Glycophorin-enriched vesicles obtained by a selective extraction of human erythrocyte membranes with a non-ionic detergent.
A method is described for isolating glycophorin-enriched vesicles from human erythrocytes by extracting membranes that were incubated for 30 min at 37 degrees C at pH 4.5 and washed at low and high ionic strength with the nonionic detergent Triton X-100. The extracts were 11.8 +/- 2.4 fold enriched in glycophorin and contained 325 +/- 69 microgram sialic acid/mg protein, which represented 61 +/- 16% of the total sialic acid. Upon removal of Triton X-100 one third of the total glycophorin forms glycophorin-enriched vesicles with coextracted, endogenous lipids as shown sedimintation, dextran-density gradient centrifugation, and electron microscopy. Addition of exogenous lipids increased the fraction of glycophorin-enriched vesicles up to 87%. The incorporation of glycophorin in the membrane was shown by hemagglutination inhibition assays using anti-M sera and by the accessibility of glycophorin to trypsin. Freeze-fractured vesicles did not reveal intramembranous particles. The selectivity of the extraction procedure is not simply due to chemical constraints introduced by disulfide cross-linkage of protein component 3, because only 20% of this protein undergo disulfide cross-linking. The selective extraction of glycophorin implies that glycophorin is segregated from protein component 3 and thus from intramembranous particles when erythrocyte membranes have been incubated at pH 4.5. This segregation may precede aggregation of intramembranous particles.
D002458:Cell Fractionation / Q000379:methods; D004910:Erythrocyte Membrane / Q000648:ultrastructure*; D004912:Erythrocytes / Q000648:ultrastructure*; D005614:Freeze Fracturing; D006021:Glycophorins / Q000302:isolation & purification*; D006023:Glycoproteins / Q000097:blood; D006801:Humans; D008565:Membrane Proteins / Q000097:blood; D011092:Polyethylene Glycols; D012795:Sialoglycoproteins / Q000302:isolation & purification*
D006021:Glycophorins; D006023:Glycoproteins; D008565:Membrane Proteins; D012795:Sialoglycoproteins; D011092:Polyethylene Glycols