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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pulmonary artery endothelial cells were isolated from bovine fetal blood vessels and used for biosynthetic studies. At confluence, cultures were incubated in minimal essential medium (MEM) without serum containing [U-14C]proline. After 24 hours, medium was removed and labeled proteins were precipitated by the addition of ammonium sulfate and fractionated by diethylaminoethyl (DEAE)-cellulose chromatography. The elution profile showed four major peaks and one minor peak. Fractions within each peak were pooled, subjected to digestion by chymotrypsin and/or collagenase, and analyzed by polyacrylamide gel electrophoresis. Peak l contained a collagen which contained approximately 6% of the 3-hydroxyproline isomer while total hydroxyproline content was approximately 45%. This material was digested by purified bacterial collagenase and had a mobility slightly slower than that of alpha 1(III) which did not change under conditions that reduce disulfide bonds. Upon digestion with chymotrypsin under conditions where native procollagens are converted to alpha-chains, this material was digested. These properties suggest that this material is type VIII or EC (endothelial cell) collagen. Peak 2 contained substantial fibronectin while peak 3 contained primarily type III procollagen. The last major peak contained a mixture of collagenous and noncollagenous material. Upon digestion with chymotrypsin, several peptides were generated which were sensitive to bacterial collagenases. The two major chymotrypsin-resistant components had mobilities slower than that of alpha(III) and were not disulfide-bonded.
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PMID:Collagen synthesis by cloned pulmonary artery endothelial cells. 671 15

Proteases have been used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion. Proteolytic digestion of membrane-proteins by pronase (1 mg/ml for 20 min at 37 degrees C) completely inhibited adhesion of baby hamster kidney (BHK) fibroblasts on fibronectin-coated plastic dishes. Various degrees of inhibition were also obtained after treatment with proteinase K, chymotrypsin, papain, subtilopeptidase A, and thermolysin. Protein synthesis was required to restore the adhesive properties of pronase-treated cells, showing the protein nature of the molecules involved in adhesion to fibronectin. A peculiar feature of these proteins was their resistance to cleavage by trypsin. After prolonged trypsin treatment (1 mg/ml for 20 min at 37 degrees C), cells adhered and spread on fibronectin-coated dishes, even when protein synthesis was inhibited by 4 microM cycloheximide. Under these conditions only three glycoproteins (gp) of molecular weight 130,000, 120,000, and 80,000 were left on the cell surface. These were precipitated by a rabbit antiserum against BHK cells that also inhibited adhesion of trypsin-treated cells. gp120 and gp80 were left at the cell surface after mild pronase digestion (0.2 mg/ml for 20 min at 37 degrees C), under conditions not affecting adhesion. These data suggest that these glycoproteins may be involved in fibronectin-mediated cell adhesion in some yet unknown way.
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PMID:Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins. 674 66

Fibronectin is a major cell-surface glycoprotein which has been reported to interact with glycosaminoglycans. A nitrocellulose filter-binding assay was developed to quantitate these interactions at physiological pH and ionic strength. Fibronectin isolated from chick embryo fibroblasts binds both hyaluronic acid and heparin; heparan sulfate is bound less efficiently, and chondroitin sulfate and glycopeptides are bound minimally. The binding of hyaluronic acid and heparin to fibronectin is saturable and reversible and occurs at separate binding sites. The binding of both molecules to fibronectin is not blocked by EDTA or by other glycosaminoglycans, and is only moderately inhibited by elevated ionic strength. Scatchard analyses revealed nonlinear, high affinity binding to fibronectin with a KD of approximately 10(-7) to 10(-8) M for these glycosaminoglycans. The affinity for heparin was utilized for the isolation of heparin-binding domains of fibronectin on heparin-agarose affinity columns. Heparin-binding proteolytic fragments with apparent molecular weights of 160,000 and 50,000 were isolated following hydrolysis of fibronectin by chymotrypsin or pronase, respectively. The possible involvement of such high affinity binding sites of fibronectin in the binding of glycosaminoglycans to the cell surface or in the organization of extracellular matrices is discussed.
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PMID:Characterization of fibronectin interactions with glycosaminoglycans and identification of active proteolytic fragments. 677 Dec 64

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.
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PMID:Alignment of biologically active domains in the fibronectin molecule. 678 65

Culture of chick-embryo sternal-cartilage chondrocytes within three-dimensional collagen gels promotes the synthesis of three low-molecular-weight collagenous polypeptides. The proportions of these novel collagens synthesized and released into the medium are markedly influenced by the presence or the absence of fibronectin in the serum supplement. Chondrocytes cultured on plastic dishes appear to synthesize only small amounts of these low-molecular-weight species. The three species (designated G, H and J) were characterized with respect to the proportion of [14C]proline incorporated into each polypeptide occurring as hydroxy[14C]proline and with respect to their susceptibilities to bacterial collagenase. On the basis of their electrophoretic mobilities under reducing conditions, the G, H and J polypeptides were calculated to have Mr 59 000, 69 000 and 84 000 respectively. Chymotrypsin digestion converted the G collagen into a species containing polypeptides of Mr 45 000, whereas the H and J polypeptides yielded a single band of Mr 53 000. The H and J polypeptides were found to occur as disulphide-linked aggregates, as was the chymotrypsin-digestion product. Peptide 'mapping' has shown that G, H and J polypeptides show no common identity and are distinct from the known interstitial collagens. Native G collagen was digested by human collagenase to discrete products, whereas H and J chains were not cleaved under identical conditions.
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PMID:Identification and partial characterization of three low-molecular-weight collagenous polypeptides synthesized by chondrocytes cultured within collagen gels in the absence and in the presence of fibronectin. 687 Aug 39

