Parietal Endoderm Cells Research Articles

Neuron-like derivatives of cultured F9 embryonal carcinoma cells express characteristics of parietal endoderm cells

Murine F9 embryonal carcinoma cells exposed to retinoic acid and dibutyryl cyclic AMP gradually arborize and acquire a neuron-like morphology in monolayer culture. Whether F9 cells can be induced to differentiate into cells with features specific to neural cells is controversial. We analyzed the intermediate filament content and pericellular matrix proteins of F9 cells after exposing them to retinoic acid, dibutyryl cyclic AMP, and nerve growth factor. In long-term cultures, a great majority of the cells appeared neuron-like, but showed intra- and pericellular laminin and type IV collagen, and frequently cytokeratin filaments as well. Several monoclonal antibodies to neurofilaments did not react with these cells in immunofluorescence or immunoblotting, though they recognize either all or individual mouse neurofilament triplet proteins. Polyclonal antibodies to neurofilament proteins gave a diffuse, nonfibrillar, vinblastine-resistant fluorescence in the morphologically neuron-like cells, but in immunoblotting failed to reveal polypeptides compatible with neurofilament triplet proteins. In long-term cultures, most of the cells appeared to have partially or totally lost the intermediate filaments. This was confirmed with anti-IFA antibodies which normally react with all intermediate filament proteins. The F9-derived cells did not respond to nerve growth factor in any detectable way. We conclude that the morphologically neuron-like derivatives of F9 cells display characteristics of modified parietal endoderm-like cells and do not show unequivocal features of neural cells.

Differentiation defective mutants of skeletal myoblasts altered in a gelatin-binding glycoprotein.

We have previously described a myoblast cell surface glycoprotein of the molecular mass 46,000 (gp46), which is associated with myoblast differentiation. In this report we demonstrate that gp46 binds specifically to gelatin-Sepharose and in this respect is similar to a glycoprotein of the molecular mass 47,000, which has earlier been described as a cell surface localized protein in mouse parietal endoderm cells and in chick embryo fibroblasts. To ascertain the relationship of gp46 to myoblast differentiation, wild-type L6 myoblasts, as well as two concanavalin A (ConA) resistant, differentiation-negative, myoblast mutants (D-1 and C-8), were examined for gp46 expression. In the mutant designated D-1, which has a defect in dolichol mannosyl transferase, both mannose incorporation into gp46 and ConA binding to gp46 was reduced compared with L6, without markedly affecting the gelatin adhesion properties of gp46. Western blotting with a monoclonal antibody against gp46 was used to show that the expression of gp46 was normal in D-1 but was reduced in mutant C-8 compared with L6. Reduction occurred both in the plasma membrane and endoplasmic reticulum fractions of C-8 compared with wild-type L6. In L6 myoblasts, the expression of gp46 remained constant during myoblast replication and fusion but decreased markedly postfusion. In the nonfusing myoblast mutants D-1 and C-8 and in wild-type L6 cells that were prevented from fusing by treatment with 5-bromo-2'-deoxyuridine, the expression of gp46 remained invariant. We suggest that collagen interactions, mediated by gp46, are important for normal rat skeletal muscle differentiation.

Identification and partial characterization of two major proteins of Mr 47,000 synthesized by bovine retinal endothelial cells in culture.

Biosynthetic experiments with cultured bovine retinal endothelial cells have identified a glycoprotein of Mr 47,000 (Gp47) as a major component secreted into the medium. Gp47 is a non-collagenous glycoprotein with a pI of 4.6-5.5, which does not bind to either gelatin-Sepharose or heparin-Sepharose but is retained by concanavalin A-Sepharose. The Mr of this species decreases to approx. 42,000 in the presence of tunicamycin, indicating that it contains asparagine-linked oligosaccharides. A second protein of Mr 47,000 (P47) is present in the cell layer/matrix of these cultured cells. The electrophoretic mobility of P47 remains unaltered when synthesized in the presence of tunicamycin. Peptide-mapping experiments using N-chlorosuccinimide and Staphylococcus aureus V8 proteinase demonstrate that Gp47 and P47 are distinct proteins, and are not related to colligin, a membrane-bound collagen-receptor protein of similar size, or to SPARC, a major secreted product of parietal endodermal cells and sparse cultures of aortic endothelial cells.

Primary structure and nuclear localization of a murine homeodomain protein.

