The NADP-linked alcohol--aldehyde/ketone oxidoreductase of Ta. brockii appears to have properties distinct from those of previously described primary- and secondary-alcohol dehydrogenases.

scholarly journals LaoABCR, a Novel System for Oxidation of Long-Chain Alcohols Derived from SDS and Alkane Degradation inPseudomonas aeruginosa

2018 ◽  
Vol 84 (13) ◽  
pp. e00626-18 ◽  
Author(s):  
Gianna Panasia ◽  
Bodo Philipp
Keyword(s):  
Gene Cluster ◽  
Distribution Systems ◽  
Sodium Dodecyl ◽  
Constitutive Expression ◽  
Opportunistic Pathogen ◽  
Alkane Degradation ◽  
Content Type ◽  
Cell Extracts ◽  
Growth Substrates ◽  
Long Chain

ABSTRACTThe opportunistic pathogenPseudomonas aeruginosastrain PAO1 is able to use a variety of organic pollutants as growth substrates, including the anionic detergent sodium dodecyl sulfate (SDS) and long-chain alkanes. While the enzymes initiating SDS and alkane degradation are well known, the subsequent enzymatic steps for degradation of the derived primary long-chain alcohols have not yet been identified. By evaluating genes specifically induced during growth with SDS, a gene cluster encoding a putative alcohol dehydrogenase (PA0364/LaoA), a probable inner membrane protein (PA0365/LaoB), and a presumable aldehyde dehydrogenase (PA0366/LaoC) was identified and designated the Lao (long-chain-alcohol/aldehyde-oxidation) system. Growth experiments with deletion mutants with SDS, 1-dodecanol, and alkanes revealed that LaoA and LaoB are involved in the degradation of primary long-chain alcohols. Moreover, detection of 1-dodecanol oxidation in cell extracts by activity staining revealed an interdependency of LaoA and LaoB for efficient 1-dodecanol oxidation. Anin silicoanalysis yielded no well-characterized homologue proteins for LaoA and LaoB. Furthermore, a gene adjacent to thelaogene cluster encodes a putative transcriptional regulator (PA0367/LaoR). AlaoRdeletion mutant exhibited constitutive expression of LaoA and LaoB, indicating that LaoR is a repressor for the expression oflaoABC. Taken together, these results showed that the proteins LaoA and LaoB constitute a novel oxidation system for long-chain alcohols derived from pollutants.IMPORTANCEThe versatile and highly adaptive bacteriumPseudomonas aeruginosa is able to colonize a variety of habitats, including anthropogenic environments, where it is often challenged with toxic compounds. Its ability to degrade such compounds and to use them as growth substrates can significantly enhance spreading of this opportunistic pathogen in hygienic settings, such as clinics or water distribution systems. Thus, knowledge about the metabolism ofP. aeruginosacan contribute to novel approaches for preventing its growth and reducing nosocomial infections. As the Lao system is important for the degradation of two different classes of pollutants, the identification of these novel enzymes can be a useful contribution for developing effective antibacterial strategies.

scholarly journals The Archaeal Small Heat Shock Protein Hsp17.6 Protects Proteins from Oxidative Inactivation

2021 ◽  
Vol 22 (5) ◽  
pp. 2591
Author(s):  
Pengfei Ma ◽  
Jie Li ◽  
Lei Qi ◽  
Xiuzhu Dong
Keyword(s):  
Heat Shock ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Small Heat Shock Protein ◽  
Phase Cell ◽  
Molecular Weights ◽  
Oxidative Inactivation ◽  
Methionine Residues ◽  
Shock Proteins ◽  
And Function

Small heat shock proteins (sHsps) are widely distributed among various types of organisms and function in preventing the irreversible aggregation of thermal denaturing proteins. Here, we report that Hsp17.6 from Methanolobus psychrophilus exhibited protection of proteins from oxidation inactivation. The overexpression of Hsp17.6 in Escherichia coli markedly increased the stationary phase cell density and survivability in HClO and H2O2. Treatments with 0.2 mM HClO or 10 mM H2O2 reduced malate dehydrogenase (MDH) activity to 57% and 77%, whereas the addition of Hsp17.6 recovered the activity to 70–90% and 86–100%, respectively. A similar effect for superoxide dismutase oxidation was determined for Hsp17.6. Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis assays determined that the Hsp17.6 addition decreased H2O2-caused disulfide-linking protein contents and HClO-induced degradation of MDH; meanwhile, Hsp17.6 protein appeared to be oxidized with increased molecular weights. Mass spectrometry identified oxygen atoms introduced into the larger Hsp17.6 molecules, mainly at the aspartate and methionine residues. Substitution of some aspartate residues reduced Hsp17.6 in alleviating H2O2- and HClO-caused MDH inactivation and in enhancing the E. coli survivability in H2O2 and HClO, suggesting that the archaeal Hsp17.6 oxidation protection might depend on an “oxidant sink” effect, i.e., to consume the oxidants in environments via aspartate oxidation

