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)

1. Experiments were performed to determine whether the specific collagenases and other metal proteinases are bound and inhibited by alpha(2)-macroglobulin, as are endopeptidases of other classes. 2. A specific collagenase from rabbit synovial cells was inhibited by human serum. The inhibition could be attributed entirely to alpha(2)-macroglobulin; alpha(1)-trypsin inhibitor was not inhibitory. alpha(2)-Macroglobulin presaturated with trypsin or cathepsin B1 did not inhibit collagenase, and pretreatment of alpha(2)-macroglobulin with collagenase prevented subsequent reaction with trypsin. The binding of collagenase by alpha(2)-macroglobulin was not reversible in gel chromatography. 3. The collagenolytic activity of several rheumatoid synovial fluids was completely inhibited by incubation of the fluids with alpha(2)-macroglobulin. 4. The collagenase of human polymorphonuclear-leucocyte granules showed time-dependent inhibition by alpha(2)-macroglobulin. 5. The collagenolytic metal proteinase of Crotalus atrox venom was inhibited by alpha(2)-macroglobulin. 6. The collagenase of Clostridium histolyticum was bound by alpha(2)-macroglobulin, and inhibited more strongly with respect to collagen than with respect to a peptide substrate. 7. Thermolysin, the metal proteinase of Bacillus thermoproteolyticus, was bound and inhibited by alpha(2)-macroglobulin. 8. It was shown by polyacrylamidegel electrophoresis of reduced alpha(2)-macroglobulin in the presence of sodium dodecyl sulphate that synovial-cell collagenase, clostridial collagenase and thermolysin cleave the quarter subunit of alpha(2)-macroglobulin near its mid-point, as do serine proteinases. 9. The results are discussed in relation to previous work, and it is concluded that the characteristics of interaction of the metal proteinases with alpha(2)-macroglobulin are the same as those of other proteinases.
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PMID:The interaction of alpha2-macroglobulin with proteinases. Binding and inhibition of mammalian collagenases and other metal proteinases. 437 31

A study of the influence of chemical modifications on the activity of Achromobacter iophagus collagenase (EC 3.4.24.8) has led to the following conclusions: a modification of 4 out of 80 COOH groups with carbodiimide led to 90% loss of enzymic activity. A 70% inactivation was found after modification of two tyrosines out of 30 with tetranitromethane. The modification of four to six tryptophans out of 16 with 2-hydroxy-5-nitrobenzyl bromide decreased enzyme activity to 36%. This inactivation is accelerated in the presence of collagen. An increase of reagent/enzyme molar ratio led to a modification of 16 tryptophan residues and denaturation of Acahromobacter collagenase. A modification of two arginines out of 18 with 1,2-cyclohexanedione and eight NH2 groups out of 24 with 2,3-dimethyl maleic anhydride does not change the collagenolytic activity. All NH2 groups become available for 2,3-dimethyl maleic anhydride after dissociation of the dimer. A possible analogy of hydrolytic site of collagenase with that of two other known bacterial metalloproteinases (thermolysin and Bacillus subtilis neutral proteinase (EC 3.4.24.4)) is discussed.
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PMID:Chemical modifications of Achromobacter collagenase and their influence on the enzymic activity. 625 92

The peptide alpha 1(III)-CB9 was prepared and purified from human liver, and its amino acid sequence was determined. Automated Edman degradation of the intact peptide and peptides derived from selective cleavage with hydroxylamine and digestions with trypsin, thermolysin, and Staph V8 protease enabled determination of the complete amino acid sequence. The peptide alpha 1(III)-CB9 represents the COOH terminus of the helical (pepsin-resistant) portion of type III collagen and terminates in a Cys-Cys sequence responsible for the intramolecular disulfide cross-linkages with other chains. The present work completes the entire amino acid sequence of the helical (pepsin-resistant) portion of human cirrhotic liver type III collagen consisting of peptides alpha 1-(III)-CB3-7-6-1-8-10-2-4-5-9. The COOH terminus of human liver alpha 1(III) contained two additional triplets which, together with the extra triplet at the NH2 terminus in alpha 1(III)-CB3, make the helical portion of type III collagen longer than alpha 1(I) by nine residues (three Gly-X-Y triplets). The helical region of human liver type III collagen, therefore, consists of 1023 amino acids or 341 triplets.
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PMID:Covalent structure of collagen: amino acid sequence of alpha 1(III)-CB9 from type III collagen of human liver. 701 80