The murine homeobox Hox 1.1 (m6) is the first of a cluster of six boxes on chromosome 6. Using probes and synthetic peptides derived from the Hox 1.1 sequence, we were able to isolate cDNAs and antibodies that allowed us to characterize the product of this homeobox-containing gene. From the open reading frame on the cDNA clone B21, a protein could be predicted, made up of 229 amino acids and having a calculated molecular weight of 25,740. A unique feature of this protein is that it has 15 glutamic acid residues as its carboxyl terminus, which gives it a very hydrophilic and acidic carboxyl terminal structure, most probably folding onto an alpha-helix. A second domain of six amino acids is present on the Hox 1.1 protein, which is conserved in other homeodomain proteins. Antibodies generated against synthetic peptides from the homeobox region were used in the immunoblotting procedure and revealed a major protein band of Mr 31,000 in extracts from 3T3 cells and F9 teratocarcinoma cells induced by retinoic acid and cAMP. The nuclear location of the protein was established by immunofluorescence. The presence of this protein in F9 cell nuclei is in faithful accordance with the kinetics established for the 2.4-kilobase Hox 1.1 transcript during differentiation into parietal endoderm cells.

Fetomodulin: Marker surface protein of fetal development which is modulatable by cyclic AMP

A novel cell surface marker of fetal development was identified in both in vivo and in vitro systems of the mouse using monoclonal antibodies against a glycoprotein of an apparent size of 133,000 Da. Two independent clones of hybridomas were isolated by fusing murine myeloma cells, NS-1, with spleen cells of a rat which was immunized with murine 3T3 fibroblast. The analysis of molecular size and tryptic peptides of the immunoprecipitate indicated that fibroblast and putative parietal endoderm cells, which were derived by induced differentiation of F9 embryonal carcinoma cells with retinoic acid and cyclic AMP, expressed apparently the same protein. Undifferentiated F9 cells and F9 cells which were treated with retinoic acid or cyclic AMP alone had little or no immunoprecipitable proteins. Analogously, parietal endoderm of in vivo embryos tested positive for this protein but visceral endoderm and embryonic ectoderm did not. The amount of this surface protein was increased in fibroblast and differentiated F9 cells by elevation of intracellular cyclic AMP concentrations. These results are consonant with a hypothesis that this surface protein plays a role in fetal development via a quantitative modulation by cyclic AMP.

The epitope of mouse embryonic antigen(s) recognized by monoclonal antibody TEC-02 is a carbohydrate carried by high-molecular-weight glycoconjugates

The expression and properties of mouse embryonic antigens, recognized by monoclonal antibody TEC-02, were analyzed in teratocarcinoma-derived cell lines. TEC-2 antigens were found in the majority of the parietal endoderm cells PYS-2 and in a fraction of cultured embryonal carcinoma cells but not in other cell lines tested. During the course of retinoic acid-induced differentiation of embryonal carcinoma cells F9, the expression of TEC-2 was transiently increased. Immunolabeling of extracts from F9 and PYS-2 cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that TEC-2 antigens are polydisperse glycoconjugates of high molecular weight (mostly > 100 000). The TEC-2 epitope was shown to be carbohydrate which in F9 cells might be attached to the same carrier as another developmentally regulated carbohydrate epitope TEC-1. The TEC-2 antigens, isolated by indirect immunoprecipitation, were degraded by extensive pronase digestion or mild alkaline treatment to mostly large products. Immunostaining of glycolipid standards suggested that TEC-2 epitope involves the GalNAc β1 → 4Gal β1 → 4R sequence. Combined data indicate that TEC-2 is a new developmentally regulated carbohydrate epitope carried in embryonal carcinoma cells predominantly on glycoprotein-bound large carbohydrates.

Structure and affinity for antithrombin of heparan sulfate chains derived from basement membrane proteoglycans.

Metabolically 35S- or 3H-labeled heparan sulfate was isolated from murine Reichert's membrane, an extraembryonic basement membrane produced by parietal endoderm cells, and from the basement membrane-producing Engelbreth-Holm-Swarm mouse tumor. The polysaccharides were subjected to structural analysis involving identification of products formed on deamination of the polysaccharides with nitrous acid. The polysaccharide from Reichert's membrane contained N- and O-sulfate groups in approximately equal proportions. It bound almost quantitatively and with high affinity to antithrombin. A high proportion of antithrombin-binding sequence was also indicated by the finding that 3-O-sulfated glucosamine residues accounted for about 10% of the total O-sulfate groups. In contrast, at least 80% of the sulfate residues in the heparan sulfate isolated from the mouse tumor were N-substituents. Only a minor proportion of this polysaccharide bound with high affinity to antithrombin, and no 3-O-sulfated glucosamine residues were detected. These results are discussed in relation to the possible functional role of heparan sulfate in basement membranes.

