Electrophoretic analysis of substrate-attached proteins from normal and virus-transformed cells

113

Evidence is presented that fibronectin is present on the platelet cell membrane and that it is a receptor for collagen in the platelet-collagen interaction. First, sodium dodecyl sulfate/acrylamide gel electrophoresis was performed on the proteins remaining attached to the surface of collagen after the removal of the remainder of the platelet by sonication. The material was relatively enriched in a glycoprotein that comigrated with cold-insoluble globulin (CIG), a form of fibronectin, and in other proteins which comigrated with myosin, actin, and tropomyosin. The presumptive presence of contractile proteins is consistent with the presence of microfibrillar proteins. Second, the collagen-attached material was shown to contain a protein that reacted'with anti-GIG serum by immunoelectrophoresis. Third, when CIG was preincubated with fibrous collagen, the platelet-collagen interaction was inhibited. Fourth, rabbit anti-human CIG stimulated human platelets to secrete the contents of their dense granules. The stimulation was not due to antibody complexes present in the solution. Fifth, a protein was extracted from wel -washed platelets and purified on affinity columns of anti-GIG antibodies. The isolated protein was found to bind to fibrous collagen. When the endothelial lining of blood vessels is breached, blood platelets come in contact with the components of connective tissue. The platelets adhere to the collagen in the connective tissue and are stimulated to release the contents of their dense granules, including ADP, Ca2+, and serotonin (1, 2). The Ca2+ and ADP are required for the subsequent platelet-platelet interactions, which lead to the formation of aggregates on a nucleus of the platelets bound to collagen (3). The aggregates form a platelet plug as an initial step in hemostasis. Because the collagen-platelet interaction is an important event in hemostasis, a great deal of interest has centered on the determination of the mechanism of the interaction. A major stimulus to interest in the collagen-platelet interaction came as a result of the investigations of Barber and Jamieson (4, 5). They presented evidence that membrane-bound glucosyl transferases on the surface of platelets bind to the galactose and glucose residues of collagen in an enzyme-substrate and enzyme-product complex, respectively. Further investigations by others tended to support this hypothesis (6, 7). However, evidence to the contrary has been presented. Menashi et al. (8) have shown that only denatured collagen can act as a substrate for the platelet glucosyl transferase, whereas only native, fibrous collagen serves as a platelet substrate in the collagen-platelet interaction (9, 10). In addition, others have shown that the carbohydrate residues of collagen can be completely destroyed by periodate oxidation without affecting the ability of the fibrous form of the oxidized collagen to interact normally with platelets (10, 11).Because the only current hypothesis as to the nature of the collagen receptor of platelets has been severe...

supporting

confidence: 52%

mentioning

confidence: 52%

Evidence that fibronectin is the collagen receptor on platelet membranes.

Bensusan¹,

Koh²,

Henry³

et al. 1978

113

“…A similar structure, referred to as a "microexudate carpet" (7), is secreted by fibroblasts during adhesion to an artificial surface. The constituents of this microexudate are not known in detail but are thought to include collagen, glycoproteins, and proteoglycans (8,9).…”

mentioning

confidence: 99%

Cell surface-associated structural proteins in connective tissue cells.

Börnstein¹,

Ash²

1977

117

“…4), but was not observed with Coomassie staining, and is probably a hyaluronate proteoglycan (6,16). Others of the glucosaminelabeled components that could be tentatively identified are reference polypeptides 2, 3, and 6 shown in Fig.…”

Section: Resultsmentioning

confidence: 93%

“…Chief among these are a glycoprotein of about 250,000 daltons (LETS) and actin (2-4). Other major differences between the glycoproteins of normal and transformed cells have been shown by the binding of iodine-labeled lectins (5), and by cell surface proteins identified as substrate-attached components (6).…”

mentioning

confidence: 99%

Polypeptides of cells transformed by RNA or DNA tumor viruses.

Strand¹,

August²

1977

Proteins solubilized from normal BALB/3T3 cells and BALB/3T3 transformed by simian virus 40 or Kirsten sarcoma virus have been analyzed by two-dimensional gel electrophoresis and tryptic peptide mapping. A large fraction of the polypeptides of the virus-transformed cells, about 30%, were different from normal cells. In contrast to the marked differences between normal and transformed cells, the polypeptides of the DNA and RNA virus-transformed cells were almost identical. These findings were observed with polypeptides stained by Coomassie Blue, or labeled with ['4C]glucosamine or [35Sjmethionine. Pulse-chase analysis showed that most of the polypeptides were stable during 20 hr of incubation. The identity of several polypeptides was confirmed by tryptic peptide mapping. Analysis of the protein content of transformed as compared to normal cells has been directed chiefly at studies of cell surface glycoproteins (for a review see ref. 1). Several proteins are found to be reduced in amount or in their association with the membrane. Chief among these are a glycoprotein of about 250,000 daltons (LETS) and actin (2-4). Other major differences between the glycoproteins of normal and transformed cells have been shown by the binding of iodine-labeled lectins (5), and by cell surface proteins identified as substrate-attached components (6).The present study was initiated to analyze the extent and specificity of change in the protein content of cells following oncogenic transformation. For these studies, we have compared the total cellular proteins of BALB/3T3 cells infected by simian virus 40 (a DNA virus) and the proteins of the same cell transformed by a completely different virus, Kirsten murine sarcoma type-C virus (an RNA virus). The results obtained by twodimensional gel electrophoresis indicate that transformation is accompanied by complex changes in the type, concentration, or properties of a large number of cellular polypeptides and that the great majority of these changes are the same irrespective of whether the cell is transformed by a DNA or RNA tumor virus. MATERIALS AND METHODSCells. The clonal line (AS1) of BALB/3T3 (7), and a derivative subclone K-234, ACi/ml (445 Ci/mmol) and without fetal bovine serum. Following incubation for 1 hr at 370 the monolayers were washed twice with warm growth medium and the cells were then either lysed (pulse) or incubated with 10 ml of growth medium at 370 for various time periods (chase).Preparation of Cell Extracts. Cells were harvested by removing the cell culture fluid and washing the monolayer twice with 10 ml each of warm Dulbecco's phosphate-buffered saline (Gibco). Labeled cells were only washed once. They were suspended in 10 ml of Dulbecco's phosphate-buffered saline containing 1 mM EDTA by incubation at 370 for 5 min, collected by centrifugation at 2000 X g for 5 min, and lysed by suspension in 0.6 ml of extraction buffer [5 mM Tris-HCl, pH 9.2/1 mM EDTA/400 mM KCI/1% (vol/vol) Triton X-100/1 mM phenylmethylsulfonyl fluoride (PMSF)]. The lysed cel...