To determine how the carbohydrate moiety of fibronectin influences the susceptibility of protein to proteolytic degradation, we compared the effects of various proteases on glycosylated and nonglycosylated fibronectins. Nonglycosylated fibronectin, from tunicamycin-treated chicken embryo fibroblasts, was degraded more rapidly to acid-soluble products than glycosylated fibronectin by pronase, thermolysin, trypsin, and chymotrypsin. The absence of carbohydrate did not markedly affect overall patterns of proteolytic fragments identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Except for the expected increases in electrophoretic mobilities of the nonglycosylated peptides, the only important difference was that of the nonglycosylated fragment corresponding to the carbohydrate-rich, collagen-binding domain, was completely digested by the proteases in 60 min at 30 degrees C. In contrast, the comparable fragment from glycosylated fibronectin was resistant to protease digestion. Heparin-binding domains that normally lack carbohydrate are equally susceptible to proteases in glycosylated and nonglycosylated fibronectin. We conclude that the carbohydrate component of fibronectin plays an important role in the stabilization of a specific domain of the protein against proteolytic degradation; however, the carbohydrate does not alter overall proteolytic specificity.
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PMID:Carbohydrates selectively protect a specific domain of fibronectin against proteases. 704 25

In rheumatoid and osteoarthritis, degradation of articular cartilage is mediated by the matrix metalloproteinases collagenase, stromelysin and gelatinase. The key event in this process is the cleavage of triple helical collagen by collagenase. We have determined the crystal structure of the catalytic domain of human recombinant fibroblast collagenase complexed with a synthetic inhibitor at 2.2 A resolution. The protein fold is similar to the amino termini of the zinc endopeptidases astacin thermolysin and elastase despite a lack of primary sequence homology. The conformation of the bound inhibitor provides a molecular basis for the design of inhibitors of collagenase and other matrix metalloproteinases. Such inhibitors should be useful in the treatment of a variety of diseases including arthritis and cancer.
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PMID:Structure of the catalytic domain of human fibroblast collagenase complexed with an inhibitor. 765 13

The basement membrane zone is important for graft adhesion and stability. The aim of the present study was to visualize the regeneration of the basement membrane and determine the sequential appearance of its constituents in the early postgrafting period of cultured human epidermal sheets. A keratinocyte single cell suspension, devoid of dermal fibroblast contamination, was obtained from human skin by a two-step tissue digestion method with thermolysin and trypsin. After culturing, epidermal sheets were generated, detached enzymatically by incubating with thermolysin (for 20-30 min) or Dispase (for 45-60 min), and deposited on a muscular graft bed of athymic mice. Immunohistochemistry and ultrastructural analyses were performed on biopsies harvested 2, 4 and 21 days postgrafting. Bullous pemphigoid antigens and laminin were detected at the dermo-epidermal junction, showing an almost continuous line 2 days postgrafting. Type IV collagen was generally absent at this time, but it was detected 4 days postgrafting. Type VII collagen was labelled as a discontinuous line of increasing intensity from 2 to 21 days postgrafting. Ultrastructural analysis revealed hemidesmosomes and a discontinuous lamina densa 2 days postgrafting, and a complete basement membrane with a continuous lamina densa, hemidesmosomes and anchoring fibrils 21 days postgrafting. The sequence of appearance of major basement membrane components was similar for cultured sheets detached with thermolysin or Dispase. However, it differed from that of other wound healing models. Results are discussed in terms of the variable keratinocyte migration requirement between various wound healing models.
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PMID:Early basement membrane formation following the grafting of cultured epidermal sheets detached with thermolysin or Dispase. 779 97

Elevated levels of lipoprotein(a), which consists of apolipoprotein(a) [apo(a)] covalently linked to a low-density lipoprotein-like moiety, is an independent risk factor for the development of atherosclerosis. We show that a recombinant form of apo(a) [r-apo(a)] binds strongly to fibronectin and fibrinogen, weakly to laminin, and not at all to von Willebrand factor, vitronectin, or collagen type IV. In contrast to the binding of plasminogen to fibronectin, r-apo(a) binding does not appear to be mediated by lysine-dependent interactions, based on the inability of epsilon-aminocaproic acid concentrations up to 0.2 mol/L to significantly decrease r-apo(a) binding to fibronectin. Plasminogen competed weakly for the binding of r-apo(a) to fibronectin, whereas r-apo(a) completely abolished plasminogen binding. The 29- and 38-kd heparin-binding thermolysin fragments of fibronectin, previously identified as the lipoprotein(a) binding domains, were digested with trypsin, and a peptide that retained the ability to bind r-apo(a) was isolated; the sequence of the peptide (AVTTIPAPTDLK) corresponds to the amino terminus of the 29- and 38-kd domains. A synthetic peptide with this sequence was able to compete effectively with fibronectin for r-apo(a) binding.
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PMID:Binding of recombinant apolipoprotein(a) to extracellular matrix proteins. 794 5

