Purification and characterization of the P-80 glycoprotein from human brain.
A glycoprotein antigen was purified from human brain by immunoaffinity chromatography using the 44D10-monoclonal IgG, and its chemical nature was investigated. The yield of antigen was estimated at 91% and a 4340-fold purification was obtained relative to the white-matter homogenate. The antigen preparation from brain was further purified by preparative SDS/polyacrylamide-gel electrophoresis (PAGE) to obtain a glycoprotein with an Mr of 80,000 consisting of a single polypeptide. Amino acid analyses revealed a composition which was high in acidic and neutral amino acids, and low in basic residues. The presence of both glucosamine and galactosamine suggested that the glycoprotein contained both N- and O-linked glycans. Neutral sugar analyses showed that fucose, galactose and mannose were present. An assay for sialic acid determined that there were approximately 20 mol of sialic acid per mol of glycoprotein. Chemical cleavage of oligosaccharides by trifluoromethanesulphonic acid followed by SDS/PAGE showed that carbohydrate accounted for 25,000 of the 80,000-Mr glycoprotein.
Girgrah
N
N
Department of Biochemistry, Hospital for Sick Children, Toronto, Ontario, Canada.
Cruz
T F
TF
Letarte
M
M
Moscarello
M A
MA
D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000302:isolation & purification; D001921:Brain / Q000276:immunology*; D002241:Carbohydrates / Q000032:analysis; D002458:Cell Fractionation; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000941:Antigens; D002241:Carbohydrates; D006023:Glycoproteins; D009419:Nerve Tissue Proteins