| Proteoglycans in polarized epithelial Madin-Darby canine kidney cells. | |
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MedLine Citation:
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PMID: 7487945 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Madin-Darby canine kidney (MDCK) cells were cultured on polycarbonate filters to study the synthesis and sorting of proteoglycans in polarized epithelial cells. Two strains of MDCK cells were used. MDCK I cells resemble distal tubule epithelial cells, and MDCK II cells share some characteristics with proximal tubule cells. Both strains were grown to confluency and labelled with [35S]sulphate for 24 h. The apical and basolateral media and the cell fractions were harvested and analysed by DEAE ion-exchange chromatography. A large portion of the [35S]sulphate-labelled macromolecules bound strongly to the ion-exchange columns, and could be eluted in three distinct peaks. The latest eluting peak was demonstrated to contain almost exclusively chondroitin sulphate, whereas peak 2 contained mostly heparan sulphate, demonstrated by using chondroitinase ABC and nitrous acid (pH 1.5) respectively to depolymerize the [35S]glycosaminoglycan chains. Peak 1 contained negligible amounts of proteoglycans. Large differences could be observed in proteoglycan sorting in MDCK I and II cells. Strain I secreted approx. 67% of the proteoglycans to the apical side and 17% to the basolateral side. The cell fraction contained 17% of the proteoglycans after 24 h of labelling. In contrast, 19% of the proteoglycans were sorted to the apical side of MDCK II cells and 61% to the basolateral side, whereas the cell fraction contained 20%. Furthermore, the level of [35S]proteoglycan biosynthesis (apical and basolateral media and cell fraction total) was higher in MDCK I cells than in strain II. Based on the amount of material degraded by chondroitinase ABC and nitrous acid respectively, and the total amounts of [35S]proteoglycans recovered from the cells, it was calculated that the MDCK I strain synthesized approx. 56% chondroitin sulphate and 44% heparan sulphate. In contrast, the MDCK II strain synthesized 69% heparan sulphate and 31% chondroitin sulphate. To further identify the [35S]proteoglycans synthesized by MDCK I and II cells, antibodies against perlecan, versican and syndecan were used. The antibody against mouse syndecan did not cross-react with any of the proteoglycans produced in MDCK I or II cells. Both MDCK I and II cells expressed perlecan; 57-61% could be recovered from the basolateral fractions and 18-34% from the apical medium. Versican was also found in both MDCK I and II cells. Compared with perlecan, a larger percentage of versican (43-53%) was found in the cell fractions. |
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Authors:
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K Svennevig; K Prydz; S O Kolset |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: The Biochemical journal Volume: 311 ( Pt 3) ISSN: 0264-6021 ISO Abbreviation: Biochem. J. Publication Date: 1995 Nov |
Date Detail:
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Created Date: 1995-12-21 Completed Date: 1995-12-21 Revised Date: 2009-11-18 |
Medline Journal Info:
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Nlm Unique ID: 2984726R Medline TA: Biochem J Country: ENGLAND |
Other Details:
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Languages: eng Pagination: 881-8 Citation Subset: IM |
Affiliation:
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Department of Biochemistry, University of Oslo, Norway. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Cell Line Cell Polarity* Chromatography, Ion Exchange Dogs Electrophoresis, Polyacrylamide Gel Ethanolamines Heparan Sulfate Proteoglycans Heparitin Sulfate / analysis, biosynthesis*, immunology Kidney Tubules, Distal / cytology*, metabolism Kidney Tubules, Proximal / cytology*, metabolism Macromolecular Substances Mice Proteochondroitin Sulfates / analysis, biosynthesis*, immunology Proteoglycans / analysis, biosynthesis*, immunology Sodium Dodecyl Sulfate Sulfur Radioisotopes |
| Chemical | |
Reg. No./Substance:
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0/Ethanolamines; 0/Heparan Sulfate Proteoglycans; 0/Macromolecular Substances; 0/Proteochondroitin Sulfates; 0/Proteoglycans; 0/Sulfur Radioisotopes; 100-37-8/2-diethylaminoethanol; 151-21-3/Sodium Dodecyl Sulfate; 9050-30-0/Heparitin Sulfate |
| Comments/Corrections | |
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