Flavobacterium meningosepticum, Elder strain (ATCC 33958), secretes into the medium a neutral zinc endoprotease as a major component of the extracellular proteins. The enzyme was purified to homogeneity in a simple two-step procedure involving ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular weight of this metalloprotease was determined to be about 27,000 (P27) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. P27 was comparable to thermolysin in the relative rates of elastin-orcein, azocasein, and azoalbumin hydrolysis. P27 and thermolysin hydrolyzed equally well 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the same primary sites that are susceptible to cleavage by vertebrate collagenases, Gly-Ile, and Gly-Leu. P27 was also capable of partially hydrolyzing Type I acid-soluble calf skin collagen and slowly hydrolyzing N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala, a bacterial collagenase substrate not cleaved by thermolysin. P27 was further differentiated from thermolysin from the inability of the former to hydrolyze N-[3-(2-furyl)acryloyl]-Gly-Leu-NH2. In addition, a vertebrate elastase substrate succinyl-Ala-Ala-Ala-p-nitroanilide was hydrolyzed by P27 but not by thermolysin. P27 is a newly described and unique enzyme from the standpoint of substrate specificity and from the fact that it is resistant to inhibition by phosphoramidon, an inhibitor of a number of zinc endopeptidases, including thermolysin.
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PMID:Purification and characterization of a neutral zinc endopeptidase secreted by Flavobacterium meningosepticum. 818 8

Cells interact with type IV collagen mainly via the integrins alpha 1 beta 1 and alpha 2 beta 1. A triple helical CNBr derived fragment CB3[IV], which contains the recognition sites for both integrins, was isolated from type IV collagen. Trypsin treatment of CB3[IV] gave rise to four smaller fragments, F1-F4, of which the smallest one, F4, contained the recognition site for alpha 1 beta 1. Further fragmentation of F4 by thermolysin treatment at 50 degrees C led to fragment TL1, which represents the C-terminal half of F4, and which was no longer able to interact with alpha 1 beta 1. Therefore the recognition site of alpha 1 beta 1 had to be located within the N-terminal half of F4, a position which was verified by electron micrographs of a crosslinked F2-alpha 1 beta 1 complex. Modification of the Arg and Asp residues, which abolished the binding activity of F4, led to the identification of Arg (461) within the alpha 2(IV) and Asp (461) within the alpha 1 (IV) chain as essential residues for the alpha 1 beta 1. The array of these two residues on the surface of the triple helix is discussed.
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PMID:The alpha 1 beta 1 integrin recognition site of the basement membrane collagen molecule [alpha 1(IV)]2 alpha 2(IV). 822 88

Inhibitory effects of some enzymatic hydrolysates of collagen and collagen-related synthetic peptides on fibrinogen/thrombin clotting were investigated. The hydrolysate of porcine skin collagen with thermolysin or bacterial collagenase inhibited fibrinogen/thrombin clotting, but did not inhibit the activity of thrombin. Although the activity was not pronounced, the hydrolysate of collagen with such proteinases as trypsin and pepsin also inhibited the clotting. Gly-Pro-Arg, which is a known inhibitor of fibrinogen/thrombin clotting, was isolated from the bacterial collagenase hydrolysate of porcine collagen by HPLC. Collagen-related synthetic peptides such as Gly-Pro-Arg-Gly, Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Gly-Pro-Ala, Gly-Pro-Arg-Gly-Pro-Pro, and Gly-Pro-Arg-Pro-Pro also inhibited the clotting, but did not inhibit the activity of thrombin. The inhibitory activity of Gly-Pro-Arg-Gly-Pro-Pro and Gly-Pro-Arg-Pro-Pro was more marked than that of Gly-Pro-Arg. However, Gly-Pro-Lys, Gly-Ala-Arg, Gly-Pro-Hyp, Ala-Gly-Pro-Arg and Gly-Pro-Ala-Gly-Pro-Arg had no inhibitory effect on the clotting.
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PMID:Inhibitory effects of enzymatic hydrolysates of collagen and collagen-related synthetic peptides on fibrinogen/thrombin clotting. 832 52


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