首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
J Takagi  H Asai  Y Saito 《Biochemistry》1992,31(36):8530-8534
Propolypeptide of von Willebrand factor (pp-vWF) binds to type I collagen, and we have reported that a binding domain exists in a 21.5/21-kDa fragment originated from the C-terminal portion [Takagi, J., Fujisawa, T., Sekiya, F., & Saito, Y. (1991) J. Biol. Chem. 266, 5575-5579]. The collagen-binding property of the 21.5/21-kDa fragment was compared with that of the intact pp-vWF. Although pp-v WF preferentially binds to native type I collagen fibrils, the isolated fragment no longer has this specificity and binds well to collagen of other types in the native and heat-denatured states. In order to determine the critical site that mediates this collagen/gelatin binding, several peptides were synthesized based on the primary structure of the 21.5/21-kDa fragment. Among these, a 25-residue peptide strongly inhibited the binding of the 125I-labeled 21.5/21-kDa fragment to collagen. Using this inhibitory effect as an index, the binding site was defined to the sequence as follows: WREPSFCALS. Furthermore, a decapeptide of this sequence bound to collagen and gelatin, indicating that this sequence is responsible for the binding of the 21.5/21-kDa fragment to collagen/gelatin.  相似文献   

2.
We compared the binding of propolypeptide and mature portions of von Willebrand factor of bovine origin to fibrillar type-I collagen obtained from bovine tendon. The propolypeptide (pp-vWF) and the mature portion (m-vWF) of human origin consist of 741 and 2050 amino acids, respectively, and are rather large proteins. The collagen-binding properties of the two proteins of bovine origin were similar in that both bound more avidly to native collagen than to heat-denatured collagen. Bindings was affected similarly by ionic strength but was not modified either by divalent cations or a synthetic peptide containing Arg-Gly-Asp. However, the binding sites in the fibrillar type-I collagen molecule for pp-vWF and m-vWF seem to be different: the two proteins did not effectively compete with each other for binding to collagen. Furthermore, pepsin treatment of fibrillar type-I collagen resulted in a drastic decrease in the binding of pp-vWF, while only a moderate decrease in the binding of m-vWF was observed after the treatment.  相似文献   

3.
Plasma fibronectin (Fn) is a constituent of cryoglobulins and has been shown to interact with immune complexes. In a previous report we demonstrated that Fn specifically bound to IgG immobilized on a solid matrix. To localize and biochemically characterize the sites on the Fn molecule involved in this interaction, Fn was enzymatically cleaved with subtilisin and subjected to IgG affinity chromatography. Three major polypeptide fragments of 16 kDa, 22 kDa, and a triplet of 26- to 29-kDa bound IgG. They were localized to three separate regions of the molecule by Western blot analysis using antisera to specific regions of the Fn molecule, by amino acid sequencing, and by their previously described heparin binding affinities. The 22-kDa fragment interacted with IgG under physiologic conditions and it is localized at the N-terminal of the Fn molecule. The 16-kDa and 26- to 29-kDa fragments bound to IgG under conditions of lower ionic strength; the former commences at residue 588, carboxyl-terminal to the collagen binding region and the latter begins at residue 1597, carboxyl-terminal to the cell binding domain. The interaction of Fn with Ig has significant implications in host defense and also in immune complex disease where basement membrane Fn may sequester immune complexes from the circulation.  相似文献   

4.
Cleavage of caldesmon with chymotrypsin yields a series of fragments which bind both calmodulin and actin and inhibit the binding of myosin subfragments to actin and the subsequent stimulation of ATPase activity. Several of these fragments have been purified by cation exchange chromatography and their amino-terminal sequences determined. The smallest fragment has a molecular mass of about 7.3 kDa and extends from Leu597 to Phe665. This polypeptide inhibits the actin-activated ATPase of myosin S-1; this inhibition is augmented by smooth muscle tropomyosin and relieved by Ca(2+)-calmodulin. The binding of the 7.3-kDa fragment to actin is competitive with the binding of S-1 to actin. Thus, this polypeptide has several of the important features characteristic of intact caldesmon. However, although an intact caldesmon molecule covers between six and nine actin monomers, the 7.3-kDa fragment binds to actin in a 1:1 complex. Comparison of this fragment with others suggests that a small region of caldesmon is responsible for at least part of the interaction with both calmodulin and actin.  相似文献   

