Alignment of biologically active domains in the fibronectin molecule

Gelatin-binding material was isolated from a human plasma cryoprecipitate by affinity chromatography on gelatin-Sepharose. Individual fragments of fibronectin with Mr = 170,000, 100,000, and 80,000 and a mixture of fragments with Mr = 205,000 and 190,000 (200K fraction) were isolated from this material. These fragments reacted with antifibronectin and with antibodies to a gelatin-binding Mr = 70,000 tryptic fragment of fibronectin. They all shared the same NH2-terminal amino acid sequence. The 205K and 190K fragments bound also to heparin-Sepharose, whereas the smaller fragments did not. The 200K fraction and the 170K fragment mediated cell attachment when used to coat plastic, whereas the 100K and 80K fragments were inactive in this assay. Further digestion of the 205K and 190K fragments with chymotrypsin yielded separate sets of smaller fragments that bound to either gelatin-Sepharose or heparin-Sepharose, as well as fragments that did not show either of these binding activities but mediated cell attachment. Since the NH2-terminal ends of the 205K, 190K, 100K, and 80K fragments are the same, the results define the order of the active sites in the fibronectin molecule as gelatin-binding site, cell attachment site, and heparin-binding site.     

Developmental regulation of a secreted gelatin-binding protein during myogenesis in vitro

Collagen has a stimulatory effect on the differentiation of skeletal muscle cells in culture. Putative collagen-binding proteins were isolated from detergent-solubilized cultures of the L6 rat muscle cell line and primary clonal cultures of human skeletal muscle satellite cells, using gelatin-Sepharose affinity chromatography. In addition to fibronectin, which has been reported by others to be synthesized by cultured muscle cells, we found that muscle cultures synthesized gelatin-binding proteins of lower apparent molecular weight. Only one of these proteins was secreted into the growth medium and bound to type I collagen. Binding of this protein to gelatin and collagen-Sepharose was resistant to repeated washing with 1 M NaCl and nonionic detergent. The secreted gelatin-binding protein had an apparent molecular weight of 63,000-72,000, depending upon the conditions of electrophoresis. The lack of reactivity of the secreted protein with polyclonal antisera against fibronectin, the lack of effect of protease inhibitors on its appearance in the medium, and the rapid de novo production of the protein during pulse labeling with radioactive methionine indicated that it was not a fibronectin fragment. The rate of synthesis of the secreted gelatin-binding protein increased markedly during the myogenesis of rat and human cultures.     

The 95000-Mr gelatin-binding protein in human serum and plasma.

A gelatin-binding 95000-Mr protein was detected in human serum and plasma by immunoblotting using antibodies against the 95000-Mr gelatin-binding protein, a major secretory component of cultured adherent human monocyte/macrophages. Serum and plasma were prepared by incubating blood at 4, 22 or 37 degrees C for different periods of time, and gelatin-binding proteins were isolated from 200 microliter portions by gelatin-Sepharose affinity chromatography. The bound material was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In protein-stained gels, fibronectin and some minor polypeptides were seen, but not the 95000-Mr protein. In immunoblotting of identical serum samples the antibodies detected apparently two closely spaced polypeptide bands at Mr95000, and in plasma samples a single band at the position of the faster-migrating one of the two above-mentioned bands. The immunoperoxidase reaction was stronger when serum and plasma were prepared by incubating for longer periods of time (up to 8 h) or at higher temperatures (up to 37 degrees C). In samples made from plasma, the immunoperoxidase reactions were weaker than in those from serum, indicating a lower quantity of the protein. The results suggest that the 95000-Mr protein is released from monocytes and granulocytes during the incubation of blood and, more likely, when they possibly interact with the blood clot and may become adherent.     