Distinct patterns of glycosylation of colligin, a collagen-binding glycoprotein, and SPARC (osteonectin), a secreted Ca2+-binding glycoprotein. Evidence for the localisation of colligin in the endoplasmic reticulum.

Mouse parietal endoderm PYS cells were labelled with [2-3H]mannose for 16-24 h. Colligin, an Mr-47000 collagen-binding protein, and SPARC, a Mr-43000 protein, highly homologous to the Ca2+-binding protein osteonectin, were isolated from labelled cell extracts and culture medium respectively. Glycopeptides obtained by exhaustive digestion with pronase were analysed by lectin-affinity, ion-exchange, and gel-filtration chromatography and by paper chromatography of high-mannose oligosaccharides after endo H release. The results show that the N-linked carbohydrate chains of colligin are exclusively the high-mannose type, of which (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2 make up 77%. This carbohydrate structure provides strong evidence that colligin is a component of the endoplasmic reticulum, and argues against a role in cell-surface interactions. By contrast to colligin, SPARC secreted by PYS cells contains predominantly a diantennary complex type of chain containing a variable number of sialic acid and core-substituted fucose residues. Similar glycosylation patterns to those discussed above were seen in colligin isolated from primary mouse embryonic parietal endoderm cells and the murine 3T3 cell line, and in SPARC secreted by bovine corneal endothelial cells. Unlike the type-IV-collagen-binding glycoprotein studied by Dennis, J., Waller, C. and Schirrmacher, V. [J. Cell Biol. 99, 1416-1423 (1984)], removal of N-linked oligosaccharides from colligin had no effect on its binding to native type IV collagen.

The role of secretory granules in the transport of basement membrane components: radioautographic studies of rat parietal yolk sac employing 3H-proline as a precursor of type IV collagen.

Biosynthesis of type IV collagen in the parietal endodermal cells of 12 day gestant Sherman rats was examined following intraconceptal injection of 3H-proline. The concepti were removed at times varying from 2 minutes to 24 hours after the injection. The parietal wall of the yolk sac, including endodermal cells and the associated basement membrane known as Reichert's membrane were processed for electron microscopic radioautography. Silver grains were counted over the organelles of endodermal cells as well as over Reichert's membrane. Radioactivity was high in endodermal cells during the first 2 hr after 3H-proline injection and later dropped to some extent, while radioactivity rose in Reichert's membrane. Examination of endodermal cell organelles showed some early labeling over rER and Golgi apparatus without a clear-cut trend, except for a drop in Golgi label at late times after 3H-proline injection. The density of silver grains over secretory granules rose significantly by 40 min, reached a high peak by 4 hr and then declined at the time when radioactivity increased over Reichert's membrane. Furthermore, the radioactively-labeled secretory granules were localized mainly at the trans Golgi face soon after injection and near the cell surface adjacent to Reichert's membrane at later times. Biochemical reports indicate that a substantial amount of the proline taken up by the 12-14.5 day rat embryo endodermal cells is incorporated into type IV collagen. Since there is high labeling of the secretory granules from 40 min to 4 hr and the labeled granules are associated with the Golgi apparatus at early times, it is proposed that collagen precursors are processed through rER and Golgi apparatus, packaged into secretory granules and then transported to the cell surface where type IV collagen or its precursors are released and subsequently deposited into Reichert's membrane.

Isolation and Characterization of the cDNAs Corresponding to mRNAs Abundant in Undifferentiated Mouse Embryonal Teratocarcinoma Stem Cells, but Not in Differentiated Mouse Parietal Endoderm Cells1