5.
Efforts to characterize the receptor recognition domain of alpha-macroglobulins have primarily focused on human alpha 2-macroglobulin (alpha 2M). In the present work, the structure and function of the alpha-macroglobulin receptor recognition site were investigated by amino acid sequence analysis, plasma clearance, and cell binding studies using several nonhuman alpha-macroglobulins: bovine alpha 2M, rat alpha 1-macroglobulin (alpha 1M), rat alpha 1-inhibitor 3 (alpha 1I3), and proteolytic fragments derived from these proteins. Each alpha-macroglobulin bound to the murine peritoneal macrophage alpha-macroglobulin receptor with comparable affinity (Kd approximately 1 nM). A carboxyl-terminal 20-kDa fragment was isolated from each of these proteins, and this fragment bound to alpha-macroglobulin receptors with Kd values ranging from 10 to 125 nM. The amino acid identity between the homologous carboxyl-terminal 20-kDa fragments of human and bovine alpha 2M was approximately 90%, while the overall sequence homology between all carboxyl-terminal fragments studied was 75%. The interchain disulfide bond present in the human alpha 2M carboxyl-terminal 20-kDa fragment was conserved in bovine alpha 2M and rat alpha 1I3, but not in rat alpha 1M. The clearance of each intact alpha-macroglobulin-proteinase complex was significantly retarded following treatment with cis-dichlorodiammineplatinum(II) (cis-DDP). cis-DDP treatment, however, did not affect receptor recognition of purified carboxyl-terminal 20-kDa fragments of these alpha-macroglobulins. A carboxyl-terminal 40-kDa subunit, which can be isolated from rat alpha 1M, bound to the murine alpha-macroglobulin receptor with a Kd of 5 nM.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

6.
We have used purified proteolytic fragments of von Willebrand factor (vWF) to characterize three related functional sites of the molecule that support interaction with platelet glycoprotein Ib, collagen, and heparin. A fragment of 116 kDa was found to be dimeric and consisted of disulfide-linked subunits which, after reduction and alkylation, corresponded to the previously described 52/48-kDa fragment extending from residue 449 to 728. Fragment III-T2, also a dimer, was composed of two pairs of disulfide-linked subunits, two 35-kDa heavy chains (residues 273-511) and two 10-kDa light chains (residues 674-728). The 116-kDa fragment, but not the constituent 52/48-kDa subunit, supported ristocetin-induced platelet aggregation and retained 20% (on a molar basis) of the ristocetin cofactor activity of native vWF; fragment III-T2 retained less than 5% activity. All three fragments, however, inhibited vWF interaction with glycoprotein Ib. Both 116-kDa and 52/48-kDa fragments inhibited vWF binding to heparin with similar potency, while fragment III-T2 had no effect in this regard. Only the 116-kDa fragment inhibited vWF binding to collagen. These results indicate that dimeric fragments containing two glycoprotein Ib-binding sites possess the minimal valency sufficient to support ristocetin-induced aggregation. The sequence comprising residues 512-673, missing in fragment III-T2, is necessary for binding to heparin and collagen and may be crucial for anchoring vWF to the subendothelium. Immunochemical and functional data suggest that the same sequence, although not essential for interaction with glycoprotein Ib, may influence the activity of the glycoprotein Ib-binding site. Only binding to collagen has absolute requirement for intact disulfide bonds. Thus, the three functional sites contained in the 116-kDa domain of vWF are structurally distinct.  相似文献   

