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 was previously reported that polystyrene substituted with the sulfonate group, PSSO3, which has anticoagulant heparin-like properties, and then coated with fibronectin supports the growth of human umbilical vein endothelial cells. On the other hand, polystyrene substituted with the amino acid sulfamide group, PSSO2-Asp, which has a higher anticoagulant activity, and then coated with fibronectin no longer supported the growth of endothelial cells. We report here that, while the affinity of fibronectin to either polymer is of the same order of magnitude, fibronectin is adsorbed onto the PSSO2-Asp polymer in a different conformation compared to the PSSO3 polymer. This was shown by a higher binding of polyclonal antifibronectin antibodies to fibronectin-coated PSSO2-Asp polymer, and by a decreased susceptibility of the coated fibronectin to proteolysis by thermolysin. This study provides evidence that a solid phase substrate with a strong heparin-like function may influence the conformation and biological properties of fibronectin.
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PMID:Conformational changes of fibronectin induced by polystyrene derivatives with a heparin-like function. 831 14

We have characterized an in vitro skin model consisting of neonatal keratinocytes and fibroblasts grown on a nylon mesh. To produce a dermal model, fibroblasts were seeded onto nylon mesh and grown for 4 weeks until a physiologic dermal-like matrix was formed. This matrix was found to consist of collagens I and III, fibronectin, and glycosaminoglycans. Keratinocytes were then seeded onto the dermal model and the co-culture was grown at the air/liquid interface. A differentiated epidermis with distinct basal, spinous, granular, and stratum corneum layers was formed. When incubated in the presence of keratinocytes, fibronectin immunofluorescence increased throughout the dermis compared to cultures incubated similarly in the absence of keratinocytes. A basement membrane zone rich in laminin, collagen IV, and heparan sulfate proteoglycan was detected. The epidermis, isolated from the co-culture by thermolysin digestion, was analyzed for differentiation markers. K1 keratin (67-kDa) and involucrin were detected by immunologic techniques. Ceramide lipids (types III and IV), thought to be important in barrier function, were detected by thin-layer chromatography. The permeability of the co-culture to a panel of compounds, including [3H]-water, was determined using Franz and side-by-side diffusion cells. The permeability coefficient for water was of the same order of magnitude as that determined for neonatal foreskin. The co-culture also showed selective permeability to a panel of compounds of differing lipid solubility. This co-culture metabolized [3H]-testosterone to a profile of metabolites similar to that of neonatal foreskin. We believe that this in vitro skin model will be useful for the study of drug permeability and metabolism.
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PMID:Characterization, barrier function, and drug metabolism of an in vitro skin model. 842 93

Divalent cation-dependent platelet adhesion to fibronectin (FN) is mediated by the integrin receptors alpha 5 beta 1 (GP Ic-IIa) and alpha IIb beta 3 (GP IIb-IIIa), which recognize the RGD (Arg-Gly-Asp) sequence in the cell-binding domain. However, FN can also support divalent cation-independent platelet adhesion. To determine which domain of FN mediates divalent cation-independent adhesion, proteolysis with thermolysin and affinity chromatography were used to isolate the cell-binding, gelatin-binding, and heparin-binding domains of FN. Unactivated and thrombin-activated platelets adhered to intact FN and the 45-Kd gelatin-binding domain in the presence of either Ca2+ or EDTA. Platelet spreading was mediated only by the 105-Kd cell-binding domain and required divalent cations. The heparin-binding domains did not support platelet adhesion. Reduction of intrachain disulfide bonds or removal of carbohydrate side chains on the gelatin-binding domain did not alter the ability to support platelet adhesion. Divalent cation-independent adhesion to the 45-Kd gelatin-binding domain was not inhibited by RGDS (Arg-Gly-Asp-Ser) synthetic peptides or monoclonal antibodies (MoAbs) directed against known platelet receptors. We conclude that platelets can adhere but not spread on the gelatin-binding domain of FN by a novel divalent cation-independent mechanism.
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PMID:Platelet interactions with fibronectin: divalent cation-independent platelet adhesion to the gelatin-binding domain of fibronectin. 846 64