As retinoic acid (RA) and dibutyryl cAMP (cAMP) treatment induces differentiation of mouse teratocarcinoma F9 cells into parietal endoderm cells in vitro, we initiated studies on the molecular mechanisms underlying early mammalian cell differentiation in this system. We constructed cDNA libraries on the poly(A)+RNAs extracted from the undifferentiated F9 cells, and screened for cDNA sequences expressed abundantly in F9 cells, but not in terminally differentiated mouse parietal endoderm PYS-2 cells. Six different cDNA clones were isolated and characterized. The levels of RNAs hybridizable to these clones were at most 5 to 24% in the PYS-2 cells when compared with those in the undifferentiated F9 cells. The six clones were classified into two groups on the basis of their responses to the RA and cAMP treatment. In F9 cells, the levels of RNAs hybridizable to the first group, which contained four clones, were decreased within 72 h after the addition of RA and cAMP, while those of the second group, which contained the remaining two clones, did not decrease significantly. One of the first group clones, named pF9-1, corresponded to the mouse "early transposon-like elements" and another, named pF9-4, hybridized to multi-size RNAs extracted from the undifferentiated F9 cells. The mouse genomic DNA sequences hybridizable to pF9-4 were repeated approximately 5,000 times, and comprise a new gene family, the expression of which is developmentally regulated in mouse F9 cells.

Murine endodermal cytokeratins Endo A and Endo B are localized in the same intermediate filament.

The intracellular distribution of extra-embryonic endodermal, cytoskeletal proteins A (Endo A) and B (Endo B) was investigated by double-label immunofluorescent microscopy and double-label immunoelectron microscopy. In parietal endodermal cells, the immunofluorescent distribution of Endo B was always coincident with that of Endo A and could be distinguished from vimentin, particularly at the periphery of the cell. At the electron microscopic level, antibodies against both Endo A and Endo B recognized both bundles and individual intermediate filaments. Double-label immunoelectron microscopy was achieved by use of two sizes of colloidal gold particles (5 nm and 20 nm) that were stabilized with secondary antibodies. These results show that Endo A and B are found in the same intermediate filament and probably co-polymerize to form such structures.

The response of embryonal carcinoma cells to retinoic acid depends on colony size

Single cells of the feeder-layer-dependent mouse embryonal carcinoma (EC) cell line, NG2, can spontaneously give rise to colonies containing a wide variety of differentiated cell types in vitro. When cultured with retinoic acid at a concentration of 10 −7 M, single NG2 cells irreversibly differentiated to parietal endoderm, as identified by morphological criteria and immunohistochemical staining. Parietal endoderm was also the first product of spontaneous differentiation. However, when retinoic acid was added to monolayer groups of NG2 cells, not all of the cells were induced to differentiate. The parietal-endoderm cells which did form were generally found at the periphery of cell colonies, as is the case during spontaneous differentiation. Differentiation in the centre of these colonies yielded a variety of cell types over a 21-day period. These results are consistent with the hypothesis that retinoic acid induces the differentiation of EC cells by accelerating cellular response to intrinsic stimuli, rather than by overriding these stimuli.

Purification and properties of N-acetylglucosaminide alpha13-fucosyltransferase from embryonal carcinoma cells

A membrane-bound alpha-L-fucosyltransferase, which is involved in the synthesis of a developmentally regulated carbohydrate antigen, SSEA-1, was purified about 2000-fold from F9 embryonal carcinoma cells. The procedures used were solubilization with Triton X-100, column chromatography on SP-Sephadex, DEAE-Sephadex, RCA-agarose and on GDP-agarose. Upon sodium dodecyl sulfate gel electrophoresis, the purified preparation gave a protein band with a relative molecular mass of 65 000. The optimum pH of the enzyme was between 6.0 and 7.0 and the Km toward N-acetyllactosamine was 0.55 mM. The enzyme was active with asialofetuin, but not with intact fetuin. Susceptibility of the product to alpha-L-fucosidase I from almond emulsin verified that the enzyme transferred fucose to C-3 hydroxyl of N-acetylglucosamine in the N-acetyllactosamine structure. Activities of beta-galactoside alpha 1----2-fucosyltransferase and N-acetylglucosaminide alpha 1----4-fucosyltransferase acting on synthetic substrates were not detected in the purified enzyme nor in the crude extract of F9 cells. PYS-2 parietal endoderm cells lacked all the fucosyltransferases mentioned above.

Identification of a novel glycopeptide species that correlates with differentiation of F9 cells

The high-molecular-weight fucosyl glycopeptides of differentiated F9 cells have been analyzed. We found that these high-molecular-weight surface structures contain two components with different molecular weights, the largest of which, peak I, has never before been reported. The material eluting in this peak seems to contain only acidic species. Removal of sialic acid from both the peak I and the peak II species does not eliminate the differences in molecular weight, indicating that the two species have more profound structural differences that can be accounted for by sialic acid. Since peak I glycopeptides were found both in differentiated F9 cells and in two parietal endoderm cell lines, we suggest that its presence is related to parietal endoderm differentiation.