7.
A single type-II domain has been isolated by limited proteolysis of the collagen-binding bovine seminal fluid protein, PDC-109. The 45-residue fragment corresponding to the second type-II domain of the parent molecule was found to have retained affinity for immobilized collagen, indicating that this minidomain carries critical regions of the collagen-binding site. Studies on various fragments of fibronectin have also implicated the two type-II units of this molecule in collagen-binding. In the present work we have found that type-II domains of human fibronectin, expressed in Escherichia coli as beta-galactosidase fusion proteins, bind specifically to immobilized collagen.  相似文献   

8.
Previous studies from this laboratory have utilized latex beads as probes of embryonic migratory pathways. After microinjection into embryos at the time of neural crest migration, uncoated latex polystyrene beads were found to translocate to ventral sites and to settle in the vicinity of endogenous neural crest derivatives. However, latex beads coated with fibronectin did not translocate ventrally, but remained associated with cells surrounding the implantation site. Fibronectin is a large glycoprotein with a variety of biological activities and multiple binding domains. Here, the binding activities which might be responsible for immobilization of the fibronectin-coated beads are examined. Latex beads were coated with three types of fragments of the fibronectin molecule representing different functional domains: (i) a 66-kDa fragment containing collagen-binding activity; (ii) a mixture of 45- and 32-kDa fragments containing heparin-binding activity; and (iii) a 120-kDa fragment containing cell-binding activity. The beads coated with fibronectin fragments were injected into the newly formed trunk somites of avian embryos. After injection, beads coated with either the heparin- or the collagen-binding domain translocated ventrally and distributed analogously to uncoated latex beads. In contrast, the majority of beads coated with the fibronectin cell-binding domain did not translocate but remained associated with dermamyotomal cells surrounding the injection site. The cell-binding fragment, however, was not as effective as the intact fibronectin molecule in preventing translocation of the beads. The results suggest that the cell-binding domain is primarily responsible for restriction of fibronectin beads from the ventral neural crest pathway. Because intact fibronectin is more effective at immobilizing beads than is the cell-binding fragment, other binding domains of fibronectin, more efficient coating with intact fibronectin, or crosslinking of intact fibronectin molecules may also play some role in immobilization of the beads at the implantation site.  相似文献   

9.
The epitope of monoclonal antibody (mAb 4A), which recognizes the alpha subunit of the rod G protein, Gt, has been suggested to be both at the carboxyl terminus (Deretic, D., and Hamm, H.E. (1987) J. Biol. Chem. 262, 10839-10847) and the amino terminus (Navon, S.E., and Fung, B.K.-K. (1988) J. Biol. Chem. 263, 489-496) of the molecule. To characterize further the mAb 4A binding site on alpha t and to resolve the discrepancy between these results limited proteolytic digestion of Gt or alpha t using four proteases with different substrate specificities has been performed. Endoproteinase Arg-C, which cleaves the peptide bond at the carboxylic side of arginine residues, cleaved the majority of alpha t into two fragments of 34 and 5 kDa. The alpha t 34-kDa fragment in the holoprotein, but not alpha t-guanosine 5'-O-(3-thiotriphosphate), was converted further to a 23-kDa fragment. A small fraction of alpha t-GDP was cleaved into 23- and 15-kDa fragments. Endoproteinase Lys-C, which selectively cleaves at lysine residues, progressively removed 17 and then 8 residues from the amino terminus, forming 38- and 36-kDa fragments. Staphylococcus aureus V8 protease is known to remove 21 amino acid residues from the amino-terminal region of alpha t, with the formation of a 38-kDa fragment. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin cleaved alpha t progressively into fragments of known amino acid sequences (38, then 32 and 5, then 21 and 12 kDa) and a transient 34 kDa fragment. The binding of mAb 4A to proteolytic fragments was analyzed by Western blot and immunoprecipitation. The major fragments recognized by mAb 4A on Western blots were the 34- and 23-kDa fragments obtained by endoproteinase Arg-C and tryptic digestion. Under conditions that allowed sequencing of the 15- and 5-kDa fragments neither the 34- nor the 23-kDa fragments could be sequenced by Edman degradation, indicating that they contained a blocked amino terminus. The smallest fragment that retained mAb 4A binding was the 23-kDa fragment containing Met1 to Arg204. Thus the main portion of the mAb 4A antigenic site was located within this fragment, indicating that the carboxyl-terminal residues from Lys205 to Phe350 were not required for recognition by the antibody. Additionally, the antibody did not bind the 38- and 36-kDa or other fragments containing the carboxyl terminus, showing that the amino-terminal residues from Met1 to Lys17 were essential for antibody binding to alpha t.  相似文献   

