Human placental (fetal) fibronectin: increased glycosylation and higher protease resistance than plasma fibronectin. Presence of polylactosamine glycopeptides and properties of a 44-kilodalton chymotryptic collagen-binding domain: difference from human plasma fibronectin

Human placental fibronectin was isolated from fresh term placenta by urea extraction and purified by gelatin affinity chromatography. A 44-kDa chymotryptic fragment, also purified by gelatin affinity chromatography, gave a broad, diffuse band on polyacrylamide gel electrophoresis, whereas the analogous 43-kDa fragment from human plasma fibronectin migrated as a defined, narrow band. Upon extended treatment with endo-beta-galactosidase from Escherichia freundii, the 44-kDa chymotryptic gelatin-binding fragment from placental fibronectin changed its behavior on gel electrophoresis and migrated as a narrower, more defined band. The carbohydrates on human placental fibronectin contained a large percentage of polylactosamine structures, part of which occurred on the gelatin-binding fragment, comprising almost twice as much carbohydrate as plasma fibronectin. NH2-terminal amino acid sequence analysis of the chymotryptic gelatin-binding fragments from both fibronectins showed the first 21 residues to be identical. Tryptic and chymotryptic peptide maps of the gelatin-binding fragment from placental fibronectin, however, showed differences including several protease-resistant domains not found in the analogous fragment from plasma fibronectin. Intact placental fibronectin contains 20,000 Da of carbohydrate, whereas plasma fibronectin contains 11,000 Da. Placental fibronectin is more protease-resistant than plasma fibronectin, possibly due to the additional carbohydrate. Polyclonal antibodies against either fibronectin completely cross-react with amniotic fluid fibronectin, placental fibronectin, and plasma fibronectin upon Ouchterlony immunodiffusion. Human fibronectins of putatively the same polypeptide structure are, therefore, glycosylated in a dramatically different fashion, depending on the tissue of expression. If the patterns of glycosylation comprise the only difference in the glycoprotein, this may confer the characteristic protease resistance found for each of the fibronectins.     

Purification and properties of different forms of modeccin, the toxin of Adenia digitata. Separation of subunits with inhibitory and lectin activity.

1. The subunits were isolated of modeccin (subsequently referred to as modeccin 4B), the toxin purified from the roots of Adenia digitata by affinity chromatography on Sepharose 4B [Gasperi-Campani, Barbieri, Lorenzoni, Montanaro, Sperti, Bonetti & Stirpe (1978) Biochem J. 174, 491-496]. They are an A subunit (mol.wt. 26 000), which inhibits protein synthesis, and a B subunit (mol.wt. 31 000), which binds to cells. Both sununits, as well as intact modeccin, gave single bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but showed some heterogeneity on isoelectric focusing and on polyacrylamide-gel electrophoresis at pH 9.5. 2. A second form of modeccin, not retained by Sepharose 4B, was purified by affinity chromatography on acid-treated Sepharose 6B: this form is subsequently termed modeccin 6B 3. Modeccin 6B has a molecular weight indistinguishable from that of modeccin 4B, and consists of two subunits of mol.wts. 27 000 and 31 000, joined by a disulphide bond. The subunits were not isolated because of their high insolubility in the absence of sodium dodecyl sulphate. 4. As compared with modeccin 4B, modeccin 6B is slightly less toxic to animals, does not agglutinate erythrocytes, and is a more potent inhibitor of protein synthesis in a lysate of rabbit reticulocytes, giving 50% inhibition at the concentration of 0.31 microgram/ml.     

Characterization of porcine plasma fibronectin and its fragmentation by porcine liver cathepsin B