Further localization of the gelatin-binding determinants within fibronectin. Active fragments devoid of type II homologous repeat modules

Digestion of a 42-kDa gelatin-binding fragment (GBF) of fibronectin with pepsin followed by affinity chromatography on gelatin-Sepharose produces three fractions, a drop-through non-binding fraction, a retarded fraction that is dominated by a 13-kDa fragment whose NH2 terminus is identical to that of 42-kDa GBF, and a binding fraction that contains a homogeneous fragment of apparent mass 21 kDa with an NH2 terminus corresponding to Arg484. This 21-kDa GBF binds repeatedly to gelatin-Sepharose, eluting near 2.6 M in a urea gradient. It also binds in the fluid phase to a fluorescent-labeled collagen peptide with Kd = 10 microM and inhibits the binding of 42-kDa GBF to the same peptide with KI = 7.3 microM. Thus, major gelatin-binding determinants of fibronectin are located within a 21-kDa region that contains two type I homologous "finger" modules and is devoid of the type II "kringle-like" modules that were previously thought to be essential for this activity.     

Characterization of a 59 kDa gelatin-binding fragment of buffalo plasma fibronectin

Limited proteolysis of buffalo plasma fibronectin (FN) by thermolysin yielded four gelatin-binding fragments of which, the major 59 kDa fragment, GBF1, was isolated by gelatin-Sepharose and heparin-Sepharose affinity columns. GBF1 appeared during early phase of thermolysin digestion and remained intact even after 4 hr of digestion. GBF1 may be similar to 56 kDa gelatin-binding fragment of FNs from human and hamster plasma. But, it is more resistant to thermolysin cleavage. The fragment binds to heparin with low affinity. On the basis of the structure of human plasma FN, the modular structure of GBF1 may be given as: 6Fn1 1Fn2 2Fn2 7Fn1 8Fn1 9Fn1 1Fn3. Biophysical properties of GBF1 suggest an expanded native conformation. The interaction of the fragment with gelatin is pH-dependent and independent of NaCl concentration.     

Mapping the collagen-binding site of human fibronectin by expression in Escherichia coli.

The collagen-binding domain of human fibronectin has been expressed as a cro/beta-galactosidase fusion protein in Escherichia coli. The hybrid polypeptide was recognized by an anti-(human plasma fibronectin) serum and bound specifically to gelatin-Sepharose. The collagen-binding region was subdivided by constructing a series of overlapping bacterial expression plasmids. The fusion proteins produced by these constructs were analysed for gelatin-binding activity. The results indicate that the binding site lies within an approximately 12.5 kd fragment of fibronectin, and show that the following 14 amino acid sequence is critical for gelatin-binding activity: Ala-Ala-His-Glu-Glu-Ile-Cys-Thr-Thr-Asn-Glu-Gly-Val-Met. This sequence links the second type II homology unit with the adjacent type I repeat in the amino-terminal third of the fibronectin molecule.     

Biochemical and immunological relationships among fibronectin-like proteins from different sea urchin species

Fibronectin-like proteins were purified from ovaries of the sea urchin species, Paracentrotus lividus (PI), Sphaerechinus granularis (Sg), Arbacia lixula (Al), Pseudocentrotus depressus (Pd), and Anthocidaris crassispina (Ac), by gelatin-Sepharose affinity chromatography. The major component had a molecular mass of 180 kDa and was eluted by 1 M NaCl or 8 M urea, depending on the species used. By substrate adhesion assay, we tested the biological activity of the 180 kDa protein purified from Paracentrotus lividus (P1-180K) and showed that it promotes the adhesion of homologous embryonic cells to the substrate. An antiserum, developed against Temnopleurus hardwickii fibronectin-like protein (Th-180K), was used in Western blots of the proteins purified from the five species. The antibody cross-reacted with Pl-180K, Pd-180K and Ac-180K. A peptide map of P1-180K, obtained by V8 protease partial digestion, was compared with those obtained from the other four proteins and showed an homology between 40 and 56%. This report confirms that fibronectin-like proteins can be purified from sea urchins on the basis of their binding to gelatin-Sepharose; the proteins differ for their binding affinity to gelatin and share different epitopes, suggesting that they are members of a sea urchin fibronectin super family.     