10.
Mechanism of fibronectin (FN)-induced chemotaxis of fibroblastic cells has not been fully understood. The present study was performed to establish a molecular nature of the chemotactic region of rat plasma FN. The chemotactic dose-response pattern of intact FN for mouse embryo fibroblastic cells, NIH-L13 cells, which was represented as a "bell-shape" curve with a maximum activity at around 50 nM, changed to a "biphasic" mode through a proteolysis with thermolysin. Two distinct chemotactic components were isolated from the thermolytic fragments. One component, a fragment with a molecular mass of 110-150 kDa, was estimated to contain the central cell-binding domain and the carboxyl-terminal heparin-binding domain of the intact FN molecule. Cell migration stimulated by the 110-150-kDa fragment increased successively in a dose-dependent manner, and the capability to promote the migration was much higher than that of the intact FN (over 2-fold). The second chemotactic component, a fragment with a molecular mass of 21 kDa, was shown to reside in the carboxyl-terminal fibrin-binding domain. The 21-kDa fragment produced a bell-shape dose-response pattern, being consistent with the intact FN, whereas a maximum response occurred at a 100-fold lower concentration (0.5 nM) than that of the intact FN molecule. At higher concentrations, this fragment revealed an inhibitory activity for the cell migration in response to the 110-150-kDa fragment. No significant molecular interaction between these two active components was observed by polyacrylamide gel electrophoresis under nondenaturing conditions, suggesting that the 21-kDa fragment may act directly on the cell to inhibit the cell migration. These results suggest that rat plasma FN contains at least two chemotactically active components that regulate cooperatively chemotactic migration of fibroblastic cells.  相似文献   

11.
Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin.  相似文献   

12.
Human plasma fibronectin aggregates in solution and is thought to form fibrils on cell surfaces, perhaps by self-associating and by interacting with other components such as proteoglycans. We have localized the self-association domains by testing the ability of various fragments of fibronectin to interact with each other. Complexation between fluorescamine-labeled fragments and unlabeled fragments or whole molecules was assessed by gel filtration high-performance liquid chromatography. The fragments studied included nonoverlapping fragments that are situated on the fibronectin polypeptide chain in the following order, beginning from the amino terminus: the 29-, 50-, 120-, 35-, and 25-kDa fragments, as well as multiple-domain fragments of 72 kDa containing the 29- and 50-kDa segments, a fragment of 150 kDa containing the 120- and 35-kDa segment, a fragment of 190 kDa containing the 120- and 35-kDa segments, a fragment of 190 kDa containing the 50-, 150-, and 25-kDa segments, and a 45-kDa fragment containing the 35-kDa segment. The amino-terminal 29-kDa fragment bound to the carboxyl-terminal heparin-binding (Hep II) 35-kDa fragment as well as the 150- and 190-kDa fragments that contain the 35-kDa segment. On the other hand, carboxyl-terminal 35- and 45-kDa Hep II containing fragments bound to each other as well as to amino-terminal 29- and 72-kDa fragments and to the 190-kDa fragment. Further, the 25-kDa carboxyl-terminal fibrin-binding fragment bound the 190-kDa fragment, the only fragment containing the 25-kDa segment.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

