Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts.
The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.
Furukawa
K
K
Minor
J E
JE
Hegarty
J D
JD
Bhavanandan
V P
VP
D000818:Animals; D002462:Cell Membrane / Q000032:analysis; D002846:Chromatography, Affinity / Q000379:methods; D037102:Lectins; D008099:Liver / Q000032:analysis; D008114:Liver Neoplasms, Experimental / Q000032:analysis; D008970:Molecular Weight; D009077:Mucins / Q000302:isolation & purification*; D051381:Rats; D011915:Rats, Inbred BUF; D012685:Sepharose / Q000031:analogs & derivatives; D012795:Sialoglycoproteins / Q000302:isolation & purification*
D037102:Lectins; D009077:Mucins; D012795:Sialoglycoproteins; C042758:wheat germ agglutinin-sepharose 6MB; D012685:Sepharose