Isolation and partial characterization of the sialoglycoprotein fraction of murine erythrocyte ghosts

With the lithium diiodosalicylate (LIS1) extraction-phenol partition method, we have isolated a sialoglycoprotein fraction from DBA/2 mouse erythrocyte ghosts. We have demonstrated that the Laemmeli system for SDS PAGE can resolve this fraction into four monomers of which two (gp- 2.1 and gp-3.1) appear to be authentic, whereas the other two (gp-2.2 and gp-3.2) are probably generated from gp-2.1 and gp-3.1, by limited proteolysis during the isolation procedure. All four components contain O-acetylated neuraminic acid residues, can be stained with Periodic acid-Schiff reagent (PAS) and with Coomassie Brilliant Blue (CB), and can be radioiodinated with the lactoperoxidase-glucose oxidase (LPO-GO) method. All monomers but especially gp-2.1 and gp-3.1 generate characteristic aggregates during solubilization in SDS. The aggregation is enhanced by boiling at high concentrations, and can be reversed by boiling at low concentrations. In addition, the fraction contains a diffuse component present also in ghosts which stains poorly with CB and with PAS and cannot be radioiodinated by the LPO-GO technique. SDS PAGE in the Steck and Yu gel system does not give an accurate separation of the sialoglycoprotein monomers.