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1.
The ectoenzyme 5'-nucleotidase purified from chicken gizzard is shown to specifically interact with laminin and fibronectin, components of the extracellular matrix, by a number of different techniques: (i) cosedimentation with laminin by sucrose gradient centrifugation; (ii) affinity adsorption to both laminin- and fibronectin-Sepharose 4-B; (iii) specific binding to both laminin and fibronectin dotted onto cellulose filters; and (iv) monoclonal antibodies against 5'-nucleotidase are shown to interfere with the interaction of 5'-nucleotidase with laminin and fibronectin. For all the techniques employed, the interactions were found to be specific, since 5'-nucleotidase did not bind to unrelated proteins such as bovine serum albumin or to monomeric actin. The interaction of purified chicken gizzard 5'-nucleotidase could be demonstrated for the hydrophobic enzyme solubilized in detergent and after its reconstitution into artificial phospholipid vesicles. The affinity adsorption experiments indicate that reconstituted enzyme binds more strongly to both laminin and fibronectin. The 5'-nucleotidase employed in this study is anchored to the plasma membrane by a glycan-phosphatidylinositol linker. After treatment with phosphatidylinositol-specific phospholipase C, the enzyme is transformed into a hydrophilic form, for which interactions with laminin and fibronectin could also be demonstrated by the dot-blot technique. Thus controlled cleavage of the phosphatidylinositol linker of 5'-nucleotidase could enable cells to rapidly alter their adhesiveness to certain components of the extracellular matrix.  相似文献   

2.
We have analysed the membrane anchorage of plasma-membrane 5'-nucleotidase, an ectoenzyme which can mediate binding to components of the extracellular matrix. We demonstrated that the purified enzyme obtained from chicken gizzard and a human pancreatic adenocarcinoma cell line were both completely transformed into a hydrophilic form by treatment with phospholipases C and D, cleaving glycosylphosphatidylinositol (GPI). These data indicate the presence of a glycolipid linker employed for membrane anchoring of the 5'-nucleotidase obtained from both sources. Incubation of plasma membranes under identical conditions revealed that about half of the AMPase activity was resistant to GPI-hydrolysing phospholipases. Investigation of the enzymic properties of purified chicken gizzard 5'-nucleotidase revealed only minor changes after removal of the phosphatidylinositol linker. However, cleavage of the membrane anchor resulted in an increased sensitivity towards inhibition by concanavalin A. After tissue fractionation, chicken gizzard 5'-nucleotidase could be obtained as either a membrane-bound or a soluble protein; the latter is suspected to be released from the plasma membrane by endogenous phospholipases. Higher-molecular-mass proteins immuno-cross-reactive with the purified chicken gizzard 5'-nucleotidase were detected as both soluble and membrane-bound forms.  相似文献   

3.
5'-Nucleotidases play an important role in the metabolism of nucleosides; for example, the hydrolysis of AMP generates adenosine, which can modulate a variety of cellular functions. We have used the membrane-bound AMPase from chicken gizzard and a secreted form of these enzymes to analyse their modification by the substrate analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). 5'-FSBA irreversibly inactivates 5'-nucleotidases by means of covalent modification of the proteins. ATP, a competitive inhibitor of chicken gizzard and snake-venom 5'-nucleotidase, abolished the inactivation by 5'-FSBA, demonstrating that the inactivation was due to the modification of amino acid residues essential for AMPase activity. We have synthesized radioactive 5'-FSBA, which was employed for the radiolabelling of chicken gizzard 5'-nucleotidase. Incorporation of radioactivity was completely abolished in the presence of ATP, which showed that 5'-FSBA acted by the selective modification of amino acid residues at the active site whereas other potential reactive residues of the protein were not attacked. Limited proteolysis of affinity-labelled chicken gizzard 5'-nucleotidase permitted the identification of digestion products containing the catalytic centre. Pseudo-first-order kinetics indicate that modification of a minimum of one amino acid side chain at the active centre is sufficient to result in inactivation of both chicken gizzard and snake-venom 5'-nucleotidases. Incorporation of the radioactive p-sulphonylbenzoyladenosine moiety parallels the inactivation of 5'-nucleotidase by 5'-FSBA and further substantiated the idea that modification of one amino acid residue at the active centre results in loss of the AMPase activity.  相似文献   