Fibronectin was isolated from porcine plasma by affinity chromatography with gelatin-linked Sepharose 4B. Porcine fibronectin had a chemical composition similar to those of human and other fibronectins and reacted with antiserum raised against human fibronectin. It showed hemagglutination activity with trypsin-treated rabbit erythrocytes, though the activity was far less than that of human fibronectin. Porcine plasma fibronectin consisted of two subunit chains of about 230,000-daltons linked by disulfide bonds(s). Limited proteolysis of this protein with porcine liver cathepsin B yielded five major fragments which were investigated by affinity chromatography with gelatin- and heparin-linked Sepharose 4B. One fragment (Mr = 50,000) was bound to gelatin but not to heparin, while the remaining four were bound to heparin but not to gelatin, suggesting that plasma fibronectin takes a discrete domain structure with respect to interaction with these two macromolecules. The three larger heparin-binding fragments, Mr = 175,000, 150,000, and 130,000 were eluted with different concentrations of a mixture of NaCl and urea from the heparin-column, suggesting that they have different interactions with heparin, the 130,000-dalton fragment being the one with the strongest interaction. After reduction with 2-mercaptoethanol, the 175,000-dalton fragment was converted to the 150,000-dalton region fragment, which, together with the unchanged 150,000-dalton fragment, appeared to be equivalent in amount to the 130,000-dalton fragment. This finding suggests that the 150,000- and 130,000-dalton fragments may have originated from different subunit chains. Since the 175,000-dalton fragment was not produced by cathepsin B digestion of fibronectin which had been treated with plasmin, it was concluded that the 175,000-dalton fragment contained interchain disulfide bond(s) which had linked the native subunit chains. These results suggest that porcine plasma fibronectin has non-identical subunit chains composed of domains which differ in interaction with heparin and in susceptibility to cathepsin B.     

Separation and analysis of the major forms of plasma fibronectin

Human plasma fibronectin exists in circulation in multiple molecular forms that are distinguishable by SDS-polyacrylamide gel electrophoresis (zone I, approx. 450 kDa dimers; zone II, 190-235 kDa; Zone III, 146-175 kDa). (Chen, A.B., Amrani, D.L. and Mosesson, M.W. (1977) Biochim. Biophys. Acta 493, 310-322). We report here on investigations of plasma fibronectin that had been purified from the 'heparin-precipitable fraction' of plasma by DEAE-cellulose chromatography using buffers containing a chaotropic salt (KSCN). Zone I fibronectin and zone II fibronectin were subsequently separated by Sepharose CL-6B chromatography in the presence of 0.3 M KSCN. Electrophoresis of reduced zone I fibronectin dimers showed the presence of three types of subunits (i.e., 220 kDa, 215 kDa, 207 kDa), evidently all having the same NH2-terminal sequence. Subunits of this size were also found in reduced zone II fibronectin, as well as another polypeptide of 190 kDa, the latter amounting to under 5% of the total. Unreduced zone I fibronectin was resolved by gel electrophoresis into a doublet. The upper component amounted to approx. 90% of the total and was comprised of 220 kDa and/or 215 kDa subunits; the lower component contained 207 kDa plus a 220 kDa or 215 kDa subunit. Scanning transmission electron microscopy indicated that under physiologic conditions zone II fibronectin molecules, like those in zone I, exist as pleiomorphic, loosely folded structures (approx. 16 X 8-12 nm) that are somewhat smaller than dimeric zone I molecules (approx. 24 X 16 nm). Circular dichroic spectral analyses suggests that both types have similarly folded local domains. Affinity chromatography experiments revealed a relative decrease in the binding of zone II fibronectin to gelatin but no difference from zone I fibronectin with respect to heparin or fibrin binding.     

Fibronectin-associated transforming growth factor

We have studied the ability of fibronectins to induce anchorage-independent growth of NRK-49F cells in serum-free medium. Cells were seeded in soft agar in the presence of various concentrations of plasma fibronectins, and colonies were counted after 10 days. It was found that, with some exceptions, human plasma fibronectins induced anchorage-independent growth at concentrations in 20-100 micrograms/ml range. The ability of exogenously supplied fibronectins to promote anchorage-independent growth of NRK cells is attributed to a transforming growth factor (TGF) activity associated with gelatin-agarose affinity purified plasma fibronectins. This TGF activity required epidermal growth factor (EGF) in our serum-free assay system. The TGF-like activity appears to either co-purify or to be associated with fibronectin at neutral pH during molecular sieve chromatography and during ultracentrifugation through sucrose density gradients. The TGF activity "dissociates" from fibronectin at extremes of pH, however, and can be separated from fibronectin by molecular sieve chromatography in 1 M acetic acid. Under these conditions, the TGF-like activity chromatographed as a single peak with an apparent molecular weight of approximately 30 kDa. The physical-chemical properties, chromatographic behavior, and biological activity of this TGF suggest that it is type-beta transforming growth factor/growth inhibitor (beta-TGF/GI). The TGF activity has been observed in fibronectin isolated from fresh human plasma as well as in fibronectins from several other species obtained from commercial suppliers. Our results would suggest that caution be applied in the interpretation of experiments in which gelatin affinity purified fibronectins are used at micrograms/ml concentrations.     