Effect of microenvironment and cell-line type on carbohydrate-binding proteins of macrophage-like cells

1988 ◽  
Vol 66 (11) ◽  
pp. 1169-1176 ◽  
Author(s):  
Hans-Joachim Gabius ◽  
Katalin Vehmeyer
Keyword(s):  
Binding Proteins ◽  
Cultured Cells ◽  
Sodium Dodecyl ◽  
Carbohydrate Binding ◽  
Intercellular Interaction ◽  
Cell Extracts ◽  
Carbohydrate Binding Proteins ◽  
Sugar Receptors

The pattern of sugar inhibition of rosette formation, a model for intercellular interaction between cultured cells and glutaraldehyde-fixed, trypsinated rabbit erythrocytes, served to infer the presence of carbohydrate-binding proteins. This profile from cell extracts for the two murine macrophage-like cell lines, P388D1 and J774A.1, was comparatively analyzed by affinity chromatography on supports with immobilized carbohydrates (lactose, L-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and maltose) or with the immobilized mannose-rich yeast glycoprotein mannan or fetuin-derived glycopeptides containing sialic acid residues. After elution with specific sugar in the absence of Ca2+ ions, the proteins were separated by sodium dodecyl sulfate – polyacrylamide slab gel electrophoresis. The composition of carbohydrate-binding proteins of the two lines clearly exhibited quantitative and qualitative differences. Moreover, the pattern of P388D1 cells was also demonstrated to change significantly in response to alterations in the conditions of the physiological environment. These alterations were imposed by in vitro growth, by subsequent in vivo growth in nude mice, and by re-adaptation of cells to culture after in vivo passage. Collectively, our observations and other physiological and biochemical reports on macrophage lectins indicate that the presence of sugar receptors with different specificities may be an indicator of macrophage differentiation, being reversibly modulated to a considerable extent by external factors, e.g., microenvironment. Extensive but selective alterations in this respect could play an important role in the control of recognition and effector mechanisms within diverse functions of macrophage subpopulations.

scholarly journals Investigations on ornithobacterium rhinotracheale in broiler flocks in elazig province located in the east of turkey

2012 ◽  
Vol 49 (No. 8) ◽  
pp. 305-311 ◽  
Author(s):  
G. Ozbey ◽  
H. Ongor ◽  
D. T Balik ◽  
V. Celik ◽  
A. Kilic ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rrna Gene ◽  
Major Band ◽  
Cell Extracts ◽  
Sds Page ◽  
Ornithobacterium Rhinotracheale

In the present study, lung, trachea and serum samples from broiler flocks slaughtered at an abattoir in Elazig province located in the East of Turkey were examined for the presence of Ornithobacterium rhinotracheale using culture and enzyme-linked immunosorbent assay (ELISA). The identity was latter proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis, and polymerase chain reaction (PCR) assays. A total of 324 serum and 250 lung and trachea samples were collected from 10 commercially reared chicken flocks showing respiratory manifestations. The samples were obtained from different flocks. The causative agent (ORT) was isolated from trachea (1.5%) of five chickens and from both lung and trachea (0.4%) of only one chicken in the bacteriological examination of tissues. The presence of antibodies against ORT was detected in 33 (10.2%) of the 324 sera by ELISA. A 784 bp fragment of the 16S rRNA gene was amplified using specific primers in the PCR. All ORT isolates that were positive by culture were also detected to be positive by the PCR. SDS-PAGE protein profiles of whole cell extracts showed a high similarity for all the isolates with a major band of the molecular weight of 33 kDa (kiloDalton). Results of Western blot analysis indicate four antigenic fractions predominantly with molecular weights of 33, 42, 52 and 66 kDa.

scholarly journals Mechanism of neutrophil chemiluminescence induced by wheat germ agglutinin: partial characterization of the antigens recognized by wheat germ agglutinin

Blood ◽  
1984 ◽  
Vol 64 (5) ◽  
pp. 1094-1102 ◽  
Author(s):  
Y Ozaki ◽  
J Iwata ◽  
T Ohashi
Keyword(s):  
Membrane Proteins ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Sodium Dodecyl ◽  
Submaxillary Gland ◽  
Binding Property ◽  
Molecular Weights ◽  
Acetylneuraminic Acid ◽  
Cell Extracts ◽  
High Concentrations

