Microfilament association of ASGP-2, the concanavalin A-binding glycoprotein of the cell-surface sialomucin complex of 13,762 rat mammary ascites tumor cells.
Microfilament-associated proteins and membrane-microfilament interactions are being investigated in microvilli isolated from 13,762 rat mammary ascites tumor cells. "Phalloidin shift" analyses on velocity sedimentation gradients of Triton X-100 extracts of [3H]-glucosamine-labeled microvilli identified a 120-kDa cell-surface glycoprotein associated with the microvillar microfilament core. The identification was verified by concanavalin A (Con A) blots of one- and two-dimensional (2D) electrophoresis gels of sedimented microfilament cores. By 2D-electrophoresis and lectin analyses the 120-kDa protein appeared to be a fraction of ASGP-2, the major Con A-binding glycoprotein of the sialomucin complex of the 13,762 cells. This identity was confirmed by immunoblot analyses using immunoblot-purified anti-ASGP-2 from anti-membrane serum prepared against microvillar membranes. Proteolysis of the microvilli with subtilisin or trypsin resulted in an increase in the amount of ASGP-2 associated with the microfilament cores. An increase was also observed with sialidase treatment of the microvilli, suggesting that negative charges, probably present on the highly sialated sialomucin ASGP-1 of the ASGP-1/ASGP-2 sialomucin complex, reduce ASGP-2 association with the microfilament core. Proteolysis of isolated microvillar membranes, which contain actin but not microfilaments, also increased the association of ASGP-2 with a Triton-insoluble, actin-containing membrane fraction. Purified ASGP-2 does not bind to microfilaments in sedimentation assays. Since the Triton-insoluble membrane residue is enriched in an actin-containing transmembrane complex, which contains a different glycoprotein, we suggest that the ASGP-2 is binding indirectly via this complex to the microfilament core in the intact microvilli.
D008841:Actin Cytoskeleton / Q000032:analysis / Q000378:metabolism*; D000230:Adenocarcinoma; D000818:Animals; D002499:Centrifugation, Density Gradient; D003208:Concanavalin A / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007525:Isoelectric Focusing; D008325:Mammary Neoplasms, Experimental; D008871:Microvilli / Q000032:analysis / Q000378:metabolism / Q000648:ultrastructure*; D055264:Mucin-4; D010590:Phalloidine; D051381:Rats; D012795:Sialoglycoproteins / Q000032:analysis / Q000378:metabolism*; D013381:Subtilisins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D055264:Mucin-4; D012795:Sialoglycoproteins; D003208:Concanavalin A; D010590:Phalloidine; D013381:Subtilisins