Epiglycanin as a membrane glycoprotein. Isolation of plasma membrane from the TA3-Ha tumor cell.
A plasma membrane fraction (M) was isolated from ascites cells of mouse TA3-Ha mammary carcinoma by the procedure of Brunette and Till [J. Membr. Biol., 5 (1971) 215], involving homogenization, slow-speed centrifugation, and finally, differential centrifugation in a two-phase system. Marker enzyme activities indicated only minimal contamination of M by the endoplasmic reticulum, a result confirmed by transmission electron microscopy. To monitor the presence of epiglycanin (a large cell-surface glycoprotein), each fraction was tested in a radioimmunoassay for epiglycanin content, by gas chromatography for carbohydrate and amino acid compositions, and by scintillation spectrometry for radioactivity. Cells had been treated with galactose oxidase, followed by reduction with sodium borotritide, prior to homogenization. Of the total recovered epiglycanin, 15% was present in M, but, as indicated by g.l.c., M also contained other glycoproteins in high concentration. A direct correlation was found between epiglycanin concentration, GalNAc content, and radioactivity. Electron microscopy of fraction M by shadow casting showed multiple filaments emanating from some of the particles. The dimensions of these filaments corresponded to those of isolated epiglycanin molecules.
D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis / Q000648:ultrastructure; D006023:Glycoproteins / Q000302:isolation & purification*; D008325:Mammary Neoplasms, Experimental / Q000032:analysis*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000302:isolation & purification*; D051379:Mice; D008854:Microscopy, Electron; D009363:Neoplasm Proteins / Q000302:isolation & purification*
D002241:Carbohydrates; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C016424:epiglycanin protein, mouse