Interaction of the 70,000-mol-wt amino-terminal fragment of fibronectin with the matrix-assembly receptor of fibroblasts

Plasma fibronectin binds saturably and reversibly to substrate-attached fibroblasts and is subsequently incorporated into the extracellular matrix (McKeown-Longo, P.J., and D. F. Mosher, 1983, J. Cell Biol., 97:466-472). We examined whether fragments of fibronectin are processed in a similar way. The amino-terminal 70,000-mol-wt catheptic D fragment of fibronectin bound reversibly to cell surfaces with the same affinity as intact fibronectin but did not become incorporated into extracellular matrix. The 70,000-mol-wt fragment blocked binding of intact fibronectin to cell surfaces and incorporation of intact fibronectin into extracellular matrix. Binding of the 70,000-mol-wt fragment to cells was partially abolished by cleavage into 27,000-mol-wt heparin-binding and 40,000-mol-wt gelatin-binding fragments and more completely abolished by reduction and alkylation of disulfide bonds. Binding of the 70,000-mol-wt fragment to cells was not blocked by gelatin or heparin. When coated onto plastic, the 70,000-mol-wt fragment did not mediate attachment and spreading of suspended fibroblasts. Conversely, fibronectin fragments that had attachment and spreading activity did not block binding of exogenous fibronectin to substrate-attached cells. These results indicate that there is a cell binding site in the 70,000-mol-wt fragment that is distinct from the previously described cell attachment site and is required for assembly of exogenous fibronectin into extracellular matrix.     

Degradation of fibronectin by human leukocyte elastase. Release of biologically active fragments

We have identified the biological activity of three polypeptides released by limited proteolysis of human plasma fibronectin by leukocyte elastase. A Mr = 140,000 peptide contains cell-spreading activity; a Mr = 60,000 peptide mediates binding to denatured collagen (gelatin), and a Mr = 29,000 peptide contains glutaminyl residues responsible for the transglutaminase (blood coagulation factor XIIIa)-catalyzed incorporation of amines. More extensive proteolysis yielded numerous peptides, including a Mr = 40,000 peptide derived from the Mr = 60,000 peptide which retains gelatin-binding activity. Quantification of the gelatin-binding peptides is consistent with two binding sites per dimeric fibronectin molecule of Mr = 440,000. Both Mr = 60,000 and 40,000 gelatin-binding peptides were enriched with half-cystine residues, containing 28 and 25, respectively, but devoid of cysteine. This, coupled with the electrophoretic behavior of both peptides, was consistent with the presence of intramolecular disulfide bonds in the gelatin-binding domain. Intact fibronectin contains 1 free cysteine residue/monomer, as recently described. This cysteine reacts with 5,5'-dithiobis(2-nitrobenzoic acid) very slowly under nondenaturing conditions but rapidly when fibronectin is denatured. The free cysteine is located in the Mr = 140,000 peptide. While the Mr = 40,000 and 60,000 gelatin-binding peptides bind to gelatin with an affinity about 30-fold and 5-fold less than intact fibronectin (based on a monomeric fibronectin Mr = 220,000), neither gelatin-binding peptide supports spreading of fibronectin-deficient test cells on gelatin or tissue culture plastic substrates. The purified Mr = 140,000 peptide supported cell spreading on plastic, retaining about one-half of the spreading activity of intact fibronectin on a weight basis. These data confirm recent results, suggesting multiple, protease- resistant domains with discrete biological functions within fibronectin. Our results, together with established data, suggest a model for the location of the transglutaminase-reactive glutaminyl residues, gelatin binding, and cell-adhesive domains in fibronectin. The release of univalent, biologically active fibronectin fragments by elastase, a major physiologically released inflammatory protease of human leukocytes, suggests a new potential mechanism for alteration of cell connective tissue interactions at sites of inflammation in vivo.     