13.
Alternative model for the internal structure of laminin   总被引:4,自引:0,他引:4  
S L Palm  J B McCarthy  L T Furcht 《Biochemistry》1985,24(26):7753-7760
A monoclonal antibody to laminin, LMN-1, was generated by immunizing rats with laminin from the EHS tumor and fusing the rat spleen cells with mouse NS-1 myeloma cells. Laminin fragments were generated by proteolytic digestion with thrombin, thermolysin, and chymotrypsin. Monoclonal antibody binding fragments were identified by immunoblotting. Fragments which bound monoclonal antibody LMN-1 included a 440-kilodalton (kDa) chymotrypsin fragment and thermolysin fragments of 440 and 110 kDa. These fragments could also be generated from within a 600-kDa thrombin fragment. Digestion of the 440-kDa chymotrypsin fragment with thermolysin generated the 110-kDa antibody binding fragment and a 330-kDa nonbinding fragment. Immunoblotting was performed on extracts of PYS-2 cells and EHS cells using polyclonal and monoclonal antibodies to laminin. Polyclonal antibodies stained the intact 850-kDa complex and the 200- and 400-kDa subunits, while monoclonal LMN-1 stained only the 400-kDa subunit and the complete molecule. Rotary shadowing of monoclonal LMN-1 bound to laminin molecules indicated that the binding site was within the long arm of laminin. Changes in the model of the internal organization of the laminin molecule are proposed, based on the binding of LMN-1 to the 400-kDa subunit and specific proteolytic fragments. The locations of the major thrombin and chymotrypsin fragments in the model are rotated 180 degrees relative to the previously described model [Ott, U., Odermatt, E., Engel, J., Furthmayr, H., & Timpl, R. (1982) Eur. J. Biochem. 123, 63-72] to include part of the 400-kDa subunit of laminin.  相似文献   

14.
Fibronectin receptors on mononuclear phagocytes are involved in the localization of monocytes at inflammatory sites and in the subsequent expression of macrophage-like phenotypes. In this study, we have investigated the hypothesis that proteolytically derived fragments of fibronectin may interfere with binding of fibronectin to monocytes in the extracellular matrix. We report on the reactivity of U937 cells with an 80-kDa tryptic fragment of fibronectin which contains the cell-binding domain but lacks the gelatin/collagen-binding domain. U937 cells attached to surfaces coated with the 80-kDa fragment as well as with intact fibronectin. Preincubation of the cells with the 80-kDa fragment inhibited attachment to both surfaces while intact fibronectin had little or no inhibitory effect. The Ki for inhibition of attachment (0.5 microM) was consistent with the Kd for binding of the 3H-labeled 80-kDa fragment (0.34 microM) to U937 cells in suspension. There were 4-5 x 10(5) 80-kDa binding sites per cell. The relatively high affinity of the 80-kDa fragment for the monocyte surface permitted the isolation and characterization of fibronectin-binding proteins from U937 cells and peripheral blood monocytes by affinity chromatography. When octylglucoside lysates of lactoperoxidase iodinated cells were applied to 80-kDa-Sepharose columns, a polypeptide complex of 152/125 kDa was eluted with the synthetic peptide GRGDSPC, but not with GRGESP. This complex resolved into a single diffuse band of 144 kDa upon reduction. Binding of the protein complex to the affinity column required divalent cations. The complex bound to wheat germ agglutinin and could be specifically eluted by N-acetylglucosamine. Similar cell-surface proteins were isolated from peripheral blood monocytes.  相似文献   