人早期胎盘绒毛纤维连接蛋白的分离纯化及鉴定

人妊娠５－８周的胎盘绒毛经匀浆后,用２ｍｏ１／Ｌｕｒｅａ－ＰＢＳ提取,通过Ｈｅｐａｒｉｎ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和柱层析,再经ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ凝胶过滤层析,得到人早期胎盘纤维连接蛋白（ｅａｒｌｙｐｌａｃｅｎｔａｆｉｂｒｏｎｅｃｔｉｎ,ｅｐＦＮ）。经还原及非还原ＳＤＳ－ＰＡＧＥ和免疫印迹电泳分析,ｅｐＦＮ分子量约５００ｋＤ,是由两个２５０ｋＤ亚基组成,与人足月胎盘纤维连接蛋白（ｔｅｒｍｐｌａｃｅｎｔａｆｉｂｒｏｎｅｃｔｉｎ,简称ｔｐＦＮ）相似,而大于人血浆纤维连接蛋白（ｐｌａｓｍａｆｉｂｒｏｎｅｃｔｉｎ,ｐＦＮ）。ｅｐＦＮ与抗人ｐＦＮ抗体及抗人羊水纤维连接蛋白（ａｍｎｉｏｔｉｃｆｌｕｉｄｆｉｂｒｏｎｅｃｔｉｎ,简称ａｍＦＮ）的三个主要功能区单抗均可发生反应。与五种植物凝集素结合力实验表明,ｅｐＦＮ在糖基组成上与ｐＦＮ和ｔｐＦＮ均不相同。     

Structural and functional comparisons of chicken and human cellular fibronectins

We have investigated the structural and functional differences between chicken and human cellular fibronectin by comparing the tryptic peptide patterns using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analyzing the binding properties of isolated trypsin-resistant polypeptide fragments. Although the overall functional organization of chicken and human cellular fibronectins was similar, the tryptic patterns obtained from these two molecules were strikingly different. For example, the tryptic digest of chicken cellular fibronectin contained two unique peptide fragments having molecular sizes of 45 and 70 kilodaltons. The previously unidentified carboxyl-terminal 45-kDa fragment is an intermediate that appears between 15 to 120 s of digestion. The 70-kDa fragment binds to gelatin, to fibrin (with unusually high apparent affinity), to heparin (at low ionic strength), and to fixed Staphylococcus aureus cells; it also contains an acceptor site for factor XIIIa (plasma transglutaminase). These results suggest that the functional domains of chicken and human fibronectins remain constant and that structural variations occur in the protease-susceptible regions of the molecule. The present findings are discussed in terms of the previously existing discrepancies in the literature on fibronectin.     

Plasma fibronectin is synthesized and secreted by hepatocytes

Primary cultures of hepatocytes of rats and hamsters were established and examined for the synthesis and secretion of fibronectin. Hepatocytes of both species secreted fibronectin as a soluble dimeric protein which could be purified by its affinity for gelatin and using specific antisera. Plasma and cellular fibronectins could be clearly resolved on two-dimensional gels. In both species, the majority of the fibronectin secreted by hepatocytes was of the plasma type, as shown by analyses on one- and two-dimensional gels. The secretion of plasma fibronectin increased with time in culture, both in absolute terms and relative to the secretion of albumin. Even during the first day of culture, the secretion of fibronectin relative to that of albumin appeared to be sufficient to account for the relative levels of these two proteins in plasma. Hepatocytes of both species secreted preferentially the chain of plasma fibronectin with higher apparent molecular weight, although the faster migrating chain was also secreted. In addition, hamster hepatocytes cultured for 2 or more days appeared to secrete a cellular form of fibronectin. Possible origins for the different chain types of cellular and plasma fibronectins are discussed.     

Direct association of fibronectin and actin molecules in vitro

Affinity chromatography with actin-Sepharose conjugates of purified human fibronectin, normal human plasma, or serum-free culture fluid from human fibroblasts showed that fibronectin molecules can directly bind to actin. A quantitative recovery of soluble human fibronectin was accomplished by chromatography on actin immobilized on Sepharose beads. Human fibronectin molecules bound to actin-Sepharose were eluted with 0.25--0.35 M potassium bromide, and these molecules competed in a species-specific radioimmunoassay for human fibronectin. The subunits of fibronectin isolated by actin-Sepharose chromatography comigrated in SDS polyacrylamide gel electrophoresis with those of electrophoretically homogeneous fibronectin purified by conventional procedures. The efficient direct binding of fibronectin to actin suggests that interactions between these proteins might also take place in vivo but further studies are needed to elucidate the biological significance of this affinity.     

