Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proteases capable of activating procollagenase from gingiva and from fibroblast and macrophage monolayer cultures were harvested from homogenates of canine tumor mast cells. The mast cell proteases lysed casein and Azocoll but not native collagen. In low salt concentrations the enzymes existed at high molecular weight complexes, which were dissociated by increasing the salt concentration above 1.0 M (NaCl, KCl). Gel filtration in 1.4 M KCl separated the protease activity into three peaks, all of which activated procollagenase. Two of the enzymes showed substrate specificities (hydrolysis of p-tosyl-L-arginine methyl ester and benzoyl-tyrosine ethyl ester) and reactive center reactivities similar to pancreatic trypsin and chymotrypsin. Based on gel filtration, apparent molecular weights of 160 000 (p-tosyl-L-arginine methyl ester esterase), 90 000 (main procollagenase activator) and 36 000 benzoyl-tyrosine ethyl ester esterase) were determined. Activation of procollagenase resulted in a 18-20 000 decrease of the molecular weight. The activation was directly related to the amount of activator added within certain limits. Further addition of activator resulted in proteolytic inactivation of collagenase.
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PMID:Activation of fibroblast procollagenase by mast cell proteases. 5 9

Sequencing of chymotrypsin, trypsin, collagenase- and hydroxylamine-derived peptides, using the automated Edman degradation procedure, yielded the complete amino acid sequence of alpha2-CB4 from calf skin collagen (321 residues). Together with the data from earlier work, an uninterrupted sequence in the helical region of the alpha2-chain from residues 1-393 is now known. Glycine is found in every third position of the peptide. Hydroxylation of proline and lysine occurs only in the Y-position of the triplet Gly-X-Y and is not complete in every position. Some residues, such as glutamic acid, leucine, phenylalanine and arginine, are distributed non-randomly between the X and Y-positions and this non-random distribution is different in the alpha1 and alpha2-chains. Comparison of the N-terminal 393 residues from the helical region of the alpha1 and alpha2-chains revealed a nearly identical distribution of charged polar residues arginine, lysine, glutamic and aspartic acids. The distribution of the triplet Gly-Pro-Hyp is simialr in both chains. The remaining residues in the alpha2-chain exhibit a high degree of substitutions when compared with those in the alpha1-chain. Approximately one in every two residues in both the X and Y-positions are substituted.
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PMID:The covalent structure of collagen. The amino-acid sequence of alpha2-CB4 from calf-skin collagen. 17 31

Despite ingenious concepts, there are no unequivocal clues as to what, when, and how some undefined biochemical factor(s) or constituent(s) that localizes in the arterial wall can precipitate a thromboatheromatous lesion or arterial disease. The present study focused on the extraction, partial purification, and characterization of a collagen-active platelet stimulator from the aortas of aged burros. The aggregator moiety in the aorta extracts invariably had a higher affinity for platelets in citrated platelet-rich plasma of human beings than for platelets of homologous burros. The platelet-aggregating factor(s) in the aorta extract was retained by incubation with alpha-chymotrypsin. Platelet-aggregating activity was rapidldy abolished after incubation with collagenase, as determined by plateletaggregometry tests. Evidence based on light microscope and polysaccharide histochemical reactions indicates a probability that the intracellular amorphous matrix (PAS-positive) and filamentous components (PTAH-positive) expelled from smooth muscle cells disrupted during homogenization of the aorta may be a principle source of a precursor collagen species which is a potent inducer of platelet aggregation.
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PMID:Platelet affinity for burro aorta collagen. 20 Nov 88

Bone explants from foetal and newborn rabbits synthesize and release a collagenase inhibitor into culture media. Inhibitor production in the early days of culture is followed first by latent collagenase and subsequently active collagenase in the culture media. A reciprocal relationship exists between the amounts of free inhibitor and latent collagenase in culture media, suggesting strongly that the inhibitor is a component of the latent form of the enzyme. Over 90% of the inhibitory activity of culture media is associated with a fraction of apparent mol.wt. 30000 when determined by gel filtration on Ultrogel AcA 44. The inhibitor blocks the action of rabbit collagenase on both reconstituted collagen fibrils and collagen in solution. It inhibits the action of either active collagenase or latent collagenase activated by 4-aminophenylmercuric acetate. Latent collagenase activated by trypsin is usually much less susceptible to inhibition. The activity of the inhibitor is destroyed by heat, by incubation with either trypsin or chymotrypsin and by 4-aminophenylmercuric acetate. Collagenase activity can be recovered from complexes of enzyme (activated with 4-aminophenylmercuric acetate) with free inhibitor by incubation with either trypsin or 4-aminophenylmercuric acetate, at concentrations similar to those that activate latent collagenase from culture media. The rabbit bone inhibitor does not affect the activity of bacterial collagenase, but blocks the action of collagenases not only from a variety of rabbit tissues but also from other mammalian species.
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PMID:Identification and partial characterization of an inhibitor of collagenase from rabbit bone. 20 52

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2-24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or alpha-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16-48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10(6) cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, alpha(1)-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.
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PMID:Proteases induce secretion of collagenase and plasminogen activator by fibroblasts. 20 72