Evidence for the existence of an early common biochemical pathway in the differentiation of F9 cells into visceral or parietal endoderm: Modulation by cyclic AMP

The addition of dibutyryl cyclic AMP (dbcAMP) to aggregate cultures of F9 cells in medium containing retinoic acid (RA) directs the pathway of differentiation into parietal endoderm instead of visceral endoderm. We examined the levels of some of the markers that characterize the two pathways and studied the time of commitment of cells to either direction of differentiation by using immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). For either pathway, the levels and patterns of laminin, type IV collagen, and fibronectin are the same on the first day of differentiation, characterized by slightly decreased levels of laminin and type IV collagen synthesis and an increased level of fibronectin synthesis. These levels reverse on the second day of culture when the pathways diverge markedly. The differentiation pathway, however, can be redirected into the alternate one; parietal endoderm cells become committed after 3 days, whereas visceral endoderm cells are able to change into parietal endoderm cells at any time. Thus, α-fetoprotein (AFP)-producing F9 embryoid bodies switched to dbcAMP-containing medium lose the capacity to synthesize AFP and start to express genes characteristic of parietal endoderm. Our results indicate that at least some visceral endoderm cells may redifferentiate into parietal endoderm cells. These phenomena thus mimic features of endoderm differentiation in the mouse embryo.

Localization of immunoglobulins in the mouse uterus, embryo, and placenta during the second half of pregnancy

Throughout the second half of pregnancy in mice there were many plasma cells containing immunoglobulins A (IgA) and G (IgG) in the uterine endometrium. There was intense staining of IgA in uterine glands at all stages, but little staining of IgG. The staining of both immunoglobulins (Igs) in the luminal epithelium was moderate to dark on day 11, slight on day 14, and increased from day 16 to term. From day 14 to term the endometrium exhibited folds or villi around each placenta. The cores of the villi contained many plasma cells of both isotypes, and the staining of extracellular Igs in the villous cores was darker than in nonvillous endometrium. Both Igs were detected in the uterine lumen, and in visceral and parietal yolk sac endoderm cells at all stages. Near term, the staining of Igs in the visceral yolk sac was darkest in the peripheral villous portion adjacent to the endometrial villi. From day 14 to term IgG was present in the visceral yolk sac mesenchyme and embryo, consistent with its transfer from the uterine lumen to the embryo via the vitelline circulation. In contrast, IgA was not detected in yolk sac mesenchyme until day 19, when only slight staining was observed, and IgA was never detected in the embryo. Most trophoblast giant cells contained both Igs on day 11. During the remainder of pregnancy, there was staining of both Igs in labyrinthine trophoblast and in a few giant cells adjacent to the parietal yolk sac on the placenta, but there was negligible staining in the spongiotrophoblast region. Our observations suggest that the local immune system in the mouse uterus may protect the embryo during the second half of pregnancy by secreting anti-microbial immunoglobulins A and G into the uterine lumen surrounding the visceral yolk sac, and may at the same time contribute to the transfer of maternal IgG to the embryo via the yolk sac and vitelline circulation.

Expression of c-fos in parietal endoderm, amnion and differentiating F9 teratocarcinoma cells

The expression of the cellular proto-oncogene, c-fos, in extra-embryonic tissues of the mouse was investigated using a v-fos DNA probe and an affinity-purified antiserum raised against a C-terminal synthetic peptide. At 13.5 days of development, parietal endoderm - a tissue not previously studied using these methods - was found to express c-fos RNA at a higher level than the amnion or placenta. The previously reported dramatic increase in c-fos RNA levels in extra-embryonic membranes during gestation was found to be confined to the amnion. The antipeptide serum specifically recovered proteins with M, values of 46,000 and 39,000 from extracts of parietal endoderm and amnion cells labelled for 15 min with 35S-methionine. On sodium-dodecyl-sulphate/polyacrylamide gel electro phoresis these proteins co-migrated with proteins immunoprecipitated using serum from rats innoculated with FBJ-MuSV-transformed cells (tumour-bearing rat serum). Pulse chasing and 32P-labelling experiments showed that the protein with an M, of 46,000 was rapidly converted into higher-molecular-weight phosphorylated derivatives. F9 teratocarcinoma stem cells differentiated into parietal-endoderm-like cells in response to treatment with retinoic acid and dibutyryl cyclic AMP. However, this differentiation was not accompanied by any large transient increase in c-fos RNA expression.