Transformed human cells release different fibronectin variants than do normal cells

Fibronectin molecules are dimers composed of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor-derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts. Furthermore, we observed that 45-65% of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparin-binding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma.     

Fibroblast cellular and plasma fibronectins are similar but not identical

Fibronectins are multimeric, adhesive glycoproteins present on cell surfaces and circulating in blood. Cellular fibronectin produced by fibroblasts in vitro and fibronectin isolated from plasma are known to be very similar immunologically and biochemically. We investigated whether or not they are identifical. Purified chicken and human cell-surface fibronectins are 150-fold more active in hemagglutination of fixed erythrocytes than plasma fibronectins. Cell-surface fibronectin is also 50-fold more active in restoring a more normal morphology to transformed cells originally missing the protein. However, in two other assays that measure cell attachment to collagen and cell spreading, cell-surface and plasma fibronectins have identical specific activities. In sodium dodecyl sulfate polyacrylamide gels, the subunits of human and chicken plasma fibronectins have significantly smaller apparent subunit molecular weights than cellular fibronectins present on cell surfaces or secreted into culture media. These differences are also present in a characteristic large subfragment of both forms of fibronectin after limited proteolysis by trypsin. We conclude that by both biological and biochemical criteria, cellular and plasma fibronectins are similar but not identical.     

Structure of the carbohydrate units of human amniotic fluid fibronectin

Human amniotic fluid fibronectin was found to contain three types of carbohydrates: complex-type N-glycosidic glycans, lactosaminoglycans, and O-glycosidic glycans. The structures of the complex-type glycans were established by carbohydrate and methylation analysis, Smith degradation, sequential exoglycosidase treatments, lectin chromatography, and DEAE-Sephadex chromatography. Lactosaminoglycans were analyzed by fast atom bombardment mass spectrometry, and the O-glycosidically-linked oligosaccharides by gas-liquid chromatography-mass spectrometry and high-pressure liquid chromatography. The results show that amniotic fluid fibronectin contains 2 mol of biantennary and 2-3 mol of triantennary, complex-type N-glycosidic glycans. Unlike the N-glycosidic glycans of human adult plasma fibronectin, which contain only traces of fucose and are completely sialylated, the glycans from amniotic fluid fibronectin are fucosylated and only partially sialylated. The complex-type N-glycosidic glycans present in amniotic fluid fibronectin also include a fractional amount (0.1 mol) of glycans with a polylactosaminyl structure. In addition, 4 mol of O-glycosidic oligosaccharides, which have not previously been described in fibronectins, were found in amniotic fluid fibronectin. The major oligosaccharides in this fraction have the structures Gal beta 1----3GalNAcol, NeuNAc alpha 2----3Gal beta 1----3GalNAcol and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcol. O-glycosidically linked oligosaccharides were also detected in human adult plasma fibronectin but in smaller amounts than in amniotic fluid fibronectin. These results show that amniotic fluid fibronectin differs from plasma fibronectin with regard to the number of glycans attached to the polypeptide and that the glycans present in these two fibronectins differ in structure.     

Comparative studies on amniotic fluid and plasma fibronectins.

Human fibronectin was isolated from second-trimester amniotic fluid, from amniotic fluid obtained at term and from adult plasma. The amniotic-fluid fibronectins had a slightly higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis than the plasma fibronectin. Early- and late-amniotic-fluid fibronectin had 9.5 and 9.6% carbohydrate respectively, whereas plasma fibronectin had 5.8%. The amniotic-fluid fibronectins had similar mannose and sialic acid contents to plasma fibronectin, but greater amounts of glucosamine, galactosamine, galactose and fucose. There were no detectable differences in the amino-acid composition of amniotic-fluid and plasma fibronectins, and the patterns of peptides obtained after tryptic digestion of fibronectin from the two sources showed extensive similarities. Fibronectins from plasma and amniotic fluid were equally active in promoting cell attachment and were immunologically indistinguishable. These results show that fibronectin from amniotic fluid is more heavily glycosylated than plasma fibronectin or previously analysed fibronectins from cultured fibroblasts. The observed differences in glycosylation may be related to cell type and/or stage of development.     

