Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It is now recognized that bacteria bind to and colonize mucosal surfaces in a highly selective manner. After the organisms penetrate the nonspecific mechanical and cleansing forces, ligands (or adhesins) on the surface of the bacteria interact in a lock-and-key (or induced fit) fashion with complementary receptors on mucosal surfaces of the host. The adhesins are usually composed of proteins in the form of fimbriae or fibrillae and the receptors of glycolipids or glycoproteins. In group-A streptococci the adhesin, lipoteichoic acid (LTA), is anchored to a protein(s) on the surface of the bacterial cells and interacts through its lipid moiety with fibronectin molecules deposited on and bound to the epithelial cells. In an attempt to locate the region of fibronectin recognized by LTA and group-A streptococci, fibronectin was cleaved with thermolysin and the fragment mixture absorbed with Staphylococcus aureus or Streptococcus pyogenes. Staphylococci adsorbed several high molecular weight fragments as well as a 28- and a 23-kdalton fragment, whereas S. pyogenes cells absorbed only the 28-kdalton fragment completely. The adsorbtion of the fragments by S. pyogenes was blocked by LTA. Antibodies raised against a synthetic peptide copying the NH2 terminus of fibronectin reacted in a Western blot with the 28-kdalton fragment, indicating that S. pyogenes and its LTA react with the NH2-terminal region of fibronectin at a site distinct from that of S. aureus. Our findings are consistent with the idea that LTA mediates the attachment of group-A streptococci to fatty acid-binding sites of fibronectin deposited on mucosal epithelial cells.
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PMID:Bacterial adherence of group A streptococci to mucosal surfaces. 268 67

Two new monoclonal antibodies (Mabs) which reacted with canine fibronectin were produced and characterized. Data supported the conclusion that the epitope recognized by Mab 1H9A4 is within the first three Type III homology repeats of the Hep 2 domain and that the epitope for Mab 13G3B7 is within the last Type III homology repeat of fibronectin. These antibodies, along with three others, Mabs IST-2, IST-7, and IST-9, produced and characterized in the laboratories of L. Zardi of Genoa, Italy, were used to characterize canine cartilage and plasma fibronectin. In addition, cartilage explants were labeled with [35S]methionine in order to characterize newly synthesized cartilage fibronectin. The following observations were made. (i) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (NaDodSO4-PAGE) of reduced canine plasma fibronectin revealed a characteristic doublet; reduced cartilage fibronectin revealed two major bands and one minor band. The lower molecular weight band was 10 kDa less than the beta subunit of plasma fibronectin. In Western blots, this band stained with Mab 1H9A4 but failed to react with Mab 13G3B7. (ii) Western blots of thermolysin and trypsin digests of cartilage fibronectin revealed cleavage patterns which differed from those obtained from digestions of plasma fibronectin. (iii) The ED-A sequence, detected by Mab IST-9, was present in less than 2% of the cartilage fibronectins. (iv) NaDodSO4-PAGE of purified and reduced 35S-labeled fibronectin revealed two major radioactive bands and one minor radioactive band which comigrated with the fibronectin from the cartilage but not with plasma fibronectin. We concluded that like "cellular" fibronectin, the ratio of alpha-type subunits to beta subunits was greater than 4 to 1 in cartilage fibronectin compared to 1.25 to 1 for plasma fibronectin; however, cartilage fibronectin was not a cellular fibronectin by the criterion of the presence of the ED-A sequence. Another difference between plasma and cartilage fibronectin was the presence in cartilage fibronectin of a subpopulation of subunits on which the last Type III homology repeat could not be detected. Biosynthetic data were consistent with the concept that cartilage fibronectin originates from local synthesis by the chondrocyte.
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PMID:Molecular and immunologic differences in canine fibronectins from articular cartilage and plasma. 291 46

Plasma fibronectin (pFN) has been shown to mediate phagocytosis of several types of artificial particles and tissue debris by macrophages. In the present investigation some of the dynamic aspects of this receptor-mediated cellular process have been studied. Plasma fibronectin did not bind specifically to fibronectin (FN)-receptors of rat peritoneal macrophages at either 4 degrees C or 37 degrees C. On the other hand, pFN aggregated on the surface of gelatin-coated latex beads (gLtx) and 125I-labeled pFN covalently coupled to latex beads (pFN-Ltx) bound strongly to macrophages at both temperatures. Both of these particles were also internalized at 37 degrees C. Treatment of macrophages by chymotrypsin, thermolysin, or trypsin in a protein-free tissue culture medium did not affect either of the above reactions; however, pronase treatment strongly reduced both the binding and internalization of the pFN-coated particles. The pronase-treated macrophage monolayers in time regained their ability to bind and internalize pFN-gLtx when incubated in fresh tissue culture medium. Such recovery, however, did not take place when the medium contained cycloheximide. On the other hand, phagocytosis of pFN-gLtx was not affected directly by cycloheximide with untreated macrophages; this suggests that the FN-receptor recycles during sustained phagocytosis. This assumption was substantiated by the observations that some of the established lysosomotropic amines--i.e., chloroquine, dansylcadaverine, and dimethyldansylcadaverine--caused total inhibition of internalization without affecting the binding of particles to macrophages. Furthermore, chloroquine protected the FN-receptors against destruction by pronase. Together these results suggest that macrophage receptors for FN are protein, present both on the cell surface and intracellularly, and recycle between the plasma membrane and intracellular sites during phagocytosis.
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PMID:Evidence for the recycling nature of the fibronectin receptor of macrophages. 295 89

