title: Hydrophobic and hydrophilic radio-iodination, crosslinking, and differential extraction of cell surface proteins in Paramecium tetraurelia cells.
abstract: We combined widely different biochemical methods to analyze proteins of the cell surface of P. tetraurelia since so far one can isolate only a subfraction of cell membrane vesicles enriched in the GPI-anchored surface antigens ("immoblization" or "i-AGs"). We also found that i-AGs may undergo partial degradation by endogenous proteases. Genuine intrinsic membrane proteins were recognized particularly with lipophilic 5-[(125)I]-iodonaphthalene-1-azide (INA) labeling which reportedly "sees" integral proteins and cytoplasmic cell membrane-associated proteins. With INA (+DTT), bands of </=55 kDa were similar as with hydrophilic iodogen (+DTT), but instead of large size bands including i-AGs, a group of 122, 104 and 94 kDa appeared. Several bands of the non i-AG type are compatible with integral (possibly oligomeric) or associated proteins of the cell membrane of established molecular identity, as we discuss. In summary, we can discriminate between i-AGs and some functionally important minor cell membrane components. Our methodical approach might be relevant also for an analysis of some related protozoan parasites.
authors.affiliation: Faculty of Biology, University of Konstanz, P.O. Box 5560, D-78434 Konstanz, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Flötenmeyer
authors.forename: M
authors.initials: M
authors.lastname: Momayezi
authors.forename: H
authors.initials: H
authors.lastname: Plattner
mesh_terms: D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D000954:Antigens, Surface / Q000737:chemistry / Q000302:isolation & purification; D001386:Azides; D002462:Cell Membrane / Q000737:chemistry; D002793:Cholic Acids; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D008970:Molecular Weight; D010247:Paramecium / Q000737:chemistry* / Q000276:immunology; D015800:Protozoan Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D012995:Solubility
chemical_list: D000953:Antigens, Protozoan; D000954:Antigens, Surface; D001386:Azides; D002793:Cholic Acids; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D015800:Protozoan Proteins; C015298:iodonaphthylazide; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate