scholarly journals The isolation and characterization of the high-molecular-weight glycoprotein from pig colonic mucus

1978 ◽  
Vol 173 (2) ◽  
pp. 569-578 ◽  
Author(s):  
T Marshall ◽  
A Allen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Quaternary Structure ◽  
Water Soluble ◽  
Isolation And Characterization ◽  
Nuclease Digestion ◽  
Non Covalent Interactions ◽  
Covalent Interactions ◽  
Equilibrium Centrifugation

1. A high-molecular-weight glycoprotein constitutes over 80% by weight of the total glycoprotein from water-soluble pig colonic mucus. 2. It was isolated from from nucleic acid and non-covalently bound protein by nuclease digestion followed by equilibrium centrifugation in a CsCl gradient. 3. The glycoprotein has the following composition by weight: fucose 10.4%; glucosamine 23.9%; galactosamine 8.3%; sialic acid 9.9%; galactose 20.8%; sulphate 3.0%; protein 13.3%; moisture about 10%. 4. The native glycoprotein has the high mol.wt. of 15×10(6). 5. Reduction of the native glycoprotein with 2-mercaptoethanol results in a glycoprotein of mol.wt. 6×10(6). 6. Pronase digestion removes 29% of the protein (3% of the glycoprotein) but none of the carbohydrate. 7. The molecular weight of the Pronase-digested glycoprotein is 1.5×10(6), which is halved to 0.76×10(6) on reduction with 2-mercaptoethanol. 8. The contribution of non-covalent interactions, disulphide bridges and the non-glycosylated peptide core to the quaternary structure of the glycoprotein are discussed and compared with the known structure of pig gastric glycoportein.

Preparation and Characterization of the High Molecular Weight [3H]Hyaluronic Acid

1992 ◽  
Vol 57 (10) ◽  
pp. 2151-2156 ◽  
Author(s):  
Peter Chabreček ◽  
Ladislav Šoltés ◽  
Hynek Hradec ◽  
Jiří Filip ◽  
Eduard Orviský
Keyword(s):  
Molecular Weight ◽  
Hyaluronic Acid ◽  
High Molecular Weight ◽  
Methyl Bromide ◽  
High Performance ◽  
Hydrogen Atoms ◽  
Isolation And Characterization ◽  
Labelling Procedure ◽  
Enzymatic Depolymerization

Two methods for the preparation of high molecular weight [3H]hyaluronic acid were investigated. In the first one, hydrogen atoms in the molecule were replaced by tritium. This isotopic substitution was performed in aqueous solution using Pd/CaCO3 as the catalyst. In the second method, the high molecular weight hyaluronic acid was alkylated with [3H]methyl bromide in liquid ammonia at a temperature of -33.5 °C. High-performance gel permeation chromatographic separation method was used for the isolation and characterization of the high molecular weight [3H]hyaluronic acid. Molecular weight parameters for the labelled biopolymers were Mw = 128 kDa, Mw/Mn = 1.88 (first method) and Mw = 268 kDa, Mw/Mn = 1.55 (second method). The high molecular weight [3H]hyaluronic acid having Mw = 268 kDa was degraded further by specific hyaluronidase. Products of the enzymatic depolymerization were observed to be identical for both, labelled and cold biopolymer. This finding indicates that the described labelling procedure using [3H]methyl bromide does not induce any major structural rearrangements in the molecule.

scholarly journals Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

2002 ◽  
Vol 68 (9) ◽  
pp. 4390-4398 ◽  
Author(s):  
S. A. F. T. van Hijum ◽  
G. H. van Geel-Schutten ◽  
H. Rahaoui ◽  
M. J. E. C. van der Maarel ◽  
L. Dijkhuizen
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Lactobacillus Reuteri ◽  
Bacterial Strains ◽  
Potential Health ◽  
Isolation And Characterization ◽  
A Cell ◽  
Encoding Gene ◽  
First Time

ABSTRACT Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>107) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.

scholarly journals Construction of soil defined media using quantitative exometabolomic analysis of soil metabolites

2017 ◽  
Author(s):  
Stefan Jenkins ◽  
Tami L Swenson ◽  
Rebecca Lau ◽  
Andrea Rocha ◽  
Alex Aaring ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Weight ◽  
Organic Carbon ◽  
Quantitative Information ◽  
Water Soluble ◽  
Gas Chromatography Mass Spectrometry ◽  
Isolation And Characterization ◽  
Defined Media ◽  
Chromatography Mass Spectrometry

Exometabolomics enables analysis of metabolite utilization of low molecular weight organic substances by soil isolates. Environmentally-based defined media are needed to examine ecologically relevant patterns of substrate utilization. Here, we describe an approach for the construction of defined media using untargeted characterization of water soluble soil metabolites. To broadly characterize soil metabolites, both liquid chromatography mass spectrometry (LC/MS) and gas chromatography mass spectrometry (GC/MS) were used. With this approach, 96 metabolites were identified, including amino acids, amino acid derivatives, sugars, sugar alcohols, mono- and di-carboxylic acids, osmolytes, nucleobases, and nucleosides. From this pool of metabolites, 25 were quantified. Water soluble organic carbon was fractionated by molecular weight and measured to determine the fraction of carbon accounted for by the quantified metabolites. This revealed that, much like soil microbial community structures, these soil metabolites have an uneven quantitative distribution, with a single metabolite, trehalose accounting for 9.9 percent of the (< 1 kDa) water extractable organic carbon. This quantitative information was used to formulate two soil defined media (SDM), one containing 23 metabolites (SDM1) and one containing 46 (SDM2). To evaluate SDM for supporting the growth of bacteria found at this field site, we examined the growth of 30 phylogenetically diverse soil isolates obtained using standard R2A medium. The simpler SDM1 supported the growth of up to 13 isolates while the more complex SDM2 supported up to 25 isolates. One isolate, Pseudomonas corrugata strain FW300-N2E2 was selected for a time-series exometabolomics analysis to investigate SDM1 substrate preferences. Interestingly, it was found that this organism preferred lower-abundance substrates such as guanine, glycine, proline and arginine and glucose and did not utilize the more abundant substrates maltose, mannitol, trehalose and uridine. These results demonstrate the viability and utility of using exometabolomics to construct a tractable environmentally relevant media. We anticipate that this approach can be expanded to other environments to enhance isolation and characterization of diverse microbial communities.

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