Multiple fibronectin subunits and their post-translational modifications

We report analyses of fibronectin subunit diversity by high resolution one- and two-dimensional gel electrophoresis. We have studied plasma and cellular fibronectins of rats and hamsters. Each form of fibronectin comprises multiple distinguishable subunits and, within each rodent species, all subunits of plasma fibronectin are resolvable from those of cellular fibronectin. Some, but not all, of this heterogeneity is caused by differential glycosylation. Thus, while glycosylated plasma and cellular fibronectins share no common subunits, nonglycosylated forms of these proteins appear to share 2-3 subunits. In addition, there are subunits unique to plasma and to cellular fibronectins in both rats and hamsters, although the pattern of diversity differs slightly between species. All size variants of fibronectin are phosphorylated to varying degrees. However, only some subunits are sulfated, apparently on tyrosine residues in the C-terminal third of the molecule. Comparison of the distribution of sulfate on the various fibronectin subunits with recent results on generation of multiple mRNAs by alternative splicing suggests that tyrosine sulfate is located in a polypeptide segment present in only certain fibronectin subunits. The results reported here provide information on the likely contributions of primary sequence differences and post-translational modifications to the heterogeneity of fibronectin subunits.     

Isolation and characterization of human placenta fibronectin

Fibronectin was isolated from human placenta tissues and compared with human plasma fibronectin. Placenta and plasma fibronectins had similar amino acid compositions, immunological properties, and cell attachment-promoting activities, but differed in apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be accounted for at least partly by the difference in carbohydrate composition. Unlike plasma fibronectin, placenta fibronectin failed to form a precipitin line with concanavalin A in a double diffusion system. The non- or low-reactivity of placenta fibronectin with this lectin was also demonstrated by affinity chromatography with concanavalin A-agarose, in which more than 90% of the radiolabeled glycopeptides derived from placenta fibronectin was not retained on the gel. The two fibronectins also differed in the reactivity with Lens culinaris agglutinin of their glycopeptide fractions. These data indicate that placenta and plasma fibronectins are different in their carbohydrate structures and, therefore, suggest the presence of a tissue- or cell-specific mechanism for processing the carbohydrates of this glycoprotein.     

Similarities and differences between the fibronectins of normal and transformed hamster cells

Fibronectins from normal and virally transformed hamster cells were compared by several criteria. The fibronectin from transformed cells was similar to that from normal cells in being an intact dimeric glycoprotein with the ability to bind to gelatin, activated thiol-Sepharose, and cells. No evidence was found for proteolytic cleavages or abnormalities in disulfide bonding of transformed cell fibronectin. This fibronectin was also shown to be active in promoting cell attachment, elongation, and alignment. Therefore, the fibronectin produced by transformed cells is not defective. However, it was shown that the transformed cells were partially deficient in their capacity to bind fibronectins from either normal or transformed cells. This deficiency has implications for the significance of the loss of fibronectin on oncogenic transformation. Partial proteolysis of the fibronectins from normal and transformed cells gave rise to the same fragments. However, the glycosylated fragments from transformed cell fibronectin appeared somewhat larger than those from normal cell fibronectin. Analysis of fibronectin glycopeptides showed that transformation leads both to more branches per core and to a higher sialylation of the asparagine-linked complex carbohydrate side chains.     

Developmental changes in the glycosylation and binding properties of human fibronectins. Characterization of the glycan structures and ligand binding of human fibronectins from adult plasma, cord blood and amniotic fluid