Human plasma fibronectin is composed of at least five distinct domains which we refer to as Hep-1/Fib-1, Gel, Cell, Hep-2 and Fib-2 depending on their affinity for heparin (Hep), gelatin (Gel), the cell surface (Cell) or fibrin (Fib). These domains are aligned from the NH2 to the COOH terminus in the above order and can be separated from each other by mild proteolytic digestion. We have studied the elution of fibronectin thermolysin digest from a hydroxyapatite column using a linear gradient (0.5-190 mM) of sodium phosphate buffer. The five major fibronectin domains were eluted from the hydroxyapatite chromatography column in the following order: Gel, Fib-2, Cell, Hep-1/Fib-1 and Hep-2. They were identified on the basis of their molecular mass, affinity to different macromolecules and reaction with domain-specific monoclonal antibodies. All domains except the Cell and Hep-2 domains eluted as single homogeneous peaks. The Cell domain eluted as two different peaks and the Hep-2 domain eluted as four different peaks. This is the first time that heterogeneity of these two domains has been observed. Since chromatography of a fibronectin thermolysin digest on a hydroxyapatite column provides a good separation of the five major fibronectin domains, we have elaborated a procedure in which each fibronectin domain is purified by no more than two steps; hydroxyapatite and molecular exclusion chromatography. Fractionation of fibronectin proteolytic digest on a hydroxyapatite chromatography column should be of great value in the comparative analysis of fibronectin from different sources and in the study of fibronectin heterogeneity. Its use in combination with molecular exclusion chromatography offers a simple and high-yield method for the purification of large amounts of fibronectin domains.
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PMID:Elution of fibronectin proteolytic fragments from a hydroxyapatite chromatography column. A simple procedure for the purification of fibronectin domains. 298 1

Using cultured human fetal kidney cortical cells as antigen, two monoclonal antibodies (moAbs) against human glomeruli were produced. One of these moAbs, H-4, recognized the cell surface of glomerular epithelial cells, and the other, H-13, recognized the extracellular matrix present in the mesangial area. Both also reacted with liver, H-4 recognizing antigen present on the hepatocyte, and H-13 recognizing antigen distributed along the sinusoid. Species specificity for these moAbs was examined using mouse, rat, guinea pig and rabbit glomeruli, which revealed that H-4 reacted with rat glomerular epithelial cells and H-13 stained guinea pig glomerular mesangium. In the human fetal kidney, H-13 reacted with the mesangium, glomerular and tubular basement membrane and Bowman's capsule, and H-4 with the glomerular and tubular epithelial cells. Dot immunobinding assay of fibronectin purified from glomerular culture supernatant and plasma revealed that H-13 recognized both plasma and cellular fibronectin. Immunoblot analysis of 2.0 M guanidine HCl extract after dissociation in sodium dodecyl sulfate and electrophoresis demonstrated binding of H-4 to a 125 kd polypeptide. Immunoblot analysis of thermolysin-digested fibronectin exhibited binding of H-13 to 145 kd and 110 kd fragments, but not to 38 kd-29 kd fragments. In renal biopsy specimens from patients with membranous nephropathy. H-13 stained the glomerular basement membrane (GBM), but not the mesangium, whereas anti-fibronectin antisera stained both the GBM and the mesangium. In those from patients with minimal change nephrotic syndrome (MCNS), IgA glomerulonephritis (IgAGN) and membranoproliferative glomerulonephritis (MPGN), the staining pattern with H-13 was similar to that with polyclonal anti-fibronectin antisera. These results indicate that H-4 recognizes a 125 kd polypeptide constituent of the glomerular epithelial cell membrane and that H-13 recognizes the cell binding domain of fibronectin as well as revealing structural alterations in the mesangium and GBM.
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PMID:Monoclonal antibodies to human glomerular antigens. 312 68

Fibronectin has been purified from baboon plasma by affinity chromatography on immobilized gelatin. Baboon fibronectin has subunit sizes, CNBr and thermolysin peptide patterns and an amino acid composition similar to human fibronectin. In Ouchterlony plates, baboon plasma fibronectin gives an immunological reaction of identity with human fibronectin, and a partial reaction with hamster fibronectin, using a goat antibody to human fibronectin. By radial immunodiffusion and rocket immunoelectrophoresis, using the same antibody, baboon fibronectin gives a dose response pattern similar to human fibronectin. Plasma concentrations of fibronectin in baboons are similar to those in humans. Fibronectin is thus another interesting protein that is closely related to its human counterpart and can be studied in subhuman primates using available antibodies to the human protein.
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PMID:Isolation and characterization of fibronectin from baboon plasma. 315 41