Two stress proteins of the endoplasmic reticulum bind denatured collagen

A differentiation-related gelatin-binding 46 kilodalton (kDa) glycoprotein in myoblasts (GP46, colligin) shares several properties with the 78-kDa glucose-regulated protein (GRP78), including location in the endoplasmic reticulum and related C-terminal sequences. These similarities extend to stress inducibility, since we find that GP46 is a heat-shock protein; its synthesis is elevated at 42 degrees C, resulting in a two- to three-fold increase in protein level. Further, GRP78 is a gelatin-binding protein; together with GP46 it is retained on gelatin-Sepharose beads. GRP78 and GP46 do not interact; each protein can be individually eluted, GP46 at low pH and GRP78 by ATP. These results suggest that the proteins have distinct roles in the synthesis of collagen and point to a simple method for purification.     

Adiponectin does not bind to gelatin: a new and easy way to purify high-molecular-weight adiponectin from human plasma

Human plasma contains three forms of adiponectin, a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We previously reported HMW adiponectin was a gelatin-binding protein of 28 kDa (GBP28), it having been purified due to its affinity to gelatin-Cellulofine (Nakano, Y., et al. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J. Biochem. 1996. 120: 803–12). Although HMW adiponectin binds to gelatin-Cellulofine, it cannot bind to gelatin-Sepharose. Gelatin-Cellulofine was made of formyl-Cellulofine and gelatin, and we found that HMW adiponectin binds to reduced formyl-Cellulofine with similar affinity as to gelatin-Cellulofine. Through only two steps using reduced formyl-Cellulofine and DEAE-Sepharose, HMW adiponectin can be effectively purified from human plasma.     

Secretion of a novel cell-adhesive protein distinct from fibronectin by mouse L.P3 cells growing in protein- and lipid-free synthetic medium

Several cell lines growing in protein- and lipid-free synthetic medium secreted cell-adhesive protein(s) into the medium. The conditioned medium (CM) of one of these cell lines, mouse L.P3, showed the highest cell attachment-promoting activity (CPA) among them. Cell-adhesive protein(s) in the CM of L.P3 cells (L.P3-CM) were separated into two types by sequential affinity column chromatography employing gelatin-Sepharose 4B and heparin-Sepharose 4B. One was a gelatin- and heparin-binding cell-adhesive protein (GCP), and was identified as a cellular form of mouse fibronectin. The other was a gelatin-non-binding and heparin-binding cell-adhesive protein (GNCP). The CPA of GNCP preparation was effective for the cell-attachment and spreading of both epithelial and fibroblastic cells. The CPA of GNCP preparation was not blocked by the antiserum and scarcely inhibited in the presence of the synthetic cell attachment-promoting peptide Gly-Arg-Gly-Asp-Ser-Pro, a competitive inhibitor of fibronectin. This suggests that the structure of the cell-attachment site of GNCP is different from that of fibronectin. The GNCP preparation showed little cross-reactivity with anti-mouse laminin antiserum in enzyme-linked immunosorbent assay (ELISA). These results demonstrate the possibility that GNCP in L.P3-CM is a novel cell-adhesive protein distinct from fibronectin or laminin. The secretion of the two types of cell-adhesive proteins by L.P3 cells is discussed.     

Isolation of an amino-terminal fibronectin-binding protein on human U937 cells and rat peritoneal macrophages.