4.
The ecto-enzyme 5'-nucleotidase isolated from chicken gizzard has previously been shown to be a potent ligand of two glycoproteins of the extracellular matrix, namely fibronectin and laminin. Using immunofluorescent labeling techniques we observed that 5'-nucleotidase codistributed with laminin during the development of chicken striated muscle. In contrast, ecto-5'-nucleotidase was only faintly detectable on cells surrounded by a matrix expressing high levels of fibronectin. This distribution pattern distinguished 5'-nucleotidase from the pluripotent extracellular matrix receptors, chicken beta 1-integrins, which are expressed equally well in muscle and connective tissue. In addition, the specific activity of striated muscle ecto-5'-nucleotidase was stable during development and increased markedly posthatching. At each age considered, this specific activity corresponded to an 80-kDa enzyme which was inhibited by alpha,beta-methyleneadenosine diphosphate or by a monoclonal antibody directed against the smooth muscle isoform of the enzyme. Previous in vitro studies have revealed that 5'-nucleotidase is involved in the spreading of various mesenchyme-derived cells, such as chicken embryonic fibroblasts and myoblasts, on a laminin substrate. A prerequisite to examining a potential in vivo role for 5'-nucleotidase as an extracellular matrix ligand was to study its distribution. In adult muscle, 5'-nucleotidase displayed a more restricted distribution than in embryo. Results show that, in vivo, 5'-nucleotidase is revealed by immunofluorescent labeling using poly- and monoclonal antibodies to chicken gizzard 5'-nucleotidase in two structures, the costameres and myotendinous junctions, which are closely related to the focal adhesion sites observed in cell culture.  相似文献   

5.
Previous studies have shown that 5'-nucleotidase, an ectoenzyme from chicken gizzard, interacts specifically with laminin and fibronectin, two glycoproteins of the extracellular matrix. Recently, we demonstrated that 5'-nucleotidase was involved in the spreading of chick embryo fibroblast on laminin. In the present communication, we report that a monoclonal antibody (CG37) raised-directed against 5'-nucleotidase inhibited the spreading of chick embryo myoblasts on laminin after their initial attachment to the substrate. Furthermore, monoclonal antibody CG37 specifically eluted 5'-nucleotidase from immobilized laminin and thus enabled its isolation from other myoblast laminin-binding proteins.  相似文献   

6.
The human interleukin-2 (IL-2) receptor was quantitatively cleaved into two large disulfide-bonded fragments by either trypsin or endoproteinase lys-C (endo lys-C). The smaller fragment contains both N-linked oligosaccharides found in the intact receptor and is derived from the amino terminus of the molecule. The larger proteolytic fragment was metabolically labeled with 32PO4 and represents the carboxy terminus. The predicted cleavage sites of both enzymes lie in the region of the molecule encoded by exon 3. This pattern of limited proteolysis provides biochemical evidence that the extracellular region of the receptor is organized into two domains. This supports a structural model of the receptor in which the regions of internal homology encoded by exons 2 and 4 form independent disulfide-bonded domains connected by a hydrophilic segment. To determine the role of these domains in IL-2 binding, [125I]IL-2 was chemically cross-linked to the proteolytically cleaved receptor on the cell surface. The 125I-labeled complex obtained displayed N-linked oligosaccharides and had an Mr consistent with one molecule of IL-2 cross-linked to the smaller proteolytic fragment of the receptor. Thus, the amino-terminal domain of the IL-2 receptor appears to form an integral part of the IL-2 binding site.  相似文献   

