A novel procedure that detects adhesive proteins in complex mixtures was used to characterize such proteins in plasma. The proteins are separated by SDS PAGE and transferred to nitrocellulose filters. Cells incubated on these filters attach to those proteins that have adhesive properties. When applied to human plasma proteins this procedure reveals, in addition to fibronectin, a cell-attachment protein with a polypeptide molecular weight of 70,000. Using a monoclonal antibody that inhibits attachment of cells to fibronectin, we show that this polypeptide is not a fragment of fibronectin and we present evidence that it is a component of the serum spreading factor. Therefore, as defined by our assay, this protein and fibronectin are the major attachment proteins for fibroblastic cells in plasma or serum.Many important biological phenomena--including morphogenetic migration of cells, wound closure, and tumor metastasis-involve the ability of cells to establish and to break adhesive interactions with the extracellular substratum or with other cells. That many normal cells must adhere and spread to survive in vitro reflects the importance of these interactions. Serum contains factors that are required for c¢11 adhesion and spreading (1). Fibronectin (2, 3), the best characterized adhesive serum protein, promotes attachment of many types of ceils (4-6). However, several observations suggest that fibronectin is not the only adhesive protein in serum or plasma (7-11). Serum has been shown to promote the attachment of chondrocytes by a fibronectin-independent mechanism (7, 8). In addition, 60,-000-80,000-dalton serum proteins that promote cell attachment and spreading have been identified (4,(9)(10)(11)(12).To analyze the occurrence and relationships of adhesive proteins in plasma, we studied the attachment of fibroblastic cells to plasma proteins that have been separated by SDS PAGE and transferred to nitrocellulose filters. We show here that this technique reveals in plasma, in addition to fibronectin, another major attachment protein with a molecular weight of 70,000. MATERIALS AND METHODS Cell CultureNormal rat kidney (NRK) ceils (13) were grown in Dulbecco's minimal essential medium (DME) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100/~g/ml streptomycin (Flow Laboratories, Inc. Rockville, MD).

We used antibodies raised against both a heparan sulfate proteoglycan purified from a mouse sarcoma and a chondroitin sulfate proteoglycan purified from a rat yolk sac carcinoma to study the appearance and distribution of proteoglycans in cultured cells. Normal rat kidney cells displayed a fibrillar network of immunoreactive material at the cell surface when stained with antibodies to heparan sulfate proteoglycan, while virally transformed rat kidney cells lacked such a surface network. Antibodies to chondroitin sulfate proteoglycan revealed a punctate pattern on the surface of both cell types. The distribution of these two proteoglycans was compared to that of fibronectin by double-labeling immunofluorescent staining. The heparan sulfate proteoglycan was found to codistribute with fibronectin, and fibronectin and laminin gave coincidental stainings. The distribution of chondroitin sulfate proteoglycan was not coincidental with that of fibronectin. Distinct fibers containing fibronectin but lacking chondroitin sulfate proteoglycan were observed. When the transformed cells were cultured in the presence of sodium butyrate, their morphology changed, and fibronectin, laminin, and heparan sulfate proteoglycan appeared at the cell surface in a pattern resembling that of normal cells. These results suggest that fibronectin, laminin, and heparan sulfate proteoglycan may be complexed at the cell surface. The proteoglycan may play a central role in the assembly of such complexes since heparan sulfate has been shown to interact with both fibronectin and laminin.

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells . These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells . Culture supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production . Fibronectins isolated from culture media of the normal and transformed cells were similar in SIDS polyacrylamide gel electrophoresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypep-

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