Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.2.2.7 (heparinase)
1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have examined the adhesion of primary Sertoli cells to a seminiferous tubule basement membrane (STBM) preparation in vitro. The STBM isolation procedure (Watanabe, T.K., L.J. Hansen, N.K. Reddy, Y.S. Kanwar, and J.K. Reddy, 1984, Cancer Res., 44:5361-5368) yields segments of STBM that retain their histotypic form in both three-dimensional tubular geometry and ultrastructural appearance. The STBM sleeves contain two laminae: a thick, inner basal lamina that was formed in vivo between Sertoli cells and peritubular myoid cells; and a thinner, outer basal lamina that was formed between myoid cells and sinusoidal endothelial cells. Characterization by immunofluorescence and SDS PAGE revealed that the isolated STBM retained fibronectin, laminin, and putative type IV collagen among its many components. When the STBM sleeves were gently shaken with an enriched fraction of primary Sertoli cells, the Sertoli cells bound preferentially to the lumenal basal lamina at the ends of the STBM sleeves. Few Sertoli cells bound to either the outer basal lamina of the STBM sleeves or to vascular extracellular matrix material which contaminated the STBM preparation. 3T3 cells, in contrast, bound to all surfaces of the STBM sleeves. Pretreatment of the STBM sleeves with proteases, 0.1 M Na metaperiodate, 4 M guanidine HCl, or heating to 80 degrees-90 degrees C inhibited lumenal Sertoli cell binding, but binding was not inhibited by chondroitinase ABC, heparinase, hyaluronidase, or 4 M NaCl. The lumenal Sertoli cell binding occurred in the presence or absence of added soluble laminin, but not fibronectin. The addition of soluble laminin, but not fibronectin, restored random binding of Sertoli cells to trypsinized STBM sleeves. Our in vitro model system indicates that Sertoli cells recognize differences in two basal laminae produced in vivo on either side of myoid cells.
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PMID:Sertoli cell binding to isolated testicular basement membrane. 352 69

We examined the ability of fibronectin, an extracellular glycoprotein that interacts with cell surfaces and matrix components, to bind to glomerular basement membrane and the effect of diabetes on this binding. 125I-labeled fibronectin binding to rat glomerular basement membrane (GBM) was dose dependent, related to time and amount of basement membrane, and inhibited by unlabeled fibronectin but not by unrelated proteins. Binding was reduced approximately 60% when GBM was pretreated with collagenase and approximately 24% when pretreated with chondroitinase plus heparinase. Treatment with NaCl had little effect on binding, whereas reduction with beta-mercaptoethanol removed approximately 25% of the bound 125I-fibronectin. Binding to samples prepared from rats with streptozocin-induced diabetes was significantly increased compared with that observed with control preparations at all concentrations of fibronectin and of basement membrane tested. The findings provide direct evidence that fibronectin binds to GBM and that this binding, which represents a biologic function of the protein, is enhanced in diabetes.
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PMID:Fibronectin binding to glomerular basement membrane is altered in diabetes. 356 74

To study the interaction between low-density lipoprotein (LDL) and granules from rat serosal mast cells in vitro, mast cells were stimulated with the degranulating agent 48/80 to induce exocytosis of the secretory granules. Subsequent incubation of the exocytosed granules with 125I-LDL resulted in binding of the labelled LDL to the granules. When increasing amounts of agent 48/80 were added to mast-cell suspensions, a dose-dependent release of granules was observed and a parallel increase in the amount of 125I-LDL bound to granules resulted. 125I-LDL bound to a single class of high-affinity binding sites on the granules. At saturation, 105 ng of LDL were bound per microgram of granule protein. The lipoprotein binding to mast-cell granules was apolipoprotein(apo)-B + E-specific. Thus 125I-LDL binding to the granules was effectively compared for by LDL (apo-B) or by dimyristoyl phosphatidylcholine vesicles containing apo-E, but not by high-density lipoprotein (HDL3) containing apo-AI as their major protein component. Neutralization by acetylation of the positively charged amino groups of apo-B of LDL or presence of a high ionic strength in the incubation medium prevented LDL from binding to the granules, indicating the presence of ionic interactions between the positively charged amino acids of LDL and negatively charged groups of the granules. It could be demonstrated that LDL bound to the negatively charged heparin proteoglycan of the granules. Thus treatment of granules with heparinase resulted in loss of their ability to bind LDL, and substances known to bind to heparin, such as Toluidine Blue, avidin, lipoprotein lipase, fibronectin and protamine, all effectively competed with LDL for binding to the granules. The results show that LDL is efficiently bound to the heparin proteoglycan component of mast-cell granules once the mast cells are stimulated to release their granules into the extracellular space.
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PMID:Low-density-lipoprotein binding by mast-cell granules. Demonstration of binding of apolipoprotein B to heparin proteoglycan of exocytosed granules. 359 8