Circular dichroism spectra, thermal transition profiles and proteolytic susceptibility showed that different regions in the multidomain protein fibronectin exhibit different sensitivity against urea denaturation. A 70-kDa fragment obtained by cathepsin D treatment which comprises the N-terminal part of the fibronectin chains, exhibited in 8M urea a spectrum, at 20 degrees C, identical to that of the native fragment and its thermal unfolding was shifted to lower temperatures by only 10 degrees C. The central portions of the fibronectin chains were remarkably unfolded under the same conditions as clearly demonstrated by the spectra and transition profiles of a cathepsin D-raised 125/140-kDa fragment which originates from this region. When fibronectin or its fragments were exposed to 4 or 8M urea at 4 degrees C and the urea subsequently dialysed off, the spectra and transition curves recorded were very similar to those of the native proteins. Nevertheless, this treatment introduced local conformational changes which resulted in the creation of three new cleavage sites for chymotrypsin. The most prominent one was found to be located in the central part of the middle region and no sites were created in the N-terminal 70-kDa region. In the conjunction with sequence information [Petersen et al. (1983) Proc. Natl. Acad. Sci. USA 80, 137-141] it may be concluded that the disulfide rich domains, made up by regions of internal homology of types I and II in the N-terminal portion of fibronectin, exhibit a remarkable conformational stability, whereas the disulfide free middle region which contains type III domains, is much less stable. Some domains in this region are particularly sensitive to urea denaturation and are irreversibly affected already by 4M urea at 4 degrees C. Therefore, the use of high urea concentrations for the elution of fibronectin from affinity columns may lead to an at least partially irreversible unfolding of some domains and a loss of functions associated with these structural elements.
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PMID:Discrimination of different domains in fibronectin on the basis of their stability against urea denaturation. 687 82

We have identified a specific actin-binding site in the adhesive glycoprotein fibronectin, isolated from chicken fibroblasts. Affinity chromatography of fragments, released from fibronectin by limited proteolysis with trypsin, chymotrypsin and subtilisin, on actin-Sepharose and other protein-Sepharose columns was used to locate the binding site. A 27 000-mol.wt. subtilisin-digest fragment bound efficiently to actin. The results suggest that the actin-binding site is close to, but not identical with, the reported collagen-binding site.
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PMID:Isolation of an actin-binding fragment of fibronectin. 703 Mar 13

To determine how the carbohydrate moiety of fibronectin influences the susceptibility of protein to proteolytic degradation, we compared the effects of various proteases on glycosylated and nonglycosylated fibronectins. Nonglycosylated fibronectin, from tunicamycin-treated chicken embryo fibroblasts, was degraded more rapidly to acid-soluble products than glycosylated fibronectin by pronase, thermolysin, trypsin, and chymotrypsin. The absence of carbohydrate did not markedly affect overall patterns of proteolytic fragments identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Except for the expected increases in electrophoretic mobilities of the nonglycosylated peptides, the only important difference was that of the nonglycosylated fragment corresponding to the carbohydrate-rich, collagen-binding domain, was completely digested by the proteases in 60 min at 30 degrees C. In contrast, the comparable fragment from glycosylated fibronectin was resistant to protease digestion. Heparin-binding domains that normally lack carbohydrate are equally susceptible to proteases in glycosylated and nonglycosylated fibronectin. We conclude that the carbohydrate component of fibronectin plays an important role in the stabilization of a specific domain of the protein against proteolytic degradation; however, the carbohydrate does not alter overall proteolytic specificity.
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PMID:Carbohydrates selectively protect a specific domain of fibronectin against proteases. 704 25

The interaction of fibronectin with native collagen during collagen fibril formation was investigated. Fibronectin prepared from serum, or from the cell surface, bound to the forming collagen fibrils while less fibronectin bound to preformed fibers. Denatured collagen competed with native collagen in binding fibronectin. Fibronectin delayed the precipitation of collagen fibrils but did not alter the total amount of fibrils formed. Fibronectin which was heated to 30 degrees C for 30 min did not promote cell adhesion but still bound to native collagen and delayed fiber formation. The collagen-binding fragment of fibronectin produced by digestion either with chymotrypsin or with neutrophil elastase had a similar effect in delaying fibril formation, but the cell-binding fragment was not active. These studies indicate that fibronectin can bind to aggregating collagen fibers probably at the same site shown previously to bind to denatured collagen. Since fibronectin inhibits the rate of collagen fibrillogenesis, it may regulate the size of collagen fibers.
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PMID:Interaction of fibronectin with collagen fibrils. 723 4

The reactivity of six monoclonal antibodies with fragments of fibronectin produced with trypsin, chymotrypsin, and Staphylococcus aureaus V8 protease is described. All these antibodies reacted with fragments derived from the C-terminal one-third of fibronectin. This region probably contains sites for the binding of fibronectin to cells, and to heparin and may also contain active sites for the reattachment, spreading, and alignment of transformed cells. Analysis of the reactivities of different sets of proteolytic fragments with the antibodies and with other ligands (eg. heparin) allows one to determine overlaps between the fragments and to locate the positions of the different binding sites for antibodies and ligands. One of the antibodies has allowed us to identify a site of structural difference between cellular and plasma fibronectins from hamsters. The site recognized by this antibody is located near to, but not at, the C-terminal end and does not involve carbohydrate groups. Because of its internal location in fibronectin, this difference suggests that there are probably different genes for cellular and plasma fibronectin. These monoclonal antibodies should be useful for further probing the functions present in the C-terminal regions of fibronectin and for determining their locations.
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PMID:Structural analysis of fibronectin with monoclonal antibodies. 732 Oct 57


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