15.
The ATPase core of a clathrin uncoating protein   总被引:33,自引:0,他引:33  
Chymotryptic digestion of bovine brain uncoating ATPase produced a 60-kDa fragment that was subsequently proteolyzed to 44 kDa. Loss of clathrin cage uncoating activity paralleled the conversion of the intact 70-kDa enzyme to the 60-kDa fragment, while clathrin binding activity was lost as the 60-kDa fragment was degraded to 44 kDa. This 44-kDa fragment has been purified to homogeneity and characterized as a clathrin-independent ATPase. The 44-kDa ATPase domain has been localized within the intact enzyme by the use of amino-terminal specific antibodies. This localization relates to the conserved nature of the 70-kDa heat shock protein family, of which bovine brain uncoating ATPase is a constitutively expressed member.  相似文献   

16.
The relations between surface hydrophobicities and binding properties of the functional domains of porcine plasma fibronectin were investigated. Porcine plasma fibronectin as well as human plasma fibronectin was adsorbed on a hydrophobic column with butyl or phenyl ligands in the presence of 0.5 M ammonium sulfate, and recovered in a single peak by decreasing the concentration of ammonium sulfate to 0 M, indicating that both fibronectins have very high surface hydrophobicities. On digestion with thermolysin, porcine plasma fibronectin yielded five fragments (140-150, 43, 25, 17, and 14 kDa) similar to those reported for human fibronectin, although porcine fibronectin was more resistant to the digestion than human fibronectin. The three heparin-binding fragments were found to have a wide range of surface hydrophobicities, the 140-150 kDa fragment having the lowest, the 25 kDa fragment a higher, and the 14 kDa fragment the highest among all the fragments. The 43 kDa collagen-binding and 17 kDa fragments had surface hydrophobicities as high as that of fibronectin. It is noteworthy that the 43 kDa collagen-binding fragment contributes to the high surface hydrophobicity of intact fibronectin in spite of the high content of carbohydrates.  相似文献   

17.
We assessed the participation of the three known heparin-binding domains of PFn (Hep I, Hep II, Hep III) in their interaction with heparin by making a quantitative comparison of the fluid-phase heparin affinities of PFn and PFn fragments under physiologic pH and ionic strength conditions. Using a fluorescence polarization binding assay that employed a PFn affinity-purified fluorescein-labeled heparin preparation, we found that greater than 98% of the total PFn heparin-binding sites exhibit a Kd in the 118-217 nM range. We also identified a minor (less than 2%) class of binding sites exhibiting very high affinity (Kd approximately 1 nM) in PFn and the carboxyl-terminal 190/170 and 150/136 kDa PFn fragments. This latter activity probably reflects multivalent inter- or intramolecular heparin-binding activity. Amino-terminal PFn fragments containing Hep I (72 and 29 kDa) exhibited low affinity for heparin under physiologic buffer conditions (Kd approximately 30,000 mM). PFn fragments (190/170 and 150/136 kDa) containing both the carboxyl-terminal Hep II and central Hep III domains retained most of the heparin-binding activity of native PFn (Kd = 278-492 nM). The isolated Hep II domain (33-kDa fragment) exhibited appreciable, but somewhat lower (2-5-fold), heparin affinity compared to the 190/170-kDa PFn fragment. Heparin binding to the 100-kDa PFn fragment containing Hep III was barely detectable (Kd greater than 30,000 nM). From these observations, we conclude that PFn contains only one major functional heparin-binding site per subunit, Hep II, that dominates the interaction between heparin and PFn.  相似文献   

18.
von Willebrand factor binds to fibrillar type I collagen in a rapid, temperature-independent, reversible, specific, and saturable manner. Evaluation of binding isotherms by Scatchard-type analysis demonstrated that 6-18 micrograms of von Willebrand factor bind per mg of collagen, with Ka between 2 and 8 X 10(8) M-1. Five distinct tryptic fragments, purified under denaturing and reducing conditions and representing over 75% of the molecular mass of the von Willebrand factor subunit, were tested for their capacity to inhibit the von Willebrand factor-collagen interaction. Complete inhibition was obtained with a 52/48-kDa fragment at a concentration of approximately 1 microM. The location of this fragment in the subunit was established to be between Val-449 and Lys-728. Fifteen monoclonal antibodies against the 52/48-kDa fragment inhibited von Willebrand factor binding to collagen. Six antibodies against other portions of the von Willebrand factor subunit had no inhibitory effect. The tryptic fragment was a competitive inhibitor of von Willebrand factor binding to collagen and, therefore, recognizes the same interaction site as the intact molecule. These studies precisely define a domain in the von Willebrand factor subunit that interacts with type I collagen.  相似文献   