Previous work has demonstrated that aortic endothelial cells (EC) produce a heparin-like inhibitor of smooth muscle cell (SMC) growth when both cell types were cultured on plastic. We have now tested the influence of the extracellular matrix on this EC-SMC interaction. Specifically, we examined: 1) the role of different substrates (plastic, fibronectin, monomeric, and fibrillar collagens I and III, and EC-derived matrices) on the growth rate and population density of SMC; 2) the heparin-sensitivity of SMC on these diverse substrates; and 3) the effect of these same substrates on EC ability to secrete heparin-like and polypeptide inhibitors of SMC growth. SMC demonstrated a sixfold difference in sensitivity to heparin when grown on different substrates, with the following rank order: EGTA matrix greater than collagens = plastic = fibronectin greater than deoxycholic acid (DOC) matrix. Maximally, we found a 10-fold difference in the potency of the inhibitory activity secreted by EC grown on different substrates, with the following order: plastic = EGTA matrix greater than fibronectin greater than collagens = DOC matrix. Treatment of the conditioned mediums with heparinase and trypsin indicated that 58% to 76% of the inhibitory activity was due to heparin-like species, and 24% to 42% was due to protein(s). When EC cultured on EGTA matrix are compared to those pleated on DOC matrix, the potency of the heparin-like and peptide inhibitory activities increased 8- and 17-fold, respectively. Hypothetically, one would predict a 60-fold change in the potency of the antiproliferative effect if the contributions of substrate to EC production of inhibitors and SMC sensitivity were additive.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of vascular smooth muscle cell growth by endothelial-synthesized extracellular matrices. 367 5

Proteins with affinities for specific glycosaminoglycans (GAC's) were used as probes for testing the potential of cell surface GAG's to mediate cell adhesive responses to extracellular matrices (ECM). Plasma fibronectin (FN) and proteins that bind hyaluronate (cartilage proteo-glycan core and link proteins) or heparan sulfate (platelet factor 4 [PF4]) were adsorbed to inert substrata to evaluate attachment and spreading of several 3T3 cell lines. Cells failed to attach to hyaluronate-binding substrata. The rates of attachment on PF4 were identical to those on FN; however, PF4 stimulated formation of broad convex lamellae but not tapered cell processes fibers during the spreading response. PF4-mediated responses were blocked by treating the PF4-adsorbed substratum with heparin (but not chondroitin sulfate), or alternatively the cells with Flavobacter heparinum heparinase (but not chondroitinase ABC). Heparinase treatment did not inhibit cell attachment to FN but did inhibit spreading. Cells spread on PF4 or FN contained similar Ca2+-independent cell-substratum adhesions, as revealed by EGTA-mediated retraction of their substratum-bound processes. Microtubular networks reorganized in cells on PF4 but failed to extend into the broadly spread lamellae, where fine microfilament bundles had developed. Stress fibers, common on FN, failed to develop on PF4. These experiments indicate that (a) heparan sulfate proteoglycans are critical mediators of cell adhesion and heparan sulfate-dependent adhesion via PF4 is comparable in some, but not all, ways to FN-mediated adhesion, (b) the uncharacterized and heparan sulfate-independent "cell surface" receptor for FN permits some but not all aspects of adhesion, and (c) physiologically compatible and complete adhesion of fibroblasts requires binding of extracellular matrix FN to both the unidentified "cell surface" receptor and heparan sulfate proteoglycans.
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PMID:Cell surface heparan sulfate mediates some adhesive responses to glycosaminoglycan-binding matrices, including fibronectin. 621 15

