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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Evidence is presented that biopsy specimens from fibroadenomas, benign cystic lesions, and carcinomas of the human breast can produce in organ culture a neutral protease capable of digesting type I collagen. This enzyme activity, measured with the use of a radioactive release assay, was characterized as true vertebrate collagenase and occurred in both active and latent (requiring trypsin activation) forms. For the two types of benign breast lesion studied, collagenase secretion was significantly higher from fibroadenomas than from benign cystic tissue. Breast carcinomas, however, exhibited a wide quantitative spectrum of collagenase secretion, encompassing the extremes observed for the benign lesions and showing no correlation with histologic type. These results, while providing a plausible mechanism for the marked collagen degradation seen in disseminating neoplasms, demonstrate that high collagenase secretory activity is not pathognomonic of invasive behavior. The findings, however, indicate disordered regulation of collagenase activity in malignant tumors.
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PMID:Collagenase secretion by human breast neoplasms: a clinicopathologic investigation. 298 53

A neutral metalloproteinase has been isolated and purified from adherent rheumatoid synovial cells in culture. This protease, named matrix metalloproteinase 3, (MMP-3) degrades gelatin, proteoglycan, fibronectin, type IV collagen, laminin, and the N propeptide of type I procollagen. It can be separated from MMP-2 (a potent gelatinase), and MMP-1, an interstitial collagenase. MMP-3 is released from cells as a proenzyme of 55 Kda. Activation by trypsin or organic mercurials produces 2 active species of 45 Kda and 28 Kda. The enzyme contains zinc as an intrinsic component and requires calcium for conformational stability. In concert, active MMP-1, -2, and -3 can destroy all significant structural proteins of joint structures.
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PMID:Matrix metalloproteinases 1, 2, and 3 from rheumatoid synovial cells are sufficient to destroy joints. 330 38

Previous studies demonstrated that the thermal stability of the procollagen triple helix can be assayed by digesting the protein for short periods with high concentrations of trypsin and chymotrypsin. Here we cleaved human type I procollagen or collagen with vertebrate collagenase to generate A fragments from the three-quarter amino termini and B fragments from the one-quarter carboxy termini of the molecules. The thermal stabilities of the fragments were then assayed by rapid trypsin/chymotrypsin digestion. Both fragments were resistant up to 36 degrees C and completely degraded between 37 degrees C and 39 degrees C. In subsequent experiments the same assay was carried out with type I procollagens synthesized by fibroblasts from two patients with lethal variants of osteogenesis imperfecta. With one, the A fragments were selectively destabilized, an observation consistent with previous data indicating that the mutation in the patient produced a deletion of 84 amino acids from the middle of the alpha 1(I) chain. With procollagen synthesized by fibroblasts from the second patient the B fragments were selectively destabilized, an observation consistent with preliminary data indicating a mutation that alters the primary structure of the carboxy-terminal region of the alpha 1(I) chain. Therefore, the procedures described here present a simple and direct method for locating mutations that destabilize the collagen triple helix.
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PMID:The A and B fragments of normal type I procollagen have a similar thermal stability to proteinase digestion but are selectively destabilized by structural mutations. 354 29

Synthesis of type I procollagen was examined in fibroblasts from a proband with a lethal perinatal variant of osteogenesis imperfecta. After trypsin digestion of the type I procollagen, a portion of the alpha 1 (I) chains was recovered as disulfide-linked dimers. Digestion of the protein with vertebrate collagenase and mapping of cyanogen bromide peptides suggested that a new cysteine residue was present between residues 551 and 775 of the alpha 1 (I) chain. Sequencing of cloned cDNAs prepared using mRNA from the proband's fibroblasts demonstrated that some of the clones contained a single base mutation that converted the glycine codon in amino acid position 748 of the alpha 1 (I) chain to a cysteine codon. About 80% of the type I procollagen synthesized by the proband's fibroblasts had a decreased thermal stability. The results, therefore, were consistent with the conclusion that normal pro-alpha 1 (I) chains and pro-alpha 1 (I) chains containing a cysteine residue in the alpha chain domain were synthesized in about equal amounts and incorporated randomly into type I procollagen. However, only about 10% of the alpha 1 (I) chains generated by trypsin digestion were disulfide-linked. Further studies demonstrated a decreased rate of secretion of type I procollagen containing the new cysteine residue and decreased processing of the protein by procollagen N-proteinase in cultures of postconfluent fibroblasts. Both parents were phenotypically normal and their fibroblasts synthesized only normal type I procollagen. Therefore, the mutation in the proband was a sporadic one and is very likely to have caused the connective tissue fragility that produced the lethal phenotype.
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PMID:A point mutation in a type I procollagen gene converts glycine 748 of the alpha 1 chain to cysteine and destabilizes the triple helix in a lethal variant of osteogenesis imperfecta. 366 99

