Isolation and partial characterization of rat duodenal-gland (Brunner's-gland) mucus glycoprotein.
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ABSTRACT:
SUBMITTER: Smits HL
PROVIDER: S-EPMC1158296 | biostudies-other | 1982 Jun
REPOSITORIES: biostudies-other
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Project description:Glycoprotein from pig small-intestinal mucus was isolated free of non-covalently bound protein and nucleic acid with a yield of over 60%. No non-covalently bound protein could be detected by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis or by equilibrium centrifugation in a density gradient of CsCl with 4 M-guanidinium chloride. The intrinsic viscosity and reduced viscosity of the glycoprotein preparations rose with the removal of non-covalently bound protein and nucleic acid from the glycoprotein, evidence that non-covalently bound protein does not contribute to the rheological properties of the glycoprotein in the mucus. The pure glycoprotein, in contrast with impure preparations, gelled at the same concentration of glycoprotein as that present in the gel in vivo. The glycoprotein was a single component, as judged by gel filtration and analytical ultracentrifugation. The distribution of sedimentation coefficients was polydisperse but unimodal with an s025,w of 14.5S and a molecular weight of 1.72 X 10(6). The chemical composition of the glycoprotein was 77% carbohydrate and 21% protein, 52% of which was serine, threonine and proline. The glycoprotein had a strong negative charge and contained 3.1% and 18.3% by weight ester sulphate and sialic acid respectively. The molar proportion of N-acetylgalactosamine was nearly twice that of any of the other sugars present, the glycoprotein had A and H blood-group activity and the average maximum length of the carbohydrate chains was deduced to be six to eight sugar residues.
| S-EPMC1162882 | biostudies-other
Project description: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 X 10(6). 5. Reduction of the native glycoprotein with 2-mercaptoethanol results in a glycoprotein of mol.wt. 6 X 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 X 10(6), which is halved to 0.76 X 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.
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Project description:We isolated monomeric gastric mucus glycoprotein from the rat stomach by applying three successive CsCl-density-gradient steps in the continuous presence of guanidinium chloride. The rat gastric mucin was pure as compared with mucin isolated without the chaotropic reagent. In addition, the presence of guanidinium chloride resulted in a better preservation of the protein moiety. The purified mucin was fractionated according to buoyant density and chemically radiolabelled on tyrosine or cysteine residues and digested with specific proteinases. Analysis of mucin fractions of various densities gave identical peptide patterns, suggesting that the fractions contain a common protein backbone. Electron-microscopic images of the individual mucin molecules were recorded using rotary shadowing. They showed large filamentous molecules with a mean length of 208 nm that, after proteolytic digestion, yielded glycopeptides with a mean length of 149 nm. Heterogeneity in buoyant density and electrophoretic mobility is located in this large glycopeptide which remains after proteolytic digestion. Metabolic labelling of the mucin with [35 S]sulphate and [3H]galactose, followed by purification and proteolytic digestion, revealed that this glycopeptide accounts for most of the mass and contains relatively little protein, but probably all the oligosaccharides and sulphate. As this protein part is masked by the oligosaccharides, detailed study by the methods described was not possible. The results indicate that rat gastric mucin is homogeneous in a major part of the protein backbone and that the heterogeneity of the molecule originates most likely from differences in sulphate and/or sugar composition.
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Project description:Newly synthesized proteoglycans of rat incisors were labelled in vivo for 6h with [35S]-sulphate in order to facilitate their detection during purification and characterization. Proteoglycans were extracted from non-mineralized portions (predentine) of rat incisors with 4M-guanidinium chloride and subsequently from dentine by demineralization with a 0.4M-EDTA solution containing 4M-guanidinium chloride. Both extractions were performed at 4 degrees C in the presence of proteinase inhibitors. Purification of proteoglycans was achieved with a procedure involving gel-filtration chromatography, selective precipitation of phosphoproteins, affinity chromatography and ion-exchange chromatography. Two proteoglycan populations were found in the initial extract (Pd-PG I and Pd-PG II), whereas only one fraction (D-PG) was obtained after demineralization. The minor proteoglycan fraction from the first extract, Pd-PG I, although not totally characterized, differed sharply from the other proteoglycans in that it had a larger molecular size with larger glycosaminoglycan chains composed of chondroitin 4- and 6-sulphate isomers. In contrast, the major proteoglycans Pd-PG II and D-PG had smaller hydrodynamic sizes with smaller glycosaminoglycan chains (but larger than those from bovine nasal cartilage proteoglycans) composed exclusively of chondroitin 4-sulphate. The major proteoglycans were incapable of interacting with hyaluronic acid. In general, the amino acid compositions of the major proteoglycans of rat incisors resembled that of bovine nasal cartilage proteoglycans, but the former had lower proline, valine, isoleucine, leucine, and higher aspartic acid, contents.
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Project description:The olfactory epithelium is continuously exposed to exogenous chemicals, including odorants. During the past decade, the enzymes surrounding the olfactory receptors have been shown to make an important contribution to the process of olfaction. Mammalian xenobiotic metabolizing enzymes, such as cytochrome P450, esterases and glutathione transferases (GSTs), have been shown to participate in odorant clearance from the olfactory receptor environment, consequently contributing to the maintenance of sensitivity toward odorants. GSTs have previously been shown to be involved in numerous physiological processes, including detoxification, steroid hormone biosynthesis, and amino acid catabolism. These enzymes ensure either the capture or the glutathione conjugation of a large number of ligands. Using a multi-technique approach (proteomic, immunocytochemistry and activity assays), our results indicate that GSTs play an important role in the rat olfactory process. First, proteomic analysis demonstrated the presence of different putative odorant metabolizing enzymes, including different GSTs, in the rat nasal mucus. Second, GST expression was investigated in situ in rat olfactory tissues using immunohistochemical methods. Third, the activity of the main GST (GSTM2) odorant was studied with in vitro experiments. Recombinant GSTM2 was used to screen a set of odorants and characterize the nature of its interaction with the odorants. Our results support a significant role of GSTs in the modulation of odorant availability for receptors in the peripheral olfactory process.
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Project description:Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 X 10(6) and 5.9 X 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).
| S-EPMC1154324 | biostudies-other
Project description:The synthesis of the polypeptide backbone of mucus glycoproteins in rat stomach was studied. CsCl centrifugation of the homogenate of [3H]serine pulse-chase labelled stomach or mucosal scrapings showed that [3H]serine was mainly incorporated into molecules having a density identical to that of proteins and that only 8-12% was incorporated into macromolecules with the density of mucus glycoproteins. [3H]-Galactose, however, was almost exclusively incorporated into macromolecules with a density identical to that of mucus glycoproteins. Electrophoretic analysis of the CsCl fraction containing the mucus glycoprotein revealed that 78% of the [3H]serine-labelled macromolecules had an electrophoretic behaviour identical to that of mucus glycoproteins. Thus, only a small portion (about 6-10%) of incorporated [3H]serine was present in the backbone of the mucus glycoprotein. Translation in a wheat germ cell-free system of total RNA derived from both whole stomach and superficial mucosal scrapings, using either [35S]methionine or [35S]cysteine as radioactive amino acid, yielded a wide range of proteins. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, one major translation product of whole stomach RNA had an apparent Mr (43000) identical to that of rat pepsinogen. As this polypeptide could not be found amongst the translation products of RNA from scrapings it probably was pepsinogen. The present data provide strong evidence that the backbone polypeptide of mucus glycoproteins only accounts for a small part of the proteins synthesized by mucus-producing cells.
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