Fibronectins from human adult plasma, fetal plasma and from amniotic fluid obtained during early and late gestation were compared with respect to (i) their reactivity with lectins, (ii) their binding to the physiological ligands gelatin and heparin, and (iii) the role of the carbohydrate residues in the binding to these two ligands. The two fibronectin isoforms displayed distinct developmental differences in both glycosylation and binding properties: (i) Proportions of tri/tetraantennary complex glycans compared to the fraction of biantennary structures, as inferred from the reactivity with concanavalin A, were highest in amniotic fluid fibronectin from late pregnancy, lower in amniotic fluid fibronectin from early gestation, and even lower in fetal and adult plasma fibronectins. Likewise, fucose (alpha 1-6) linked to the innermost N-acetylglucosamine of the chitobiosyl core, defined by reactivity with Lens culinaris agglutinin (LCA), was present primarily in amniotic fluid fibronectin, and decreased in content during gestation from the 2nd. to the 3rd. trimenon. Both fetal and adult plasma fibronectins were only weakly reactive with LCA, indicating a low content of (alpha 1-6) linked fucose residues. After prior treatment with sialidase, both plasma and amniotic fluid fibronectins strongly reacted with erythrocyte phytohaemagglutinin (E-PHA), indicating that both fibronectin isoforms contain bisecting (beta 1-4) N-acetylglucosamine residues. Amniotic fluid fibronectins showed much greater reactivity than adult and fetal plasma fibronectins with wheat germ agglutinin; binding of this lectin to amnion fluid fibronectins was not decreased by desialylation indicating the presence of poly(N-acetyllactosamine) units. Whereas amniotic fluid fibronectins were strongly reactive with peanut agglutinin, neither adult nor fetal plasma fibronectins did bind to this lectin unless after prior desialylation. Hence, both fibronectin isoforms contain O-glycan residues that are fully sialylated in fetal and adult plasma fibronectins, but only partly sialylated in amniotic fluid fibronectins. According to these differences, glycosylation of plasma and amniotic fluid fibronectins is under developmental regulation. (ii) Amniotic fluid fibronectins had a significantly lower binding activity for both heparin and gelatin than plasma fibronectins. Moreover, amnion fibronectin from late gestation displayed a significantly lower binding to these two ligands than amnion fibronectin from early gestation. Fetal plasma fibronectins had a lower binding activity for gelatin than adult plasma fibronectin. (iii) Treatment of fibronectins with sialidase, fucosidase and removal of N-glycans with endoglycosidases H and F did not affect binding to gelatin and heparin, indicating that the interaction of plasma and amnion fibronectin with these two ligands is not influenced by their oligosaccharide moieties.     

Purification of human plasma fibronectin using immobilized gelatin and Arg affinity chromatography

This protocol describes a method for purification of fibronectin (Fn) from human plasma based on a combination of gel filtration and affinity chromatography steps. Clarified plasma is first loaded onto a Sepharose CL-4B column and unbound material is sequentially purified on columns containing covalently coupled gelatin and Arg. The elution conditions are optimized to obtain a homogeneous preparation of Fn on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although the Fn yield is expected to be lower than that obtained using other methods, affinity adsorbents based on gelatin and Arg and gentle elution steps offer advantages including a high purity of the preparation and a correctly folded protein. The preparation can be useful for interaction studies and analysis of biological and immunological activities of Fn.     

Two-dimensional peptide mapping of fibronectins from bovine aortic endothelial cells and bovine plasma

We have made a comparison between plasma and endothelial cell fibronectin, since these cells are in intimate contact with plasma $\text{in vivo}$. Cellular and secreted fibronectins were purified from cloned lines of adult bovine aortic endothelial cells, and compared to purified bovine plasma fibronectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional peptide mapping. When unreduced, all three fibronectins migrated on gels as single bands with M_r 440,000. After reduction, cellular and secreted fibronectins migrated on gels as single bands with M_r 220,000, but plasma fibronectin migrated as two bands with M_r 220,000 and 210,000. All three fibronectins, including the two subunits of plasma fibronectin, had identical structures by peptide mapping analysis.     

Inhibition of protein synthesis in vitro by proteins from the seeds of Momordica charantia (bitter pear melon).

1. A haemagglutinating lectin was purified from the seeds of Momordica charantia by affinity chromatography on Sepharose 4B and on acid-treated Sepharose 6B. It has mol.wt. 115 000 and consists of four subunits, of mol.wts. 30 500, 29 000, 28 500 and 27 000. 2. The lectin inhibits protein synthesis by a rabbit reticulocyte lysate with an ID50 (concentration giving 50% inhibition) of approx. 5 micrograms/ml. Protein synthesis by Yoshida ascites cells is partially inhibited by the lectin at a concentration of 100 micrograms/ml. 3. From the same seeds another protein was purified which has mol.wt. 23 000 and is a very potent inhibitor of protein synthesis in the lysate system, with an ID50 of 1.8 ng/ml. This inhibitor has no effect on protein synthesis by Yoshida cells, and has no haemagglutinating properties. 4. Artemia salina ribosomes preincubated with the lectin or with the inhibitor lose their capacity to perform protein synthesis. The proteins seem to act catalytically, since they inactivate a molar excess of ribosomes. 5. The lectin and the inhibitor are somewhat toxic to mice, the LD50 being 316 and 340 micrograms/100 g body wt. respectively.