Expression of cellular oncogenes in teratoma-derived cell lines

The expression of ten proto-oncogenes was studied in cell lines derived from transplantable mouse teratomas. The cell lines represent different forms of early embryonic cell specialization. The analysis included two embryonal carcinoma (EC) lines (PCC3 and F9), and four differentiated cell lines derived from teratocarcinoma, namely trophoblastoma (3-TDM), parietal endoderm (PYS-2), visceral endoderm (PSA5-E) and skeletal myoblasts (Cl10). The expression of c-oncogenes was studied by analysing poly(A)+RNA for complementary sequences by dot blot and Northern blot hybridization. The results were related to the rate of cell multiplication and the state of differentiation by examining [ 3H]thymidine incorporation, growth curves and tissue-specific differentiation markers. Expression of c- myc and c- Ki- ras was found in all cell lines. In dot blot assays, poly(A)+RNA from all cell lines also hybridized with v- abl and v- sis probes. A marked decrease in c- myc expression was found in teratoma-derived myoblasts differentiating into myotubes. A similar reduction was found when ‘nullipotent’ F9 cells were induced by retinoic acid (RA) to form primitive endoderm. However, reduction of the growth rates of the parietal and visceral endodermal cell lines were not accompanied by decreased expression of c- myc or c- Ki- ras. Hybridization signals obtained with a v- sis probe was low in all teratoma-derived cell lines tested, except for the myogenic cell line Cl10. Both in exponentially growing and differentiated cultures of this line, two size classes of transcripts hybridized strongly to the v- sis probe. However, these transcripts, 7 and 3 kb, most likely represent endogenous retroviral transcripts and not c- sis transcripts. Expression of c- myb, c- mos, c- fes, c- src and c- erb A and c- erb B could not be detected in any of the cell lines studied.

Effects of pulsed electromagnetic field on growth and differentiation of embryonal carcinoma cells.

A murine embryonal carcinoma cell line (F9) was used to examine the effect of a pulsed electromagnetic field on the growth and differentiation of malignant cells. The cells can be induced to differentiate into parietal endodermal cells by treatment with retinoic acid. The pulsed electromagnetic field (1 Gauss and 10 Gauss) promoted the growth of embryonal carcinoma cells in both the presence and absence of retinoic acid. The pulsed electromagnetic field was also found to inhibit retinoic acid-induced differentiation, when the degree of differentiation was based on morphological criteria or on the production of plasminogen activator.

A membrane glycoprotein involved in teratocarcinoma cell adhesion to substratum

An antiserum was raised against glycoproteins isolated from teratocarcinoma OTT6050 by affinity chromatography on Ricinus communis agglutinin-1 (RCA-1). The antiserum inhibited attachment and spreading of the teratocarcinoma cells on plastic tissue culture plates. Embryonal carcinoma cells (F9 and N4-1) and PYS-2 parietal endodermal cells were also affected. The antiserum was also effective in preventing adhesion of trophoblasts to substratum. Furthermore, the antiserum caused rounding and detachment of the cells previously spread on the substrates, and the process could be reversed by removing the antiserum. Fab fragments isolated from the antiserum also inhibited cell-substratum adhesion, but not cell-cell adhesion, indicating that the factor involved is specific for cell-substratum adhesion. The antiserum inhibited adhesion of F9 and PYS-2 cells not only to plastic dishes but also to those coated with plasma fibronectin, laminin, and plant lectins such as concanavalin A (conA). Therefore, it is likely that the antiserum inhibited cell-substratum adhesion by blocking the function of an intrinsic component of cell-substratum adhesion rather than interfering with specific receptors for fibronectin or laminin. The glycoprotein with the antibody-blocking activity was partially purified from particulate fraction of teratocarcinoma OTT6050 by extraction with Triton X-100 and affinity chromatography on RCA-1-agarose and conA-agarose Upon Western blot analysis of the glycoprotein preparation, the antibody reacted only with a glycoprotein of the apparent molecular weight (MW), 125 000 (GP-125). An antibody preparation was isolated by affinity chromatography on Sepharose 4B coupled with the glycoprotein fraction and was shown to have all of the activities described above. Molecules resembling GP-125 could be also isolated from cultured F9 and PYS-2 cells by solubilization with Triton X-100 and indirect immunoprecipitation.