Solubilization and fractionation of glycoproteins and glycolipids of KB cell membranes.
1. A fraction enriched in plasma membranes of human tumour KB cell line, a permissive cell for adenovirus type 5, was obtained. 2. Electrophoresis of the membranes in polyacrylamide gels with buffers containing sodium dodecyl sulphate showed that the membranes after reduction with 2-mercaptoethanol contained over 20 polypeptide species. Three polypeptides were glycosylated and had apparent mol.wts. of 92000, 72000 and 62000. 3. The glycoproteins and the specific receptors responsible for adenovirus adsorption to the membranes were readily extracted into solutions containing low concentrations of Triton X-100. Glycolipids and proteins were also made soluble. A membranous residue obtained after Triton X-100 extraction was enriched in several proteins that appeared to consist of polypeptides of lower molecular weight than the average of KB membrane polypeptides. 4. Sphingomyelin, cholesterol and triglycerides were similarly concentrated in the insoluble residue remaining after successive extractions of KB membranes with Triton X-100. Further, ceramide trihexoside was significantly less easily extracted from KB membranes than lactosyl ceramide. 5. The differences noted in the ease of extraction of membrane components are discussed. 6. The components of membranes made soluble by detergent extraction and containing the large part of the KB membrane glycoproteins were subjected to chromatography on Sepharose 6B and DEAE-cellulose and to isoelectric focusing in the presence of buffers containing Triton X-100. In general, the degree of separation into fractions enriched in individual glycoproteins was disappointing. Possible reasons for the poor fractionation of membrane components by chromatographic systems conveniently used for purification of proteins and glycoproteins of non-membranous origin are briefly discussed.
D000256:Adenoviridae; D002250:Carbon Radioisotopes; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis*; D002518:Ceramides / Q000032:analysis; D002784:Cholesterol / Q000032:analysis; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006017:Glycolipids / Q000032:analysis*; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D008623:Mercaptoethanol; D008854:Microscopy, Electron; D008970:Molecular Weight; D009303:Nasopharyngeal Neoplasms; D012967:Sodium Dodecyl Sulfate; D013109:Sphingomyelins; D013462:Sulfur Radioisotopes; D013501:Surface-Active Agents; D014280:Triglycerides / Q000032:analysis; D014316:Tritium
D002250:Carbon Radioisotopes; D002518:Ceramides; D006017:Glycolipids; D006023:Glycoproteins; D013109:Sphingomyelins; D013462:Sulfur Radioisotopes; D013501:Surface-Active Agents; D014280:Triglycerides; D014316:Tritium; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D002784:Cholesterol