7.
Inflammatory cells are capable of degrading extracellular matrix macromolecules in vivo in the presence of proteinase inhibitors. We and others have hypothesized that such proteolysis is permitted in large part by mechanisms operative in the immediate pericellular environment, especially at zones of contact between inflammatory cells and insoluble matrix components. To further test this hypothesis in vitro, we have used a model system in which viable polymorphonuclear neutrophils (PMN) are allowed to contact a surface coated with proteinase-sensitive substrate, and in which PMN interaction with the surface can be modulated. We have evaluated proteolysis of the surface-bound protein in the presence and absence of proteinase inhibitors. Our results were: (a) In the presence (but not in the absence) of proteinase inhibitors, proteolysis was confined to sharply marginated zones subjacent to the cells; (b) opsonization of the surface enhanced spreading of the PMN, (c) opsonization diminished the effectiveness of alpha-1-proteinase inhibitor (alpha-1-PI) and alpha-2-macroglobulin as inhibitors of proteolysis of surface-bound protein; (d) anti-oxidants did not alter the effectiveness of alpha-1-PI in inhibiting proteolysis of opsonized substrate by PMN; and (e) PMN could restrict entry of alpha-1-PI into zones of contact with opsonized surfaces. We conclude that: (a) In the presence of proteinase inhibitors, PMN can express sharply marginated and exclusively pericellular proteolytic activity; (b) locally high proteinase concentrations and/or exclusion of proteinase inhibitors from pericellular microenvironments may be important mechanisms for pericellular matrix degradation by PMN; and (c) these observations may have general relevance to extracellular matrix remodeling by a variety of inflammatory and other cell types.  相似文献   

8.
Membrane-type 1 matrix metalloproteinase (MT1-MMP), a transmembrane proteinase with a short cytoplasmic domain and an extracellular catalytic domain, controls a variety of physiological and pathological processes through the proteolytic degradation of extracellular or transmembrane proteins. MT1-MMP forms a complex on the cell membrane with its physiological protein inhibitor, tissue inhibitor of metalloproteinases-2 (TIMP-2). Here we show that, in addition to extracellular proteolysis, MT1-MMP and TIMP-2 control cell proliferation and migration through a non-proteolytic mechanism. TIMP-2 binding to MT1-MMP induces activation of ERK1/2 by a mechanism that does not require the proteolytic activity and is mediated by the cytoplasmic tail of MT1-MMP. MT1-MMP-mediated activation of ERK1/2 up-regulates cell migration and proliferation in vitro independently of extracellular matrix proteolysis. Proteolytically inactive MT1-MMP promotes tumor growth in vivo, whereas proteolytically active MT1-MMP devoid of cytoplasmic tail does not have this effect. These findings illustrate a novel role for MT1-MMP-TIMP-2 interaction, which controls cell functions by a mechanism independent of extracellular matrix degradation.  相似文献   

9.
Laminin and fibronectin, but not collagen, affect the AMPase activity of the purified transmembrane protein 5'-nucleotidase. Laminin stimulates whereas fibronectin inhibits the AMPase activity of this ectoenzyme. The AMPase-modulating effects by these components of the extracellular matrix require a preincubation period of several hours when detergent-solubilized 5'-nucleotidase is employed, they can, however, instantaneously be elicited with liposome-incorporated 5'-nucleotidase.  相似文献   

10.
The smooth muscle cells of chicken gizzard harbor the ectoenzyme 5'-nucleotidase. The purified enzyme was reconstituted into 3H-labeled proteoliposomes which were used as a model to study the association of a membrane protein with fibronectin. We demonstrated that the binding process between proteoliposomes and fibronectin has the qualities of a receptor-ligand interaction, i.e., is saturable and specific. In contrast to the association of fibronectin with integrins, the interaction with 5'-nucleotidase does not require divalent metal ions. Synthetic peptides containing the RGD-sequence or a monoclonal antibody interfering with binding of other receptors to the cell-binding domain of fibronectin did not abolish the interaction with 5'-nucleotidase. This indicates that the RGDS-sequence does not represent the major contact site for the AMPase and that the 5'-nucleotidase belongs to a separate class of fibronectin receptors with distinct properties as compared to the integrins.  相似文献   