Differentiation of ST-13 preadipocytes into adipocytes was inhibited almost completely by addition of rat plasma fibronectin (FN) (approximately 100 micrograms/mL), but was reversed by GRGDSP cell recognition peptide (1.5 mM) and anti-alpha 5 beta 1. On the contrary, the thermolysin digest of FN stimulated adipocyte differentiation in a dose-dependent manner, in which remarkable increases in the values of the differentiation indexes, the number of adipocytes (8-fold above the control), glycerophosphate dehydrogenase (GPD) activity (12-fold), and triacylglycerol content (5-fold), were observed by inclusion of the thermolysin digest (100 micrograms/mL). The increase in GPD activity by the thermolysin digest was inhibited remarkably (about 70% inhibition) by an antibody directed to the amino-terminal fibrin-binding (Fib 1) domain of FN and slightly (about 15%) by an antibody directed to the central cell-binding (Cell) domain, but not by anti-gelatin-binding domain and anti-carboxy-terminal fibrin-binding domain. Treatment of ST-13 cells by a purified 24K fragment (100 micrograms/mL) derived from the Fib 1 domain caused an over 20-fold augmentation of the GPD activity, accounting for a major part of the differentiation stimulatory activity of the thermolysin digest. The differentiation stimulatory effect of the 24K Fib 1 fragment was not affected by either GRGDSP peptide or anti-alpha 5 beta 1. Thus, FN can regulate adipose development of ST-13 cells by its two antipodal, inhibitory and stimulatory, activities, the latter of which is expressed only upon fragmentation. Proteolytic cleavage of FN may play an important role in controlling the action of FN on adipocyte differentiation.
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PMID:An amino-terminal fibronectin fragment stimulates the differentiation of ST-13 preadipocytes. 850 92

The lambda-toxin of Clostridium perfringens type B NCIB10691 was purified by ammonium sulfate precipitation, followed by size exclusion, anion-exchange, and hydrophobic interaction chromatography. The purified toxin had an apparent molecular mass of 36 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The toxin possessed casein-hydrolyzing activity, which was inhibited specifically by metal chelators, indicating that the toxin is a metalloprotease. The gene encoding the lambda-toxin (lam), which was shown by Southern analysis to be located on a 70-kb plasmid, was cloned into Escherichia coli cells. Nucleotide and N-terminal amino acid sequencing revealed that the lam gene encodes a 553-amino-acid protein, which is processed into a mature form, the molecular mass of which was calculated to be 35,722 Da. The deduced amino acid sequence of the mature enzyme contains an HEXXH motif characteristic of zinc metalloproteases and is homologous to other known enzymes belonging to the thermolysin family. The purified toxin degraded various biologically important substances, such as collagen, fibronectin, fibrinogen, immunoglobulin A, and the complement C3 component. It caused an increase in vascular permeability and hemorrhagic edema on injection into the dorsal skin of mice. These results suggest that the toxin contributes to the pathogenesis of histolytic infection by lambda-toxin-producing C. perfringens.
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PMID:Purification, characterization, and primary structure of Clostridium perfringens lambda-toxin, a thermolysin-like metalloprotease. 855 45

Plasma fibronectin incubated with a low concentration of SH reagent under physiological conditions without cells formed a multimer which retained the ability of heparin-binding and cell-binding but lost gelatin affinity [Sakai, K., Fujii, T., and Hayashi, T. (1994) J. Biochem. 115, 415-421]. The conformation of the multimeric fibronectin, as observed by ultraviolet circular dichroism and fluorescence spectroscopy was different from that of dimeric plasma fibronectin. Monitoring the change in ellipticity indicated that conformational change was mostly accomplished within the first 3 h of incubation with 0.5 mM dithiothreitol at 37 degrees C. In contrast, multimers became detectable after 4 h of incubation. The results indicate that the overall reaction of multimerization of plasma fibronectin consists of two steps: the initial step of conformational change of dimeric fibronectin, and the later polymerization step of the polypeptide in an altered conformation. The initial step, involving the conformational change of fibronectin, depended on temperature: it proceeded at 37 degrees C but not at 25 degrees C. In contrast the second step took place at 25 degrees C at a low, yet significant rate. Proteolytic susceptibility of the fibronectin to thermolysin or m-calpain changed within 3 h of incubation with dithiothreitol at 37 degrees C in accordance with the conformational change detected by circular dichroism. Namely, the fibronectin in an altered conformation appeared to be less susceptible to thermolysin, but more susceptible to m-calpain. The changes in enzymatic susceptibilities tended to be localized in the amino- and carboxyl-terminal regions, which are consistent with the implications from the spectroscopic analysis.
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PMID:Conformational change precedes the formation of multimeric fibronectin. 890 76

