Isolation and characterization of plasma membrane proteins of cultured human retinal pigment epithelium.
Two-dimensional gel electrophoresis (2-D) was used to resolve the plasma membrane proteins from cultured human retinal pigment epithelial cells. The cells were metabolically labeled either with [35S]methionine to reveal proteins in general or with [3H]glucosamine or [3H]fucose which are more specific for glycoprotein visualization. The cell surface proteins were also iodinated, using the lactoperoxidase--glucose oxidase technique. These labeled membranes were separate into plasma membrane-enriched fractions by subjecting the water-shocked postnuclear supernatant to a discontinuous sucrose-density gradient. The five resulting membrane fractions were assayed for protein, RNA (microsomes), galactosyltransferase (Golgi membranes), 5'-nucleotidase (plasma membranes), and succinate dehydrogenase (mitochondrial membranes) and were examined by electron microscopy. The plasma membranes were enriched with minimal contamination at the 0.6-0.85 M (F2) and 0.85-1.0 M (F3) sucrose interfaces based on these biochemical and morphological criteria. Examination of 2-D autoradiographic profiles of F2 and F3 showed that approximately 180 proteins or protein subunits had incorporated [35S]methionine. Certain proteins were also labeled by [3H]glucosamine and [3H]fucose, and surface-labeled by iodination. This was especially true of 17 different high-molecular-weight (43-139 X 10(3) MW) very acidic glycoproteins which formed a constellation of spots. These glycoproteins, as well as others, were also seen in the whole-cell acidic glucosamine-labeled 2-D profiles, where about 150 proteins were detected. A total of 39 proteins were catalogued, of which 34 were detectable in the plasma membrane-enriched fractions. The results show that the use of subcellular fractionation, specific precursors, and labeling techniques aids in the detection and characterization of minor proteins in 2-D gels.
D000328:Adult; D000368:Aged; D002462:Cell Membrane / Q000648:ultrastructure; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D005136:Eye Proteins / Q000302:isolation & purification*; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification*; D008854:Microscopy, Electron; D008875:Middle Aged; D008970:Molecular Weight; D010857:Pigment Epithelium of Eye / Q000032:analysis* / Q000648:ultrastructure; D012313:RNA / Q000032:analysis; D013347:Subcellular Fractions / Q000032:analysis
D005136:Eye Proteins; D006023:Glycoproteins; D008565:Membrane Proteins; D012313:RNA; D004247:DNA