Hydrophobic and hydrophilic radio-iodination, crosslinking, and differential extraction of cell surface proteins in Paramecium tetraurelia cells.
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.
Flötenmeyer
M
M
Faculty of Biology, University of Konstanz, P.O. Box 5560, D-78434 Konstanz, Germany.
Momayezi
M
M
Plattner
H
H
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
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