Several cell-mediated activities for the amino terminus of fibronectin have been documented. In the present study we describe a macrophage surface protein with binding activity directed to the amino terminus of the fibronectin molecule. The binding of a 29-kDa amino-terminal fibronectin fragment to macrophages reached steady state by 30 min and was half-maximal at approximately 2 x 10(-8) M. This binding was specifically inhibited by excess unlabeled 29-kDa fragment or intact fibronectin but not by a 180-kDa fibronectin fragment which lacks the amino terminus. Competitive binding studies of the 70-kDa amino-terminal fibronectin fragment to macrophages revealed a single binding site with KD = 7.14 x 10(-8) M and approximately 8 x 10(4) binding sites/cell. Radiolabeled surface proteins extracted from rat peritoneal macrophages and from the human U937 cell line were applied to an affinity column comprised of the 70-kDa amino-terminal fragment of fibronectin coupled to a solid support. A single trypsin-sensitive radiolabeled protein of 67 kDa, from either cell type, was eluted from this column with urea. This protein showed no immunologic identity with fibronectin, fibrin(ogen), or albumin. The 67-kDa protein exhibited identical apparent molecular weight under reducing and nonreducing conditions, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. We have localized the fibronectin binding activity of this protein to within the 29-kDa amino-terminal domain of fibronectin. The 67-kDa protein eluted from the 70-kDa column failed to bind to a column comprised of the 45-kDa gelatin-binding fragment of fibronectin. Additionally, the 67-kDa protein was specifically eluted from the 70-kDa column by the 29-kDa amino-terminal fragment but not by the 45-kDa gelatin-binding fragment. These data suggest that this 67-kDa protein is a macrophage cell surface binding protein for the amino terminus of fibronectin.     

Localization of viral-envelope-glycoprotein-binding sites in fibronectin.

Purified viral-envelope glycoproteins from influenza A virus were found to bind to two fragments of the fibronectin molecule. Human plasma fibronectin was digested by leucocyte cathepsin G, and three different fragments, of Mr 30000, 40000 and 12000-140000, with specific binding functions were isolated. Micelles of radiolabelled influenza A glycoprotein were allowed to bind to these fragments immobilized on polystyrene micro-titre wells. The C-terminal 120000-14000-Mr fragments that carry the cell-binding activity bound viral proteins most efficiently, whereas the 40000-Mr gelatin-binding fragment bound considerably less. The N-terminal 30000-Mr Staphylococcus aureus-binding fragment was negative in the assays. Laminin, a basement-membrane protein, also bound viral proteins, though less effectively than fibronectin. The binding was abolished if laminin or fibronectin fragments were pretreated with neuraminidase. This suggests that the sialic acids in the sugar moieties of these glycoproteins are involved in the binding. The affinity of viral-envelope glycoproteins for certain domains of fibronectin and for laminin may play a role in virus-cell interactions.     

Highly purified, functionally active human fibronectin preparation

Fibronectin has been purified by gelatin-Sepharose affinity chromatography from fresh frozen human plasma. The bound fibronectin was eluted with 3 M urea. The purity of the fibronectin obtained has been checked on (immunoelectrophoresis, polyacrylamide gel electrophoresis, FPLC). Biological activity of the purified molecule has been monitored by means of three assays: quantitation of the gelatin-binding activity by ELISA, quantitation of the fibronectin-mediated attachment of fibroblasts on plastic and evaluation of the opsonic activity (uptake of gelatin latex particles by a murine macrophage line). When deep-frozen, fibronectin retains all of its properties. This highly purified and functional fibronectin fulfills the basic requirements for a standard reagent. It will allow to investigate physicochemical and functional alterations of various fibronectins.     

Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes

In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.     

Cardiac alpha-crystallin. I. Isolation and identification

A water soluble protein, a major component of the cytosolic fraction of rat heart cells, was purified using either reverse phase HPLC or antibodies affinity chromatography procedures and characterized. The protein has an apparent Mr of 24 k, as judged by SDS-gel electrophoresis. Under non-denaturing conditions, however, the protein occurs as a homomultimer (Mr between 400 and 650 k) of the monomeric 24 kDa species and could be selectively enriched by fractionation of the cytosolic fraction on 10 to 40% sucrose gradients. Polyclonal antibodies, raised against the denatured 24 kDa protein, were used to investigate its tissue distribution. Besides the heart, where it is very abundant, the 24 kDa protein is expressed also in other red muscles and in kidneys, but was not detectable in stomach, thymus, liver, and brain. The amino acid composition of the protein and the partial amino acid sequence of various proteolytic fragments was determined. A search for homologies of the primary structure of known proteins has shown that the 24 kDa protein is strikingly similar, if not identical to alpha-B-crystallin. In fact, the two proteins were found to be indistinguishable also by immunological criteria. This study demonstrates that the lens protein alpha B-crystallin is a major cytosolic component of heart cells.     