Glomerular localization of heparan sulfate proteoglycan (HS-proteoglycan) has been studied immunohistochemically with a highly purified antiserum to bovine aorta HS-proteoglycan core protein. The specificity of the antiserum was enhanced by consecutive fibronectin and chondroitin sulfate-dermatan sulfate proteoglycan (CS-DS proteoglycan) affinity chromatography. The affinity-purified HS-proteoglycan antibody lacked cross-reactivity by enzyme-linked immunosorbent assays (ELISA) with CS-DS proteoglycan, fibronectin, laminin, and Type IV collagen. Reactivity of the antiserum with HS-proteoglycan antigen by ELISA was inhibited by HS core protein derived from CsCl density gradient centrifugation after heparinase treatment of the HS-proteoglycan. Immunofluorescent reactivity of the HS-proteoglycan antiserum was observed with bovine glomerular basement membrane, renal interstitium, Bowman's capsule, renal arterioles, and bovine aorta. No staining was seen with rat, mouse, or human glomeruli.
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PMID:Renal localization of heparan sulfate proteoglycan by immunohistochemistry. 622 57

The pericellular matrix fibers of cultured human fibroblasts contain fibronectin, other glycoproteins, and heparan and chondroitin sulfate proteoglycans. In the present study, cell-free pericellular matrices were isolated from metabolically labeled fibroblast cultures. The isolated matrices were digested with heparinase from Flavobacterium heparinum, and then analyzed for sulfated glycosaminoglycans (GAGs). Nitrous acid degradation was used to distinguish the N-sulfated GAGs (heparan sulfate) from chondroitin sulfate. Fibronectin and the other major matrix polypeptides were studied using gel electrophoresis, enzyme immunoassay and immunofluorescence. Upon heparinase digestion, greater than 95% of sulfated GAGs were degraded in the matrix without detectable release of fibronectin or other matrix polypeptides or alteration of the fibrillar matrix structure. We conclude that in fibroblast cultures the integrity of the fibrillar matrix is independent of sulfated GAGs. Together with earlier observations, this suggests that filamentous polymerization of fibronectin forms the backbone of early connective tissue matrix.
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PMID:Integrity of the pericellular fibronectin matrix of fibroblasts is independent of sulfated glycosaminoglycans. 637 Jun 87

Human angiogenin is an excellent substrate for the adhesion of HT-29 human colon adenocarcinoma cells. These cells adhere more quickly to human angiogenin than to fibronectin, laminin, collagen I, and collagen IV. Anti-angiogenin antibodies and the angiogenesis inhibitors platelet factor-4 and placental ribonuclease inhibitor prevent adhesion of HT-29 cells to angiogenin. Calcium and magnesium ions are not required for adhesion and Arg-Gly-Asp-Ser has no effect, indicating that the interaction is integrin-independent. Instead, adhesion seems to involve a heparan/chondroitin sulfate proteoglycan. Treatment of the cells with heparinase or heparitinase decreases HT-29 cell adhesion onto angiogenin but not onto collagen I. Moreover, cell adhesion is decreased by the presence of heparin or chondroitin sulfates and by preincubation of the cells with inhibitors of proteoglycan synthesis or secretion. In addition, angiogenin binds tightly to heparin-Sepharose, requiring 0.78 M NaCl for elution. Angiogenin-affinity chromatography of a 35S-, 3H-labeled HT-29 cell fraction enriched in cell-surface proteoglycans yields a single, heparinase-sensitive component of apparent molecular mass > 200 kDa, as detected by autoradiography after SDS-polyacrylamide gel electrophoresis. These results suggest that angiogenin could be an effective substrate for tumor cell adhesion during metastasis and may provide a basis for the design of inhibitors of this process.
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PMID:A cell-surface proteoglycan mediates human adenocarcinoma HT-29 cell adhesion to human angiogenin. 751 Jun 98