19.
The complete amino acid sequence of a DNA- and heparin-binding domain isolated by limited thermolysin digestion of human plasma fibronectin has been obtained. The domain contains 90 amino acids with a calculated molecular weight of 10,225. The apparent molecular mass of this domain is 14 kDa when analyzed by sodium dodecyl sulfate-gel electrophoresis. The anomalously high molecular size estimation may be due to the inaccuracy of this method in the low range. The structure was established from microsequence analysis of the chymotryptic, tryptic, and Staphylococcus aureus protease peptides. The molecular ion of each of the chymotryptic peptides was obtained by fast atom bombardment mass spectrometry. The domain has a preponderance of basic residues with a net charge of +5 at neutral pH. The basic nature of the domain may account for its affinity for the polyanions, DNA and heparin. The predicted secondary structure is beta-sheet, in common with all of the type III internal sequence homology structures obtained for fibronectin so far. The location of the domain in fibronectin was made possible by limited thermolysin digestion and identification of the fragments and by comparison of the sequence of the 14-kDa fragment with the partial structure of bovine plasma fibronectin. The domain comprises residues 585-675 and defines a region immediately adjacent to the collagen-binding domain. Numbering domains beginning at the amino terminus, this domain is Domain III after the fibrin/heparin/actin/S. aureus binding Domain I and the collagen-binding Domain II. The domain was obtained from a larger precursor (56 kDa) which bound heparin, DNA, and gelatin. Further digestion of the 56-kDa fragment gave rise to a 40-kDa fragment which only bound gelatin, and a 14-kDa fragment which only bound heparin or DNA. The 14-kDa fragment (Domain III) marks the beginning of the type III homology region in fibronectin, for there may be up to 15 repeats of 90 amino acids. The size of this domain corresponds to one repeat of 90 amino acids and it has some sequence homology to the other type III sequences found thus far in fibronectin.  相似文献   

20.
The membrane-bound form and a solubilized and purified form of the Ca2+-ATPase from human erythrocyte have been proteolyzed under controlled conditions by highly purified Ca2+-dependent neutral cysteine-protease, calpain I, in the absence and in the presence of the calmodulin-calcium complex. In the absence of calmodulin the 136-kDa enzyme was transformed into a group of fragments of 125-124 kDa, followed by the slower formation of a second group of fragments of 82-80 kDa. These heterogeneous fragments were capable of forming an acylphosphate intermediate. The 125- and 82-kDa minor components of each heterogeneous group of fragments (125-124 and 82-80 kDa) were capable of binding calmodulin, whereas the 124- and the 80-kDa major components did not. In the presence of calmodulin, however, the native enzyme was transformed into a 127-kDa fragment followed by the slower formation of an 85-kDa fragment. Both fragments (127 and 85 kDa) formed an acylphosphate intermediate and were capable of binding calmodulin. The presence of calmodulin during calpain action effectively protected the Ca2+-ATPase from proteolytic activation (K.K.W. Wang, A. Villalobo, and B.D. Roufogalis (1988) Arch. Biochem. Biophys. 260, 696-704) and prevented the formation of the calmodulin-insensitive 124- and 80-kDa fragments. Smaller fragments not capable of forming the acylphosphate intermediate were also produced, in particular a 39-37 kDa doublet band retaining the capacity to bind calmodulin. In contrast to the membrane-bound form, the purified form of the Ca2+-ATPase was proteolyzed by calpain at a slower rate.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号