scholarly journals Isolation and physical characterization of the MUC7 (MG2) mucin from saliva: evidence for self-association

1998 ◽  
Vol 334 (2) ◽  
pp. 415-422 ◽  
Author(s):  
Ravi MEHROTRA ◽  
David J. THORNTON ◽  
John K. SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Peptide Mapping ◽  
Minor Component ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Average Molecular Mass ◽  
Self Association ◽  
A Minor

Saliva contains two major families of mucins (MG1 and MG2); the polypeptide of the smaller of these glycoproteins (MG2) has been assigned as the product of the MUC7 gene. In this study we have devised a rapid two-step procedure that recovers this glycoprotein essentially free of other components and in sufficient quantity to enable physical and self-interaction studies. Raw saliva was solubilized in 4 M guanidinium chloride and thereafter subjected to Sepharose CL-4B chromatography. The MG2-rich fraction was recovered free from the larger MG1 glycoproteins and also smaller proteins/glycoproteins (molecular mass less than 100 kDa). MG2 glycoproteins were finally purified by anion-exchange chromatography on Mono Q. The purity of the preparation was assessed by SDS/PAGE after radiolabelling of the molecules with [14C]acetic anhydride. Peptide mapping, N-terminal sequencing and amino acid analysis verified the polypeptide of the mucins as the MUC7 gene product. The isolated molecules were examined by electron microscopy and appeared as short flexible worm-like structures 30–120 nm in length. The distribution was heterogeneous, containing a major component with number-average and weight-average lengths of 52 and 55 nm respectively and a minor component with number-average and weight-average lengths of 94 and 98 nm respectively. We propose that the two differently sized populations represent monomeric and dimeric species of the mucins. Gel chromatography performed in 0.2 M NaCl indicated the presence of monomers, dimers and tetramers; an average molecular mass for the preparation was 192 kDa. However, in 4 M guanidinium chloride the molecular mass was 158 kDa and a similar molecular mass (155 kDa) was determined for the mucin preparation after reduction. These results suggest that the mucins might self-associate via a protein-mediated interaction. On the basis of the results a model is proposed for the self-association of the MUC7 mucin, which might be important for its biological function.

scholarly journals Low-Mr iron isolated from guinea pig reticulocytes as AMP-Fe and ATP-Fe complexes

1989 ◽  
Vol 261 (3) ◽  
pp. 787-792 ◽  
Author(s):  
J Weaver ◽  
S Pollack
Keyword(s):  
Guinea Pig ◽  
Anion Exchange ◽  
Dominant Species ◽  
Exchange Resin ◽  
Anion Exchange Resin ◽  
Minor Component ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fe Complexes ◽  
A Minor

Guinea pig reticulocytes were pulse-labelled with 59Fe bound to transferrin. Haemolysates prepared from these reticulocytes were subjected to rapid (NH1)2SO1 precipitation and then chromatography on an anion-exchange resin. ATP-bound 59Fe was the dominant species in the reticulocyte cytosol; 2,3-bisphosphoglycerate and GTP iron complexes were not detected despite the fact that these were stable with (NH1)2SO1 precipitation and readily detected with anion-exchange chromatography. AMP-bound Fe was a minor component of the cytosol following rapid (NH1)2SO4 precipitation, and the major component when iron was released from transferrin by haemolysates. We speculate that ATP-Fe may be degraded in the cell to permit utilization of its iron for haem synthesis.

scholarly journals A high-affinity inositol 1,3,4,5-tetrakisphosphate receptor protein from brain is specifically labelled by a newly synthesized photoaffinity analogue, N-(4-azidosalicyl)aminoethanol(1)-1-phospho-d-myo-inositol 3,4,5-trisphosphate

1991 ◽  
Vol 280 (2) ◽  
pp. 533-539 ◽  
Author(s):  
G Reiser ◽  
R Schäfer ◽  
F Donié ◽  
E Hülser ◽  
M Nehls-Sahabandu ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Anion Exchange ◽  
Protein Band ◽  
Anion Exchange Chromatography ◽  
Bovine Brain ◽  
Exchange Chromatography ◽  
Receptor Protein ◽  
Radioactive Label ◽  
Binding Reaction ◽  
High Affinity

A photolabile arylazido analogue of Ins(1,3,4,5)P4 selectively substituted at the 1-phosphate group was synthesized by coupling 2-aminoethanol(1)-1-phospho-D-myo-inositol 4,5-bisphosphate with N-hydroxysuccinimidyl-4-azidosalicylic acid [Schäfer, Nehls-Sahabandu, Grabowsky, Dehlinger-Kremer, Schulz & Mayr (1990) Biochem. J. 272, 817-825] and subsequently phosphorylating the product by bovine brain Ins(1,4,5)P3 3-kinase. The product, N-(4-azidosalicyl)-aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-trisphosphate [AsaIns(1,3,4,5)P4] was radioiodinated and purified by anion-exchange chromatography. AsaIns(1,3,4,5)P4 bound to a high-affinity Ins(1,3,4,5)P4 receptor from pig cerebellum with an affinity only 3-fold lower than that of Ins(1,3,4,5)P4. Photoirradiation of 125I-AsaIns(1,3,4,5)P4 in the presence of the receptor preparation revealed that the radioactive label was specifically associated with a protein band of apparent molecular mass 42 kDa, which Donié & Reiser [(1991) Biochem. J. 275, 453-457] had previously tentatively assigned to the Ins(1,3,4,5)P4 receptor protein. The radioactive label was displaced from the receptor when the binding reaction with 125I-AsaIns(1,3,4,5)P4 was carried out in the presence of 5 microM-Ins(1,3,4,5)P4.

