Abstract
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.
Highlights
Human placental fibronectin was isolated from fretshhe characteristic protease resistanfcoeund for eachof term placenta by urea extraction and purifiebdy gel- the fibronectins
The carbohydrateson human placental fibronec- Fibronectins from tissueandplasma inseveral species tin contained a large percentage of polylactosamine possess differing chemical, physicala,nd biological properties
It habseen reported that hamfrom placental fibronectin, showed differstefribronectin glycopeptides fromplasma nd cellular ences includingseveral protease-resistant domainsnot sources differ onlyin thepresence of fucose and a diminution found in the analogous fragment from plasma fibro- of sialic acid on the cellular form ( 5 )
Summary
Treated human plasma by gelatin-affinity chromatography according stacking and 7.5 or 12% resolving gels.Protein samples were prepared to the method of Hopper et al [35] as modified by Engvall and by heating at 100 "C for 2 min in pH6.8,0.05 M Tris-HC1, containing. We obtain the highest yields of fibronectin using Yamada and Weston's [38] urea extraction procedure by adding enough urea to make the final concentration 2 M including the volume of placenta. Digestion of Plasma and Placental Cellular Fibronectin with Chymotrypsin and Separation of Gelatin-bindingFragments-After passing through a Sephadex G-10 column to remove urea, pFnl and cFnl (1mg/ml) were digested by chymotrypsin in pH 7.0, 0.05 M Tris-HC1 buffer containing 0.15 M NaCl and 0.01 M CaClz at an enzyme/.
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