1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4 degrees C and prolonged preincubation at 25 degrees C. Its activation often followed that of the procollagenase present in the same media. 3. Activation of neutral proteinase (as does that of procollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes (60 000-70 000) by 20 000-30 000, as determined by gel filtration of media after removal of heparin. 6. The latency of both enzymes could be due either to a zymogen or to an enzyme-inhibitor complex. A thermostable inhibitor of both enzymes was found in some media. However, combinations of either enzyme with that inhibitor were not reactivated by trypsin, indicating that this inhibitor is unlikely to be the cause of the latency.
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PMID:The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen. 20 18

Biphasic kinetic data were obtained when trypsin (EC 3.4.21.4) which had previously been complexed with a thiol-containing inhibitor (present in Ehrlich ascites tumour cells) was incubated with incremental additions of periodate. At low concentrations of periodate the trypsin was re-activated whilst at higher concentrations of periodate the trypsin was irreversibly inhibited. This biphasic reactivation followed by inhibition was also demonstrated when trypsin was first inhibited by dithiothreitol and followed by incremental addition of periodate. Similar results were obtained with chymotrypsin (EC 3.4.21.1). Incremental additions of either dithiothreitol or periodate caused inhibition of both these enzymes. The biphasic kinetic data can be explained in terms of reduction and oxidation of a significant disulphide bond in both trypsin and chymotrypsin which can be cleaved by thiols in a disulphide exchange reaction [1]. This bond is thought to maintain the active centres of each of these enzymes in a conformation sterically favourable for enzymic cleavage of specific peptide bonds in the protein substrates (polymeric collagen fibrils and casein) employed in this study.
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PMID:Inhibition of trypsin and chymotrypsin by thiols. Biphasic kinetics of reactivation and inhibition induced by sodium periodate addition. 22 48

The collagenase from the larvae Hypoderma lineatum, with a molecular weight of 24 000 and isoelectric point of 4.1, was obtained in homogeneous form by ion-exchange chromatography. It is stoichiometrically inhibited by diisopropylfluorophosphate. On the other hand it is unaffected by ethylenediaminetetraacetate, p-chloromercuribenzoate, dithiothreitol, N-tosyllysine chloromethyl ketone, N-tosylphenylalanine chloromethyl ketone and ovomucoid trypsin inhibitor. The enzyme which degrades native collagen in its helical parts, has a specific activity on thermally reconstituted collagen fibrils of 150 micrograms collagen degraded x min-1 x (mg enzyme)-1 at 37 degrees C. It hydrolyses casein but has no esterolytic activity characteristic of trypsin, chymotrypsin nor elastase. It has no action on the synthetic peptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-L-glycyl-L-prolyl-D-arginine. The amino acid composition of Hypoderma collagenase indicates a distinct similarity with the serine proteinases of the trypsin family and with another athropode serine collagenase, that of the fiddler crab Uca pugilator. This suggests that eucaryotic collagenases with digestive rather than morphogenic function represent a new category of members of the trypsin family.
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PMID:Chemical and enzymatic characterization of the collagenase from the insect Hypoderma lineatum. 23 30

Elastolytic enzyme was purified and crystallized from culture fluid of Flavobacterium immotum No. 9-35. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was determined by Sephadex G-100 gel filtration to be 13,000. The isoelectric point was between pH 8.3 and 8.9. The optimum pH of the enzyme was 7.2 for elastolytic activity. The purified enzyme showed not only elastolytic activity, but also non-specific proteolytic activity against various other proteins. Milk-clotting activity was also observed. The enzyme did not act on keratin, collagen, or fourteen amino acid esters, including N-benzoyl-L-alanine methyl ester, N-benzoyl-L-arginine ethyl ester, and N-acetyl-L-tyrosine ethyl ester, which were typical substrates of pancreatic elastase [EC 3.4.21.11], trypsin [EC 3.4.21.4], and chymotrypsin [EC 3.4.21.1], respectively. However, the enzyme selectively hydrolyzed elastin when both elastin and albumin were present in the reaction mixture. The enzyme was inhibited by o-phenanthroline and various heavy metals such as cadmium, lead, zinc, and mercury. Various inhibitors, such as diisopropyl phosphofluoridate, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, trypsin inhibitor, iodoacetamide, etc., had no effect on the elastolytic activity.
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PMID:Purification and properties of elastolytic enzyme from Flavobacterium immotum. 23 95

We have identified and purified a polypeptide region containing the collagen-binding site of the adhesive glycoprotein fibronectin. Chicken cellular fibronectin isolated from cultured embryonic fibroblasts was permitted to bind to gelatin coupled to agarose beads and was then digested extensively with chymotrypsin. A prominent 40,000-dalton fragment of fibronectin consisting of a single polypeptide chain was detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis of material remaining bound to the gelatin-agarose. This fragment appeared within 10 min after the digestion was initiated and persisted for more than 20 hr. This proteolytic fragment was isolated in electrophoretically pure form and retained its affinity for collagen. Plasma fibronectins from chicken and human blood also contained collagen-binding proteolytic fragments of similar size. This finding suggest that the collagen-binding sites of cellular and plasma fibronectins are homologous.
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PMID:Identification and isolation of a collagen-binding fragment of the adhesive glycoprotein fibronectin. 28 1


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