scholarly journals Hyaluronic acid in cartilage and proteoglycan aggregation

1974 ◽  
Vol 139 (3) ◽  
pp. 565-581 ◽  
Author(s):  
Timothy E. Hardingham ◽  
Helen Muir
Keyword(s):  
Hyaluronic Acid ◽  
Buoyant Density ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Keratan Sulphate ◽  
Sulphate Content ◽  
Lower Protein ◽  
Proteoglycan Aggregates

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.

Fractionation of Human Urine by Gel Chromatography

1970 ◽  
Vol 16 (3) ◽  
pp. 201-206 ◽  
Author(s):  
C A Burtis ◽  
Gerald Goldstein ◽  
Charles D Scott
Keyword(s):  
High Resolution ◽  
Anion Exchange ◽  
Ultraviolet Light ◽  
Human Urine ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Gel Chromatography

Abstract High-resolution anion-exchange chromatography of human urine can resolve more than 150 constituents that absorb ultraviolet light. Prefractionation of urine on Sephadex G-10 helped us identify the constituents and simplified the anion-exchange chromatogram. The material associated with the 13 peaks obtained by gel chromatography was pooled and concentrated into six fractions, which were subsequently analyzed by high-resolution anion-exchange chromatography. Each of these fractions contained from 13 to 63 ultraviolet-absorbing constituents.

scholarly journals Glutathione S-transferases of the yeast Yarrowia lipolytica have unusually large molecular mass

1998 ◽  
Vol 333 (3) ◽  
pp. 839-845 ◽  
Author(s):  
Vivienne FOLEY ◽  
David SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Yarrowia Lipolytica ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Post Translational Modification ◽  
Native Molecular Mass ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
Yeast Yarrowia Lipolytica

Two similar glutathione S-transferases (GSTs), which do not bind to glutathione– or S-hexylglutathione–agarose affinity resins, have been purified from the yeast Yarrowia lipolytica. An approx. 400-fold purification was obtained by a combination of DEAE-Sephadex, phenyl-Sepharose, hydroxyapatite and Mono-Q anion-exchange chromatography. The native molecular mass of both proteins was estimated as approx. 110 kDa by both Superose-12 gel-filtration chromatography and non-denaturing electrophoresis. SDS/PAGE indicated a subunit mass of 50 kDa. Reverse-phase HPLC of purified proteins gave a single, well-resolved, peak, suggesting that the proteins are homodimers. Identical behaviour on HPLC, native electrophoresis and SDS/PAGE, N-terminal sequencing, sensitivity to a panel of inhibitors and identical specific activities with 1-chloro-2,4-dinitrobenzene as substrate suggest that the two isoenzymes are very similar. The enzymes do not immunoblot with antisera to any of the main GST classes, and N-terminal sequencing suggests no clear relationship with previously characterized enzymes, such as that of the fungus, Phanerochaete chrysosporium [Dowd, Buckley and Sheehan (1997) Biochem. J. 324, 243–248]. It is possible that the two isoenzymes arise as a result of post-translational modification of a single GST isoenzyme.

Production, purification, and characterization of an extracellular endo-β-1, 3-glucanase from a monokaryon of Schizophyllum commune ATCC 38548 defective in exo-β-1, 3-glucanase formation

1994 ◽  
Vol 40 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Andreas Prokop ◽  
Peter Rapp ◽  
Fritz Wagner
Keyword(s):  
Molecular Mass ◽  
Schizophyllum Commune ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Glucanase Activity ◽  
Sds Page ◽  
S 100 ◽  
Reduced Activity

Production of extracellular β-1, 3-glucanase activity by a monokaryotic Schizophyllum commune strain was monitored and results indicated that the β-glucanase activity consisted of an endo- β-1, 3-glucanase activity, besides a negligible amount of β-1, 6-glucanase and β-glucosidase activity. Unlike the β-1, 3-glucanase production of the dikaryotic parent strain S. commune ATCC 38548, the β-1, 3-glucanase formation of the monokaryon was not regulated by catabolite repression. The endo- β-1, 3-glucanase of the monokaryon was purified from the culture filtrate by lyophilization, anion exchange chromatography on Mono Q, and gel filtration on Sephacryl S-100. It appeared homogeneous on SDS-PAGE with a molecular mass of 35.5 kDa and the isoelectric point was 3.95. The enzyme was only active toward glucans containing β-1, 3-linkages, including lichenan, a β-1, 3-1, 4-D-glucan. It attacked laminarin in an endo-like fashion to form laminaribiose, laminaritriose, and high oligosaccharides. While the extracellular β-glucanases from the dikaryotic S. commune ATCC 38548 degraded significant amounts of schizophyllan, the endo- β-1, 3-glucanase from the monokaryon showed greatly reduced activity toward this high molecular mass β-1, 3-/β-1, 6-glucan. The Km of the endoglucanase, using laminarin as substrate, was 0.28 mg/mL. Optimal pH and temperature were 5.5 and 50 °C, respectively. The enzyme was stable between pH 5.5 and 7.0 and at temperatures below 50 °C. The enzyme was completely inhibited by 1 mM Hg2+. Growth of the monokaryotic S. commune strain was not affected by its constitutive endo- β-1, 3-glucanase formation.Key words: endo- β-1, 3-glucanase, Schizophyllum commune, monokaryon, constitutive endo- β-1, 3-glucanase formation.