Abstract Wheat germ agglutinin (WGA) stimulated neutrophils to produce significant levels of luminol-dependent chemiluminescence (CL). Since WGA is known to bind N-acetylglucosamine (GlcNAc) oligomers and N- acetylneuraminic acid (NANA), we attempted to determine which binding property of WGA is essential for induction of CL. The succinylated form of WGA (SuWGA), which is no longer able to bind NANA, was still able to induce CL. N-Acetylglucosamine at a concentration of 20 mmol/L almost completely inhibited WGA-induced CL production by neutrophils, whereas bovine submaxillary gland mucin, a potent blocker of NANA binding of WGA, failed to inhibit CL production. Lectins with the GlcNAc-binding property were examined for their ability to induce CL. Those that have higher valences and have a tendency to bind GlcNAc oligomers in the internal portion of glycoconjugates were able to induce CL, whereas those that have low valences and bind terminal GlcNAc of glycoconjugates failed to induce CL even at high concentrations. Attempts were made to characterize the neutrophil membrane proteins recognized by WGA. Glycoproteins with a molecular weight of 25,000 daltons were identified by a 50 mmol/L GlcNAc elution of WGA gels loaded with 125I-labeled neutrophil membrane proteins. Elution with 500 mumol/L GlcNAc trimer produced several glycoproteins of different molecular weights in addition to the glycoproteins of 25,000 daltons. 125I-labeled WGA and SuWGA were used for autoradiographic analysis of cell extracts of the neutrophils separated on sodium dodecyl sulfate polyacrylamide gels. WGA recognized multiple glycoproteins of different molecular weights, whereas SuWGA bound only a few of them. Glycoproteins of 25,000 daltons, probably corresponding to those identified by 50 mmol/L GlcNAc elution, were also recognized.

Regulation of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) in Aspergillus nidulans

1983 ◽  
Vol 217 (1208) ◽  
pp. 243-264 ◽  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Aspergillus Nidulans ◽  
Structural Gene ◽  
Aldehyde Dehydrogenase ◽  
Gel Electrophoresis ◽  
Regulatory Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Cell Extracts

There is a single major alcohol dehydrogenase (ADH) and a single major aldehyde dehydrogenase (AldDH) in Aspergillus nidulans . Both ADH and AldDH are induced by ethanol and by acetaldehyde and both are subject to carbon catabolite repression. ADH and AldDH are necessary for the utilization of ethanol and of threonine, indicating that both compounds are utilized via acetaldehyde. ADH and AldDH each give a single major activity band on gel electrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell extracts shows at least two similar ADH polypeptides of approximate relative molecular mass (r. m. m.) 41000 and two similar AldDH polypeptides of approximate r. m. m. 57000. The in vitro translation of mRNA from induced, carbon derepressed wild-type cells gives up to three ADH polypeptides in the r. m. m. range 39000-43000 and an AldDH polypeptide of approximate r. m. m. 57000. The mRNA from uninduced, carbon repressed wild-type cells does not direct the synthesis of the ADH and AldDH polypeptides. This indicates that the regulation of ADH and AldDH is at the level of transcription and/or post-transcriptional modification. The probable explanation of the multiple ADH polypeptides is post-transcriptional modification of the mRNA. Allyl alcohol mutants were made by using diepoxyoctane and γ-rays as mutagens. There are two classes, alcA and alcR . Neither class can utilize ethanol or threonine as a carbon source. The alcA mutants lack normal ADH and are recessive. Of the 47 alcA mutants examined 39 do not make the ADH polypeptides while eight do so. Therefore alcA is the structural gene for ADH. The two alcA mutants tested do not make functional mRNA for ADH. The alcR mutants lack both ADH and AldDH and are recessive. No alcR mutants make the ADH or the AldDH polypeptides. The three alcR mutants tested do not make functional ADH or AldDH mRNA. The mutant alcR 125 is a nonsense mutant, which establishes that alcR codes for a protein. The alcA and alcR genes are adjacent on chromosome VII and a preliminary fine-structure map of the alcA gene has been made. Three mutants that cannot utilize ethanol or threonine and have ADH, but lack AldDH, define a gene AldA on chromosome VIII. The aldA 23 mutant makes the AldDH polypeptides, the other two aldA mutants do not. Therefore aldA is probably the structural gene for AldDH. Our current hypothesis is that alcA and aldA are the structural genes for ADH and AldDH respectively and alcR is a transacting regulatory gene coding for a protein whose function is necessary for the expression of the alcA and aldA genes.

Cofactor specificity engineering of a long-chain secondary alcohol dehydrogenase from Micrococcus luteus for redox-neutral biotransformation of fatty acids

2019 ◽  
Vol 55 (96) ◽  
pp. 14462-14465 ◽  
Author(s):  
Eun-Ji Seo ◽  
Hye-Ji Kim ◽  
Myeong-Ju Kim ◽  
Jeong-Sun Kim ◽  
Jin-Byung Park
Keyword(s):  
Fatty Acids ◽  
Alcohol Dehydrogenase ◽  
Micrococcus Luteus ◽  
Secondary Alcohol ◽  
Cofactor Specificity ◽  
Secondary Alcohol Dehydrogenase ◽  
Long Chain

Structure-based cofactor specificity engineering of an alcohol dehydrogenase (mLSADH) enables a redox-neutral biotransformation of C18 fatty acids into C9 fatty acids.

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