The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.     

Novel hyperglycosylated weak gelatin-binding fibronectin from human fetal placenta. Fractionation of a high poly(N-acetyllactosamine) fragment by tomato lectin affinity chromatography

A novel hyperglycosylated fraction of human term fetal placental fibronectin was detected by long-term affinity binding to gelatin-Sepharose. An 18-h batch-wise gelatin-binding step was necessary to obtain a very low-affinity binding fraction, characterized by especially high N-acetylglucosamine and galactose content, and diffuse, poorly stained Coomassie bands on SDS/polyacrylamide electrophoretograms. The presence of a high proportion of long 7-10-kDa poly(N-acetyllactosamine)-containing N-linked carbohydrate chains was confirmed by their gel permeation behavior, susceptibility to endo-beta-galactosidase and by methylation analysis. Our previous results suggest that 4.5-7-kDa poly(N-acetyllactosamine) structures reduce the binding of fibronectin and its chymotryptic Ala260-Trp599 subdomain GB44 to gelatin [Zhu, B. C. R. & Laine, R. A. (1985) J. Biol. Chem. 260, 4041-4045]. Based on a gradient of urea used to dissociate gelatin-bound GB44, in the present study, fractions containing the novel 7-10-kDa carbohydrates showed significantly weaker binding to gelatin. Weak gelatin-binding characteristics of this novel hyperglycosylated fraction suggest that extended poly(N-acetyllactosamine) N-linked chains can significantly weaken heterotropic binding functions of fetal glycoproteins. The combined properties of weak Coomassie staining and weak gelatin binding have caused the novel hyperglycosylated fibronectin to be overlooked in previous investigations.     

Domain structure of human plasma and cellular fibronectin. Use of a monoclonal antibody and heparin affinity to identify three different subunit chains

The domain structure of human plasma fibronectin was investigated by using heparin-binding and antibody reactivity of fibronectin and its proteolytically derived fragments. Digestion of human plasma fibronectin with a combination of trypsin and cathepsin D produced six major fragments. Affinity chromatography showed that one fragment (Mr 45 000) binds to gelatin and three fragments (Mr 31 000, 36 000, and 61 000) bind to heparin. The 31K fragment corresponds to NH2-terminal fragments isolated from other species. The 36K and 61K fragments are derived from a region near the C-terminus of the molecule and appear to be structurally related as demonstrated by two-dimensional peptide maps. A protease-sensitive fragment (Mr 137 000), which binds neither gelatin nor heparin but which has been shown previously to be chemotactic for cells [Postlethwaite, A. E., Keski-Oja, J., Balian, G., & Kang, A. H. (1981) J. Exp. Med. 153, 494-499], separates the NH2-terminal heparin- and gelatin-binding fragments from the C-terminal 36K and 61K heparin-binding fragments. A monoclonal antibody to fibronectin that recognized the 61K heparin-binding fragment was used to isolate a sixth fragment (Mr 34 000) that did not bind to heparin or gelatin and that represents a difference between the 61K and 36K heparin-binding fragments. Cathepsin D digestion produced an 83K heparin-binding, monoclonal antibody reactive fragment that contains the interchain disulfide bond(s) linking the two fibronectin chains at their C-termini. The data indicate that plasma fibronectin is a heterodimeric molecule consisting of two very similar but not identical chains (A and B). In contrast, enzymatic digestion of cellular fibronectin produced a 50K heparin-binding fragment lacking monoclonal antibody reactivity which suggests that the cellular fibronectin subunit is similar to the plasma A chain in enzyme susceptibility but contains a larger heparin-binding domain. A model relating the differences in the three fibronectin polypeptides to differences in published cDNA sequences is presented.