scholarly journals Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver

1991 ◽  
Vol 273 (2) ◽  
pp. 415-422 ◽  
Author(s):  
M Lyon ◽  
J T Gallagher
Keyword(s):  
Molecular Mass ◽  
Proteinase Inhibitors ◽  
Gradient Centrifugation ◽  
Heparan Sulphate ◽  
Exchange Chromatography ◽  
Guanidinium Chloride ◽  
Apparent Homogeneity ◽  
Core Proteins ◽  
Sds Page ◽  
Triton X

Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.

scholarly journals One of the major sulphated proteins secreted by rat hepatocytes contains low-sulphated chondroitin sulphate

1990 ◽  
Vol 272 (1) ◽  
pp. 113-118 ◽  
Author(s):  
E M Sjöberg ◽  
E Fries
Keyword(s):  
Molecular Mass ◽  
Chondroitin Sulphate ◽  
Alkali Treatment ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Exchange Chromatography ◽  
Secretory Proteins ◽  
Gel Chromatography ◽  
Chondroitinase Abc ◽  
Oligosaccharide Chain

When isolated hepatocytes are incubated with 35SO4(2-), a specific set of secretory proteins is labelled. One of these proteins is electrophoretically heterogeneous, with an apparent molecular mass of 35-45 kDa [Marcks von Würtemberg & Fries (1989) Biochemistry 28, 4088-4093]. Here we report that treatment with chondroitinase ABC converted the broad electrophoretic band of this protein, with a 50-60% loss of radioactivity, into a relatively homogeneous band with a molecular mass of 28 kDa. Size determination by gel chromatography of the protein's oligosaccharide chain (released by alkali treatment) indicated that it contained about 40 hexose units. Similar analysis of the enzyme-resistant oligosaccharide chain remaining linked to the protein after chondroitinase ABC treatment indicated a size of between six and eight hexose units. These observations suggest that the protein's oligosaccharide chain carries only three or four sulphate groups, of which one or two are located close to the polypeptide chain. Consistent with this hypothesis, the free oligosaccharide behaved like a low-sulphated glycosaminoglycan upon ion-exchange chromatography.

scholarly journals Comparison of the activities of a multiple inositol polyphosphate phosphatase obtained from several sources: a search for heterogeneity in this enzyme

1995 ◽  
Vol 305 (2) ◽  
pp. 491-498 ◽  
Author(s):  
A Craxton ◽  
N Ali ◽  
S B Shears
Keyword(s):  
Molecular Mass ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Polyclonal Antiserum ◽  
Apparent Molecular Mass ◽  
Size Exclusion ◽  
Rat Tissues ◽  
Inositol Polyphosphate ◽  
Phosphatase Activities ◽  
Specific Activities

A multiple inositol polyphosphate phosphatase (formerly known as inositol 1,3,4,5-tetrakisphosphate 3-phosphatase) was purified approx. 22,000-fold from rat liver. The final preparation migrated on SDS/PAGE as a doublet with a mean apparent molecular mass of 47 kDa. Upon size-exclusion chromatography, the enzyme was eluted with an apparent molecular mass of 36 kDa. This enzyme was approximately evenly distributed between the ‘rough’ and ‘smooth’ subfractions of endoplasmic reticulum. There was a 20-fold range of specific activities of this phosphatase in CHAPS-solubilized particulate fractions prepared from the following rat tissues: liver, heart, kidney, testis and brain. However, each of these extracts contained different amounts of endogenous inhibitors of enzyme activity. After removal of these inhibitors by MonoQ anion-exchange chromatography, there was only a 2.5-fold range of specific activities; kidney contained the most and brain contained the least. We prepared and characterized polyclonal antiserum to the hepatic phosphatase, which immunoprecipitated 85-100% of both particulate and soluble phosphatase activities. The antiserum also immunoprecipitated, with equivalent efficacy, CHAPS-solubilized phosphatase activities from heart, kidney, testis, brain and erythrocytes (all prepared from rat). Our data strengthen the case that the function of the mammalian phosphatase is unrelated to the metabolism of Ca(2+)-mobilizing cellular signals. The CHAPS-solubilized phosphatase from turkey erythrocytes was not immunoprecipitated by the polyclonal antiserum, and is therefore an isoform that is structurally distinct, and possibly functionally unique.

Sign in / Sign up

Export Citation Format

Share Document