The existing forms of collagenase [EC 3.4.24.7] in the human uterine cervix were examined. The latent collagenase extracted by homogenization in 0.25% Triton X-100 containing 0.01 M CaCl2 was indicated to be a complex of collagenase with alpha 2-macroglobulin by the behavior of the fraction of this enzyme before and after treatment with NaSCN on Sephadex G-150 column chromatography and an immunodiffusion method. The active collagenase was extracted by rehomogenization in 50 mM Tris-HCl buffer, pH 7.4, containing 0.1 M M CaCl2 from the insoluble residue at 0 degrees C. Another latent collagenase was extracted from the insoluble fraction in the same buffer by heating at 60 degrees C for 4 min and this enzyme was activated by 4-aminophenylmercuric acetate or trypsin. The molecular weights of the active and the latent forms were approximately 7.3 x 10(4) and 9.4 x 10(4), respectively. This indicates that the latency is due to the formation of a low molecular weight inhibitor enzyme complex. These results clarified that the human uterine cervix contains three existing forms (alpha 2-macroglobulin complex, active form and low molecular weight inhibitor complex) of collagenase under these experimental conditions.
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PMID:The existing forms of collagenase in the human uterine cervix. 624 99

Neutral collagenase (EC 3.4.24.7) has been detected in human liver biopsies, baboon liver for the first time. The reaction mixtures were composed of collagen in solution and total liver homogenate in 0.5 M Tris-HCl, pH 7.5 at 25 degree C/0.2 M NaCl/10 mM CaCl2/3 mM p-chloromercuribenzoic acid. Collagenase activity was found by directly subjecting the reaction mixtures to viscometric assay and the activity was confirmed to be due to neutral collagenase by identifying the products using disc gel electrophoresis. It proved necessary to use p-chloromercuribenzoic acid in order to reveal collagenase activity in total liver homogenates from these species. The p-chloromercuribenzoic acid served to inhibit thiol proteinases and all other signs of nonspecific collagenolysis on disc gel electrophoresis, and it was able to activate latent collagenase which trypsin could not.
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PMID:Direct measurement of neutral collagenase activity in homogenates from baboon and human liver. 626 Feb 7

The enzyme collagenase (EC 3.4.24.7), a key mediator in biological remodeling, can be induced in early-passage fibroblasts by a wide variety of agents and conditions. In contrast, at least some primary tissue fibroblasts are incompetent to synthesize collagenase in response to many of these stimulators. In this study, we investigate mechanisms controlling response to two of the conditions in question: (i) trypsin or cytochalasin B, which disrupt actin stress fibers, or (ii) phorbol 12-myristate 13-acetate (PMA), which activates growth factor signaling pathways. We demonstrate that collagenase expression stimulated by trypsin or cytochalasin B is regulated entirely through an autocrine cytokine, interleukin 1 alpha (IL-1 alpha). The IL-1 alpha intermediate also constitutes the major mechanism by which PMA stimulates collagenase expression, although a second signaling pathway(s) contributes to a minor extent. Elevation of the IL-1 alpha level in response to stimulators is found to be sustained by means of an autocrine feedback loop in early-passage fibroblast cultures. In contrast, fibroblasts freshly isolated from the tissue are incompetent to activate and sustain the IL-1 alpha feedback loop, even though they synthesize collagenase in response to exogenous IL-1. We conclude that this is the reason why tissue fibroblasts are limited, in comparison with subcultured fibroblasts, in their capacity to synthesize collagenase. Activation of the IL-1 alpha feedback loop, therefore, seems likely to be an important mechanism by which resident tissue cells adopt the remodeling phenotype.
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PMID:Competence for collagenase gene expression by tissue fibroblasts requires activation of an interleukin 1 alpha autocrine loop. 762 17

