scholarly journals Bacterial Lipid II Analogs: Novel In Vitro Substrates for Mammalian Oligosaccharyl Diphosphodolichol Diphosphatase (DLODP) Activities

Molecules ◽  
2019 ◽  
Vol 24 (11) ◽  
pp. 2135 ◽  
Author(s):  
Ahmad Massarweh ◽  
Michael Bosco ◽  
Isabelle Chantret ◽  
Thibaut Léger ◽  
Layla Jamal ◽  
...  
Keyword(s):  
Solid Phase ◽  
Extraction Procedure ◽  
Lipid Ii ◽  
Liver Membrane ◽  
Solid Phase Extraction Procedure ◽  
Bacterial Lipid ◽  
First Time ◽  
Layer Chromatography ◽  
Hydrolysis Of

Mammalian protein N-glycosylation requires the transfer of an oligosaccharide containing 2 residues of N-acetylglucosamine, 9 residues of mannose and 3 residues of glucose (Glc3Man9 GlcNAc2) from Glc3Man9GlcNAc2-diphospho (PP)-dolichol (DLO) onto proteins in the endoplasmic reticulum (ER). Under some pathophysiological conditions, DLO biosynthesis is perturbed, and truncated DLO is hydrolyzed to yield oligosaccharyl phosphates (OSP) via unidentified mechanisms. DLO diphosphatase activity (DLODP) was described in vitro, but its characterization is hampered by a lack of convenient non-radioactive substrates. Our objective was to develop a fluorescence-based assay for DLO hydrolysis. Using a vancomycin-based solid-phase extraction procedure coupled with thin layer chromatography (TLC) and mass spectrometry, we demonstrate that mouse liver membrane extracts hydrolyze fluorescent bacterial lipid II (LII: GlcNAc-MurNAc(dansyl-pentapeptide)-PP-undecaprenol) to yield GlcNAc-MurNAc(dansyl-pentapeptide)-P (GM5P). GM5P production by solubilized liver microsomal proteins shows similar biochemical characteristics to those reported for human hepatocellular carcinoma HepG2 cell DLODP activity. To conclude, we show, for the first time, hydrolysis of lipid II by a eukaryotic enzyme. As LII and DLO are hydrolyzed by the same, or closely related, enzymes, fluorescent lipid II analogs are convenient non-radioactive substrates for investigating DLODP and DLODP-like activities.

Measurement of heroin and its metabolites by isotope-dilution electron-impact mass spectrometry

1993 ◽  
Vol 39 (4) ◽  
pp. 670-675 ◽  
Author(s):  
B A Goldberger ◽  
W D Darwin ◽  
T M Grant ◽  
A C Allen ◽  
Y H Caplan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Enzyme Inhibitor ◽  
Solid Phase ◽  
Extraction Procedure ◽  
Gas Chromatography Mass Spectrometry ◽  
Detector Response ◽  
Heroin Use ◽  
Solid Phase Extraction Procedure ◽  
Electron Impact Mass ◽  
Hydrolysis Of

Abstract A solid-phase extraction procedure was developed for the isolation of heroin, 6-acetylmorphine, and morphine from blood, plasma, saliva, and urine with subsequent assay by gas chromatography/mass spectrometry. Aprotic solvents, mild elution conditions, and an enzyme inhibitor were used to ensure maximum analyte stability. Samples were extracted and the extract was divided into two equal portions. One portion was assayed directly for heroin; detector response was linear over a concentration range of 1.0 to 250 micrograms/L. The second part of the extract was reacted with N-methyl-bis-trifluoroacetamide and assayed for the trifluoroacetyl derivatives of 6-acetylmorphine and morphine; detector response was linear over a concentration range of 1.0 to 500 micrograms/L. The limit of sensitivity was 1.0 microgram/L for each analyte. Hydrolysis of heroin to 6-acetylmorphine during extraction and analysis was < 5%. The method can be used to corroborate heroin use and to study the pharmacological effects of heroin and its metabolites.

Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2257-2266 ◽  
Author(s):  
Helmuth Adelsberger ◽  
Christian Hertel ◽  
Erich Glawischnig ◽  
Vladimir V. Zverlov ◽  
Wolfgang H. Schwarz
Keyword(s):  
Extracellular Enzymes ◽  
Enzyme System ◽  
Thermophilic Bacterium ◽  
Recombinant Enzymes ◽  
Type Mode ◽  
Complete Set ◽  
First Time ◽  
Hydrolysis Of

Four extracellular enzymes of the thermophilic bacterium Clostridium stercorarium are involved in the depolymerization of de-esterified arabinoxylan: Xyn11A, Xyn10C, Bxl3B, and Arf51B. They were identified in a collection of eight clones producing enzymes hydrolysing xylan (xynA, xynB, xynC), β-xyloside (bxlA, bxlB, bglZ) and α-arabinofuranoside (arfA, arfB). The modular enzymes Xyn11A and Xyn10C represent the major xylanases in the culture supernatant of C. stercorarium. Both hydrolyse arabinoxylan in an endo-type mode, but differ in the pattern of the oligosaccharides produced. Of the glycosidases, Bxl3B degrades xylobiose and xylooligosaccharides to xylose, and Arf51B is able to release arabinose residues from de-esterified arabinoxylan and from the oligosaccharides generated. The other glycosidases either did not attack or only marginally attacked these oligosaccharides. Significantly more xylanase and xylosidase activity was produced during growth on xylose and xylan. This is believed to be the first time that, in a single thermophilic micro-organism, the complete set of enzymes (as well as the respective genes) to completely hydrolyse de-esterified arabinoxylan to its monomeric sugar constituents, xylose and arabinose, has been identified and the enzymes produced in vivo. The active enzyme system was reconstituted in vitro from recombinant enzymes.

scholarly journals THE PRODUCTION OF MONOCLONAL ANTIBODIES TO TETRASACCHARIDE - SYNTHETIC ANALOGUE OF THE CAPSULAR POLYSACCHARIDE OF STREPTOCOCCUS PNEUMONIAE OF SEROTYPE 14 AND THEIR IMMUNOCHEMICAL CHARACTERIZATION

2018 ◽  
pp. 26-31
Author(s):  
I. V. Yakovleva ◽  
E. A. Kurbatova ◽  
E. A. Akhmatova ◽  
E. V. Sukhova ◽  
D. V. Yashunsky ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Capsular Polysaccharide ◽  
Solid Phase ◽  
Synthetic Analogue ◽  
Immunochemical Characterization ◽  
Carbohydrate Epitopes ◽  
First Time

Aim. Production of monoclonal antibodies (mAb) to synthetic tetrasaccharide - repeating unit of the capsular polysaccharide (CP) of Streptococcus pneumoniae serotype 14 and their immunochemical characterization. Materials and methods. In order to generate the hybridoma producing mAb, mice were immunized with synthetic tetrasaccharide conjugated with bovine serum albumin (BSA) with following hybridization of B lymphocytes with mouse myeloma cells. Antibodies were obtained in vitro andin vivo. Immunochemical characterization of mAb to tetrasaccharide was carried out using a variety of ELISA options. Results. For the first time obtained mouse hybridoma, producing IgM to tetrasacchride. The IgM titer of anti-tetrasacharide antibodies in supernatants of clones and in the ascitic fluid of mice in ELISA detected by biotinylated tetrasaccharide and synthetic CP adsorbed on the solid phase was higher compared to the use of bacterial CP as well cover antigen. In the reaction of inhibition of the ELISA, the mAb recognized the corresponding carbohydrate epitopes of the bacterial CP of S. pneumoniae serotype 14 dissolved in the liquid phase better than tetrasaccharide ligand and synthetic CP. Conclusion. To detect mAb to tetrasaccharide in ELISA preferably to use synthetic analogues of the CP as solid phase antigens. The obtained mAb to tetrasaccharide can be used to determine the representation of the protective tetrasaccharide epitope of CP in the development of pneumococcal vaccines.

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