Vascular endothelial cell (EC) wound healing was characterized on an EC-synthesized extracellular matrix (ECM) previously treated with enzymes and antibodies specific for ECM components. Using a computer-assisted video-microscope recording system capable of automatic EC recognition, we learned whether components of the EC-synthesized matrix influenced post-injury migration and wound healing in vitro. Localization of actin and its encoded mRNA using isoform-specific antibodies and labeled cDNA probes allowed for a direct correlation of living-cell behavior with cytoskeletal form and distribution. Results of these studies indicate that the computer-assisted EC tracking system allows for an automatic and reproducible analysis of EC behavior following injury in vitro. EC migrate fastest immediately following injury and then achieve a new, slower migration rate that is maintained until EC from one edge of 200- to 300-microns-wide wound zone contact EC from the other wound face. Treatment of EC-synthesized matrices with antibodies against fibronectin and laminin has no effect on EC migration following injury (-0.25 microns/min) or on cytoskeletal array. Similarly, digestion of these matrices with heparinase and hyaluronidase has no effect on wound healing rates. Slowly spreading EC cytoplasm, which borders the intact and antibody-treated EC matrices, is rich in actin but lacks myosin II. Two different preparations of collagenase (bacterial and mammalian) each potentiate EC wound healing in vitro. Bacterial collagenase treatment of the EC-synthesized matrices potentiates EC migration fivefold (1 micron/min) while treatment of EC-matrices with mammalian cell collagenase stimulates EC migration following injury some twofold (0.4 micron/min) over control values. Whereas EC on control matrices migrate in unison as a tissue-like sheet, EC on the collagenase-treated EC matrices migrate as individuals. Concomitant with the increased rates of migration following injury on the collagenase-treated EC-matrices is a two- to fourfold increase in the steady-state levels of beta-actin mRNA. This increase in actin mRNA abundance is observable by its preferential localization (seen by in situ hybridization) in the lamellae bordering the wound edge in association with beta-actin, which is exclusively localized there. Because beta-actin and its encoded mRNA are positioned together in association with the plasma membrane in regions of moving cytoplasm, it seems likely that beta-actin filament assembly is required for motility following endothelial injury.
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PMID:Molecular mechanisms regulating the vascular endothelial cell motile response to injury. 752 70

Cytotactin/tenascin is a multidomain extracellular matrix protein that inhibits both cell spreading and intracellular alkalinization. The protein has multiple different domains which are homologous to regions in epidermal growth factor, fibronectin, and fibrinogen. In previous studies, we produced nonoverlapping fusion proteins corresponding to these domains and examined their effects on cell attachment and spreading. Based on their ability either to promote or to inhibit cell attachment, two of these fusion proteins were shown to be adhesive and two were shown to be counteradhesive. To determine how the adhesive and counteradhesive activities of different cytotactin/tenascin domains alter intracellular pH (designated pHi), we have measured pHi, in NIH3T3 and U251MG cells in the presence of the cytotactin/tenascin fusion proteins and intact cytotactin/tenascin, as well as fibronectin. Cells incubated in the presence of intact cytotactin/tenascin or of the counteradhesive fusion proteins had a pHi lower than control cells. In contrast, the presence of the adhesive fusion proteins or of fibronectin caused cells to have higher pHi values than control cells. When two fragments were simultaneously presented, one of which alone increased pHi and the other of which alone decreased pHi, the predominant effect was that of lowered pHi. Incubation with an RGD-containing peptide derived from the cytotactin/tenascin sequence inhibited alkalinization promoted by the adhesive fragment containing the second through sixth fibronectin type III repeats that was known to bind to integrins. Incubation of the cells with heparinase I or III inhibited the intracellular alkalinization of cells plated in the presence of the other adhesive fusion protein containing the fibrinogen domain, suggesting that heparan sulfate proteoglycans were involved in these pHi changes. The activity of protein kinase C appeared to be important for the changes in pHi mediated by all of the proteins. The protein kinase C inhibitor Calphostin C blocked the rise in pHi elicited by the adhesive fusion proteins and by fibronectin. Moreover, activation of protein kinase C by the addition of phorbol esters increased the pHi in cells plated on cytotactin/tenascin or counteradhesive fusion proteins and reversed their effects. The results of this study support the hypothesis that cytotactin/tenascin can bind to multiple cell surface receptors and thereby elicit different physiological responses. Decreases in pHi are correlated with the phenomenon of counteradhesion whereas the ability to increase pHi is associated with cell attachment via at least two different types of cell surface receptors. The data raise the possibility that binding of cytotactin/tenascin may influence primary cellular processes such as migration and proliferation through the differential regulation of pHi.
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PMID:Differential effects of cytotactin/tenascin fusion proteins on intracellular pH and cell morphology. 752 16


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