Fibronectin (FN) has a complex pattern of alternative splicing at the mRNA level. One of the alternatively spliced segments, EDA, is prominently expressed during biological processes involving substantial cell migration and proliferation, such as embryonic development, malignant transformation, and wound healing. To examine the function of the EDA segment, we overexpressed recombinant FN isoforms with or without EDA in CHO cells and compared their cell-adhesive activities using purified proteins. EDA+ FN was significantly more potent than EDA- FN in promoting cell spreading and cell migration, irrespective of the presence or absence of a second alternatively spliced segment, EDB. The cell spreading activity of EDA+ FN was not affected by antibodies recognizing the EDA segment but was abolished by antibodies against integrin alpha5 and beta1 subunits and by Gly-Arg-Gly-Asp-Ser-Pro peptide, indicating that the EDA segment enhanced the cell-adhesive activity of FN by potentiating the interaction of FN with integrin alpha5beta1. In support of this conclusion, purified integrin alpha5beta1 bound more avidly to EDA+ FN than to EDA- FN. Augmentation of integrin binding by the EDA segment was, however, observed only in the context of the intact FN molecule, since the difference in integrin-binding activity between EDA+ FN and EDA- FN was abolished after limited proteolysis with thermolysin. Consistent with this observation, binding of integrin alpha5beta1 to a recombinant FN fragment, consisting of the central cell-binding domain and the adjacent heparin-binding domain Hep2, was not affected by insertion of the EDA segment. Since the insertion of an extra type III module such as EDA into an array of repeated type III modules is expected to rotate the polypeptide up to 180 degrees at the position of the insertion, the conformation of the FN molecule may be globally altered upon insertion of the EDA segment, resulting in an increased exposure of the RGD motif in III10 module and/or local unfolding of the module. Our results suggest that alternative splicing at the EDA exon is a novel mechanism for up-regulating integrin-binding affinity of FN operating when enhanced migration and proliferation of cells are required.
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PMID:Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment. 931 47

We have previously reported that (-)-epigallocatechin gallate (EGCg) inhibited lung carcinoma cell adhesion to fibronectin (FN) and demonstrated its interaction with FN. In the present work, we studied the interaction between thermolysin fragments of FN and EGCg. An amino acid sequence analysis of the fragment bound by EGCg-agarose provided its identification as a carboxyl-terminal heparin-binding domain. Thus, the inhibition of cancer cell adhesion to FN by EGCg is not caused by its direct binding to the cell-binding domain containing an Arg-Gly-Asp-sequence.
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PMID:Interaction between the carboxyl-terminal heparin-binding domain of fibronectin and (-)-epigallocatechin gallate. 964 40

Burkholderia cenocepacia ZmpA is expressed as a preproenzyme typical of thermolysin-like proteases such as Pseudomonas aeruginosa LasB and Bacillus thermoproteolyticus thermolysin. The zmpA gene was expressed using the pPRO-EXHTa His(6) tag expression system, which incorporates a six-His tag at the N-terminal end of the protein, and recombinant ZmpA was purified using Ni-nitrilotriacetic acid affinity chromatography. Upon refolding of the recombinant His(6)-pre-pro-ZmpA (62 kDa), the fusion protein was autoproteolytically cleaved into 36-kDa (mature ZmpA) and 27-kDa peptides. Site-directed mutagenesis was employed to infer the identity of the active site residues of ZmpA and to confirm that the enzyme undergoes autoproteolytic cleavage. Oligonucleotide mutagenesis was used to replace H(465) with G(465) or A(465), E(377) with A(377) or D(377), or H(380) with P(380) or A(380). Mutagenesis of H(465), E(377), or H(380) resulted in the loss of both autocatalytic activity and proteolytic activity. ZmpA with either substitution in H(380) was not detectable in B. cenocepacia cell extracts. The activity of the recombinant ZmpA was inhibited by EDTA and 1,10 phenanthroline, indicating that it is a zinc metalloprotease. ZmpA, however, was not inhibited by phosphoramidon, a classical inhibitor of the thermolysin-like proteases. The refolded mature ZmpA enzyme was proteolytically active against various substrates including hide powder azure, type IV collagen, fibronectin, neutrophil alpha-1 proteinase inhibitor, alpha(2)-macroglobulin, and gamma interferon, suggesting that B. cenocepacia ZmpA may cause direct tissue damage to the host or damage to host tissues through a modulation of the host's immune system.
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PMID:Functional analysis of the Burkholderia cenocepacia ZmpA metalloprotease. 1596 51

Superfibronectin (sFN) is a fibronectin (FN) aggregate that is formed by mixing FN with anastellin, a fragment of the first type III domain of FN. However, the mechanism of this aggregation has not been clear. In this study, we found that anastellin co-precipitated with FN in a ratio of approximately 4:1, anastellin:FN monomer. The primary binding site for anastellin was in the segment (III)1-3, which bound three molecules of anastellin and was able to form a precipitate without the rest of the FN molecule. Anastellin binding to (III)3 caused a conformational change in that domain that exposed a cryptic thermolysin-sensitive site. An additional anastellin binds to (III)11, where it enhances thermolysin digestion of (III)11. An engineered disulfide bond in (III)3 inhibited both aggregation and protease digestion, suggesting that the stability of (III)3 is a key factor in sFN formation. We propose a three-step model for sFN formation: 1) FN-III domains spontaneously unfold and refold; 2) anastellin binds to an unfolded domain, preventing its refolding and leaving it with exposed hydrophobic surfaces and beta-sheet edges; and 3) these exposed elements bind to similar exposed elements on other molecules, leading to aggregation. The model is consistent with our observation that the kinetics of aggregation are first order, with a reaction time of 500-700 s. Similar mechanisms may contribute to the assembly of the native FN matrix.
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PMID:Domain unfolding plays a role in superfibronectin formation. 1619 31


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