11.
The Drosophila eggshell provides an in vivo model system for extracellular matrix assembly, in which programmed gene expression, cell migrations, extracellular protein trafficking, proteolytic processing, and cross-linking are all required to generate a multi-layered and regionally complex architecture. While abundant structural components of the eggshell are known and are being characterized, less is known about non-abundant structural, regulatory, and enzymatic components that are likely to play critical roles in eggshell assembly. We have used sensitive mass spectrometry-based analyses of fractionated eggshell matrices to validate six previously predicted eggshell proteins and to identify eleven novel components, and have characterized the expression patterns of many of their mRNAs. Among these are several putative structural or regulatory (non-enzymatic) proteins, most larger in mass than the major eggshell proteins and often showing preferential expression in follicle cells overlying specific structural features of the eggshell. Of particular note are the putative enzymes, some likely to be involved in matrix cross-linking (two yellow family members previously implicated in eggshell integrity, a heme peroxidase, and a small-molecule oxidoreductase) and others possibly involved in matrix proteolysis or adhesion (proteins related to cathepsins B and D). This work provides a framework for future molecular studies of eggshell assembly.  相似文献   

12.
Proteases play fundamentally important roles in normal physiology and disease pathology. Methods for detection of active proteolysis may greatly aid in the diagnosis of disease progression, and suggest modes of therapeutic intervention. Most assays for proteolytic potential are limited by a lack of specificity and/or quantification. We have developed a solid-phase activity assay for members of the matrix metalloproteinase (MMP) family that is specific and can be used to quantify active enzyme concentration. The assay has two principal components: a capture antibody that immobilizes the MMP without perturbing the enzyme active site, and a fluorescence resonance energy transfer substrate for monitoring proteolysis at low enzyme concentrations. The assay was standardized for MMP-1, MMP-3, MMP-13, and MMP-14. The efficiency of the assay was found to be critically dependent upon the quality of the antibodies, the use of substrates exhibiting high specific activities for the enzymes, and enzyme samples that are fresh. The assay was applied to studies of constitutive and induced MMP activity in human melanoma cells. Analysis of several melanoma cell lines, and comparison with prior studies, correlated higher constitutive MMP-13 activity with higher levels of the cell surface receptor CD44. Ligands to two different melanoma cell surface receptors (the alpha2beta1 integrin or CD44) were found to induce different proteolytic profiles, suggesting that the extracellular matrix can modulate melanoma invasion. Overall, the solid-phase MMP activity assay was found to be valuable for analysis of protease activity in cellular environments. The solid-phase assay is suitably flexible to allow studies of virtually any proteolytic enzyme for which appropriate substrates and antibodies are available.  相似文献   

13.
ARIA, or acetylcholine receptor-inducing activity, is a polypeptide that stimulates the synthesis of acetylcholine receptors in skeletal muscle. Here we demonstrate that the ability of ARIA to induce phosphorylation of its receptor in muscle is blocked by highly charged glycosaminoglycans. ARIA constructs lacking the NH2-terminal portion, containing an immunoglobulin-like domain, are fully active and are not inhibited by glycosaminoglycans. Limited proteolysis of ARIA with subtilisin blocks the glycosaminoglycan interaction by degrading this NH2-terminal portion, but preserves the active, EGF-like domain. We also show that ARIA can be released from freshly dissociated cells from embryonic chick spinal cord and cerebellum by either heparin, high salt or limited proteolysis with subtilisin, suggesting that ARIA is bound to the extracellular matrix through charged interactions. We present a model of how ARIA may be stored in extracellular matrix at developing synapses and how its release may be mediated by local proteolysis.  相似文献   

14.
Polyclonal and monoclonal antibodies raised against chicken gizzard 5'-nucleotidase were tested in adhesion assays of embryonic chicken fibroblasts (CEF) for their ability to interfere with the adhesion process of these cells on either laminin or fibronectin substrata. The initial attachment process of CEF on fibronectin and laminin substrata was not influenced by preincubating these cells with antibodies against chicken gizzard 5'-nucleotidase. However, the subsequent spreading process of these cells was found to be inhibited for at least 2 h on a laminin substratum. This effect was obtained with a polyclonal antibody as well as with one from 12 monoclonal antibodies raised against the native enzyme purified from chicken gizzard. In vitro assays demonstrated a competition of laminin and this monoclonal antibody for the binding site on purified 5'-nucleotidase. Spreading-arrested and rounded CEF do not develop prominent intracellular stress-fibers like control cells, instead they seem to concentrate their available actin in areas of presumptive initial contact with the laminin substratum.  相似文献   