It was reported in our previous studies that the "spongy degeneration-like" changes of human tumor cells were detected by coculture with human embryonic fibroblasts in vitro. It was discovered that the degenerative changes were mediated by a factor secreted from human embryonic fibroblasts. This factor was named the tumor-degenerating factor (TDF). The present study found that fibronectin inhibited TDF activity while TDF inhibited cell attachment mediated by fibronectin. It was possible that these mutual inhibitions were due to the direct binding of the TDF molecule to the fibronectin molecule. Since it is well known that fibronectin is composed of multiple domains which differ in their biological activities, this study also attempted to clarify which domain(s) inhibit TDF activity, through the use of trypsin, thermolysin and 2-nitro-5-thiocyanobenzoic acid (NTCB). It is concluded that multiple domains of fibronectin are required for the inhibition of TDF activity.
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PMID:Mutual inhibitory activities of tumor-degenerating factor (TDF) and fibronectin. 324 45

Oleic acid binds in a saturable fashion to human plasma fibronectin (FN). Analysis of the binding indicated the presence of a high affinity binding site with nKa approximately equal to 10 uM-1. Furthermore, it was found that binding of sodium oleate to FN modulated its susceptibility to degradation by various proteinases. FN saturated with sodium oleate was hydrolysed at a higher rate by trypsin, cathepsin D, thermolysin and pancreatic elastase than native FN. In contrast, sodium oleate inhibits the activity of two human granulocyte proteinases, human leucocyte elastase (HLE) and cathepsin G on either FN or on their respective specific synthetic substrates (at concentrations ranging from 10(-6) mM to 10 mM). Cathepsin G inhibition was non-competitive and gave a Ki in the 10 uM range similar to the previously reported inhibitory constant of oleic acid toward HLE.
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PMID:Effect of sodium oleate on the hydrolysis of human plasma fibronectin by proteinases. 329 75

In this report, we present evidence to suggest that streptococci and lipoteichoic acid (LTA) interact with a fatty acid binding site located near the NH2-terminus of fibronectin. The evidence is based on the following observations. Antibodies directed against a synthetic peptide (residues 1-30 of the amino-terminus of fibronectin) reacted with the two thermolysin-generated peptides (24 and 28 kilodaltons [kDa]) that were adsorbed by and eluted from streptococci. The adsorption of the 24- and 28-kDa peptides to streptococci was inhibited by LTA. The two monoclonal antibodies that inhibited the binding of LTA to fibronectin reacted only with the 24- and 28-kDa fragments of fibronectin. Conversely, LTA, as well as lauric acid and oleic acid, blocked the binding of the same monoclonal antibodies to fibronectin. LTA had no effect on the binding of hybridoma antibodies directed against the collagen or cell-binding domain.
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PMID:Hybridoma antibodies to the lipid-binding site(s) in the amino-terminal region of fibronectin inhibits binding of streptococcal lipoteichoic acid. 329 57

It is now recognized that bacteria bind to and colonize mucosal surfaces in a highly selective manner. After the organisms penetrate the nonspecific mechanical and cleansing forces, ligands (or adhesins) on the surface of the bacteria interact in a lock-and-key (or induced-fit) fashion with complementary receptors on mucosal surfaces of the host. The adhesins are usually composed of proteins in the form of fimbriae or fibrillae and the receptors of glycolipids or glycoproteins. In group A streptococci the adhesin, lipoteichoic acid (LTA), is anchored to one or more proteins on the surface of the bacterial cells and interacts through its lipid moiety with fibronectin molecules deposited on and bound to the epithelial cells. In an attempt to locate the region of fibronectin recognized by LTA and group A streptococci, fibronectin was cleaved with thermolysin and the fragment mixture adsorbed with Staphylococcus aureus or Streptococcus pyogenes. Staphylococci adsorbed several high-molecular-weight fragments as well as a 28-kilodalton and a 23-kilodalton fragment, whereas S. pyogenes cells adsorbed only the 28-kilodalton fragment completely. The adsorption of the fragments by S. pyogenes was blocked by LTA. Antibodies raised against a synthetic peptide copying the NH2 terminus of fibronectin reacted in a western blot with the 28-kilodalton fragment; this result indicated that S. pyogenes and its LTA react with the NH2-terminal region of fibronectin at a site distinct from that at which S. aureus reacts. Our findings are consistent with the idea that LTA mediates the attachment of group A streptococci to fatty acid binding sites of fibronectin deposited on mucosal epithelial cells.
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PMID:Bacterial adherence: the attachment of group A streptococci to mucosal surfaces. 331 44


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