Isolation and partial characterization of sulfated glycoproteins synthesized by corneal epithelium.
The corneal epithelia isolated from 19-day-old chick embryos were incubated with [35S]sulfate and [3H]glucosamine. More than 95% of the isotope-labeled sulfated glycoconjugates were extracted with 4 M guanidine/HCl, 50 mM sodium acetate, pH 6.0, and 0.5% Triton X-100 and separated on DEAE-Sepharose CL-6B into two major fractions, i.e. sulfated glycoprotein and sulfated proteoglycan fractions. Mild alkali treatment of the sulfated proteoglycan fraction and characterization of the sulfated glycosaminoglycans enabled the identification of heparan sulfate ([35S], 81%; [3H], 91%) and chondroitin sulfate ([35S], 19%; [3H], 9%). Fractionation of the sulfated glycoprotein fraction on Sepharose CL-6B resulted in the separation of four sulfated glycoproteins with apparent molecular weights of 70,000, 130,000, 280,000, and over 2,000,000. The products of alkaline borohydride treatment of the sulfated glycoproteins separated into three fractions on Sephadex G-75, two fractions contained sulfated oligosaccharides, and the third one contained inorganic sulfate and N-acetyl-galactosaminitol. Incorporation of [35S]sulfate into higher molecular weight fraction of the two fractions was selectively inhibited by tunicamycin, whereas that incorporated into the smaller molecular weight fraction was not inhibited by the drug and, rather, was activated. These results suggest that the former contains N-glycosidically linked sulfated glycopeptides, and the latter is derived from O-glycosidically linked sulfated oligosaccharide side chains.
D000818:Animals; D002642:Chick Embryo; D003315:Cornea / Q000378:metabolism* / Q000648:ultrastructure; D004848:Epithelium / Q000378:metabolism / Q000648:ultrastructure; D006025:Glycosaminoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D007700:Kinetics; D008854:Microscopy, Electron; D009844:Oligosaccharides / Q000302:isolation & purification; D013462:Sulfur Radioisotopes; D014316:Tritium; D014415:Tunicamycin / Q000494:pharmacology
D006025:Glycosaminoglycans; D009844:Oligosaccharides; D013462:Sulfur Radioisotopes; D014316:Tritium; D014415:Tunicamycin