15.
The endolysosomal cysteine endoprotease cathepsin L is secreted from cells in a variety of pathological conditions such as cancer and arthritis. We compared the secretome composition and extracellular proteolytic cleavage events in cell supernatants of cathepsin L-deficient and wild-type mouse embryonic fibroblasts (MEFs). Quantitative proteomic comparison of cell conditioned media indicated that cathepsin L deficiency affects, albeit in a limited manner, the abundances of extracellular matrix (ECM) components, signaling proteins, and further proteases as well as endogenous protease inhibitors. Immunodetection corroborated that cathepsin L deficiency results in decreased abundance of the ECM protein periostin and elevated abundance of matrix metalloprotease (MMP)-2. While mRNA levels of MMP-2 were not affected by cathepsin L ablation, periostin mRNA levels were reduced, potentially indicating a downstream effect. To characterize cathepsin L contribution to extracellular proteolysis, we performed terminal amine isotopic labeling of substrates (TAILS), an N-terminomic technique for the identification and quantification of native and proteolytically generated protein N-termini. TAILS identified >1500 protein N-termini. Cathepsin L deficiency predominantly reduced the magnitude of collagenous cleavage sites C-terminal to a proline residue. This contradicts cathepsin L active site specificity and indicates altered activity of further proteases as a result of cathepsin L ablation.  相似文献   

16.
The structural features of vasoactive intestinal peptide (VIP) and of its Gln16-diaminopropane derivative (VIP-DAP) in solution were investigated by limited proteolysis experiments with trypsin and thermolysin. The proteolysis of the native peptide by both proteinases takes place near the residues in positions 12 and 21/22, suggesting that these amino acids are embedded in segments more flexible than the rest of the molecule. VIP-DAP appears to be more resistant to the proteolytic attack of trypsin, indicating that the derivatization in position 16 is able to stabilize the structure of the peptide. Moreover, the analysis of the mass spectra of the proteolytic mixtures supports the evidence that the derivatization is also able to protect Met17 against oxidation. From these data it can be concluded that VIP in solution under physiological conditions is characterized by the presence of segments with secondary structure, linked together by "hinge" regions that confer flexibility to the peptide, whereas VIP-DAP is embedded in a more rigid conformation, more suitable to receptor interaction.  相似文献   

17.
Drosophila acetylcholinesterase (EC 3.1.1.7) is a 150-kDa glycoprotein anchored in plasmic membranes via a glycolipid. It is composed of two active subunits which are themselves made of two noncovalently linked polypeptides of 18 and 55 kDa resulting from the proteolysis of a single precursor of 75 kDa. Active Drosophila acetylcholinesterase can be expressed in Xenopus oocytes as an excreted protein. We have identified some of the amino acids essential in post-translational modifications of the protein by site-directed mutagenesis and expression of mutants in this system. The intersubunit disulfide bond involves cysteine at position 615. Cleavage of the 75-kDa precursor, as observed in Drosophila, originates from a hydrophilic peptide (in position 148 to 180) which does not exist in cholinesterase sequences from vertebrates. This cleavage is associated with excretion out of the cell. Drosophila acetylcholinesterase exhibits four effective sites of asparagine-linked glycosylation in positions 126, 174, 331, and 531. We show that glycosylations and dimerization protect the protein against proteolytic digestion. In contrast, none of these post-translational modifications significantly affects the activity of acetylcholinesterase or affinity for its substrate.  相似文献   

18.
Aggrecanases have been characterized as proteinases that cleave the Glu373-Ala374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0-9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology.  相似文献   

19.
Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.  相似文献   

20.
5'-Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps. It is shown that the purification scheme can be applied to 5'-nucleotidase from other sources, such as rat liver. On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa. The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data [Bailyes, E.M., Soos, M., Jackson, P., Newby, A. C., Siddle, K. & Luzio, J.P. (1984) Biochem. J. 221, 369-377). Gel filtration, using non-denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa). Antibodies raised against the enzyme purified from chicken gizzard bind only 5'-nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver. The existence of tissue specific variants of 5'-nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5'-nucleotidase. They also differ in their mode of interaction with actin. The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F-actin than the enzyme isolated from rat liver.  相似文献   

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