The precursor of matrix metalloproteinase 9 (pro-MMP-9, progelatinase B) noncovalently binds to tissue inhibitor of metalloproteinases (TIMP)-1 through the C-terminal domain of each molecule. We have isolated the proMMP-9.TIMP-1 complex from the medium of human fibrosarcoma HT-1080 cells and investigated the activation processes of the complex by 4-aminophenylmercuric acetate, trypsin, and matrix metalloproteinase 3 (MMP-3, stromelysin 1). The treatment of the proMMP-9.TIMP-1 complex with 4-aminophenylmercuric acetate or trypsin converts proMMP-9 to lower molecular weight species corresponding to active forms, but no gelatinolytic activity is detected. The lack of enzymic activity results from binding of TIMP-1 to the activated MMP-9. The treatment of the proMMP-9.TIMP-1 complex with a possible physiological proMMP-9 activator, MMP-3, does not reveal any gelatinolytic activity unless the molar ratio of MMP-3 to the complex exceeds 1. This is due to the inhibition of MMP-3 by TIMP-1 forming a ternary proMMP-9.TIMP-1.MMP-3 complex. The formation of the ternary complex weakens the interaction between proMMP-9 and TIMP-1, resulting in partial dissociation of the complex into proMMP-9 and the TIMP-1.MMP-3 complex. When MMP-3 is in excess, the propeptide is completely processed, and the full activity of MMP-9 is detected. Similarly, the proMMP-9.TIMP-1 complex inhibits MMP-1 (interstitial collagenase) and in turn renders the proMMP-9 activable by a catalytic amount of MMP-3. These results suggest that formation of the proMMP-9.TIMP-1 complex regulates extracellular matrix breakdown in tissue by switching the predominant MMP activity from one type to another.
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PMID:Steps involved in activation of the pro-matrix metalloproteinase 9 (progelatinase B)-tissue inhibitor of metalloproteinases-1 complex by 4-aminophenylmercuric acetate and proteinases. 762 79

Histological studies have previously demonstrated an association between mast-cell activation/degranulation and areas of connective-tissue lysis in vivo; in addition, mast-cell extracts have been shown to activate latent forms of collagenase and stromelysin. In the present study we have examined the potential roles of rat mast-cell proteinase (RMCP) I and RMCP II as activators of the precursors of matrix metalloproteinase (MMP)-1 (interstitial collagenase), MMP-2 (gelatinase A) and MMP-3 (stromelysin 1). Both RMCPs I and II activated proMMP-3 by converting the 57 kDa precursor into a 45 kDa polypeptide. The N-terminal amino acid of 45 kDa MMP-3 activated by RMCP II was identified as Phe83. By contrast, only RMCP II activated the 52 kDa proMMP-1 by converting it into a 41 kDa protein and generating the new N-termini, namely Gln80 and Val82. The collagenolytic activity which resulted from this cleavage was only 35% of the full activity, but this could not be augmented by subsequent treatment with MMP-3, the latter being a crucial enzyme for the generation of the fully active MMP-1 with Phe81 at the N-terminus, in conjunction with other serine proteinases. Thus RMCP II activates proMMP-1 via a mechanism different from that reported for the stepwise processing by combinations of other trypsin-like enzymes and MMP-3. ProMMP-2 (pro-gelatinase A) was not activated by either RMCP I or RMCP II, despite processing to smaller products.
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PMID:Activation of precursors for matrix metalloproteinases 1 (interstitial collagenase) and 3 (stromelysin) by rat mast-cell proteinases I and II. 782 45

We studied the in vivo effect of long-term doxycycline treatment combined with NSAID on human interstitial collagenases, other matrix metalloproteinases, serine proteinases, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and lactoferrin from saliva and serum during the course of acute reactive arthritis (ReA). Collagenase activity and serine proteases (elastase-like, cathepsin G-like and trypsin-like activities) of saliva (n = 10) and gelatinase, lactoferrin and TIMP-1 of saliva (n = 10) and serum (n = 10) samples before and after 2 months doxycycline treatment, combined with NSAID, were studied by quantitative SDS-PAGE assay, ELISA assay and by spectrophotometric assay. The cellular source and molecular forms of salivary collagenase were characterized by immunoblotting using specific antisera. We found that activities of total and endogenously active interstitial collagenase reduced significantly. The salivary collagenase was found to originate from neutrophils. No fragmentation of either pro 75-kD and active 65-kD MMP-8 was detected after 2 months doxycycline treatment. However, during 2 months doxycycline and NSAID treatment no reduction of salivary and serum gelatinase, lactoferrin and TIMP-1-levels and salivary serine protease activities were detected. The in vivo inhibition of collagenase (MMP-8) activity during long-term doxycycline therapy in human saliva containing inflammatory exudate of ReA patients may contribute to the reduced tissue destruction observed in recent clinical and animal model studies in arthritides during long-term doxycycline/tetracycline treatment.
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PMID:In vivo inhibition of human neutrophil collagenase (MMP-8) activity during long-term combination therapy of doxycycline and non-steroidal anti-inflammatory drugs (NSAID) in acute reactive arthritis. 792 79


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