Topography and microfilament core association of a cell surface glycoprotein of ascites tumor cell microvilli.
Membrane-microfilament interactions are being investigated in microvilli isolated from 13762 rat mammary ascites tumor cells. These microvilli are covered by a sialomucin complex, composed of the sialomucin ascites sialoglycoprotein-1 (ASGP-1) and the associated concanavalin A (Con A)-binding glycoprotein ASGP-2. Limited proteolysis of the microvilli releases large, highly glycosylated fragments of ASGP-1 from the microvilli and increases the association of ASGP-2 with the Triton-insoluble microvillar microfilament core (Vanderpuye OA, Carraway CAC, Carraway, KL: Exp Cell Res 178:211, 1988). To analyze the topography of ASGP-2 in the membrane and its association with the microfilament core, microvilli were treated with proteinase K for timed intervals and centrifuged. The pelleted microvilli were extracted with Triton X-100 for the preparation of microfilament cores and Triton-soluble proteins or with 0.1 M carbonate, pH 11, for the preparation of microvillar membranes depleted of peripheral membrane proteins. These microvilli fractions were analyzed by dodecyl sulfate gel electrophoresis, lectin blotting with Con A and L-phytohemagglutinin, and immunoblotting with anti-ASGP-2. The earliest major proteolysis product from this procedure was a 70 kDa membrane-bound fragment. At longer times a 60 kDa released fragment, 30-40 kDa Triton-soluble fragments, and 25-30 kDa membrane- and microfilament-associated fragments were observed. Phalloidin shift analysis of microfilament-associated proteins on velocity sedimentation gradients indicated that the 25-30 kDa fragments were strongly associated with the microfilament core. From these studies we propose that ASGP-2 has a site for indirect association with the microfilament core near the membrane on a 15-20 kDa segment.
Sheng
Z
Z
Department of Biochemistry, University of Miami School of Medicine, Florida 33101.
Vanderpuye
O A
OA
Hull
S R
SR
Carraway
C A
CA
Carraway
K L
KL
D008841:Actin Cytoskeleton / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000032:analysis*; D019286:Endopeptidase K; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D015151:Immunoblotting; D008840:Microfilament Proteins / Q000378:metabolism; D008871:Microvilli / Q000032:analysis* / Q000378:metabolism; D055264:Mucin-4; D010447:Peptide Hydrolases / Q000378:metabolism; D051381:Rats; D011952:Receptors, Concanavalin A / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D012795:Sialoglycoproteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured
D006023:Glycoproteins; D008840:Microfilament Proteins; D055264:Mucin-4; D011952:Receptors, Concanavalin A; D012795:Sialoglycoproteins; C027774:concanavalin A-binding glycoproteins; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; D019286:Endopeptidase K