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Topography and microfilament core association of a cell surface glycoprotein of ascites tumor cell microvilli
Author(s) -
Sheng Zeqi,
Vanderpuye Oluseyi A.,
Hull Steven R.,
Carraway Coralie A. Carothers,
Carraway Kermit L.
Publication year - 1989
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240400406
Subject(s) - microfilament , glycoprotein , microbiology and biotechnology , cell , ascites , chemistry , biology , cytoskeleton , medicine , biochemistry
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.