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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pure collagenase (clostridiopeptidase A, EC 3.4.24.3) having a molecular weight of 70 000 was obtained from the culture medium of Clostridium histolyticym by a combination of ultrafiltrations, molecular sieve, affinity and hydrophobic chromatography. The value of its specific activity is the highest of those described previously but 6-times lower than that of the collagenase from Achromobacter iophagus (EC 3.4.24.8). Its amino acid composition differs from previous data, namely by the presence of cysteine, methionine, tryptophan and O-phosphoserine residues. In contrast to Achromobacter collagenase it does not dissociate in subunits during the deactivation by EDTA or LiCl/glycine buffer at pH 10.5. Existence of multiple forms of Clostridium collagenase previously described is discussed as being due to autolysis of a single molecular species or to a different degree of phosphorylation.
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PMID:Chemical characterization of the homogeneous collagenase from Clostridium histolyticum. 626 87

A new method for the simultaneous determination of newly synthesized collagen and noncollagen proteins has been developed. Because tryptophan is not found in collagen noncollagen proteins were specifically labeled with [3H]tryptophan. [14C]Proline was used to label both groups of proteins. To calculate the 14C-labeled noncollagen protein the 3H radioactivity of the protein mixture was divided by the ratio of 3H:14C in noncollagen protein of a representative sample. This value was obtained by collagenase digestion. The remaining 14C radioactivity in the protein mixture was attributed to [14C]collagen. There was a very good correlation between the dual label method and the widely used collagenase digestion method for the measurement of collagen and noncollagen protein production and for the calculation of the relative rate of collagen synthesis. This new method provides a simple and accurate analysis of collagen production, and it is suitable for rapid processing of a large number of biological samples.
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PMID:[3H]tryptophan-[14C]proline dual label method for the simultaneous determination of collagen and noncollagen protein production. 652 75

The expression of the matrix-degrading enzymes collagenase and stromelysin is modulated by a variety of biologic and pharmacologic agents. IFN-gamma has potent effects on metalloproteinase production and therefore may play an important role in preventing excessive connective tissue degradation during inflammation and repair. We investigated the mechanisms of collagenase and stromelysin regulation by IFN-gamma in human dermal fibroblasts. IFN-gamma (300 U/ml) prevented the stimulation of metalloproteinase gene expression by IL-1 beta. In addition, incubation of fibroblasts with IFN-gamma resulted in a marked increase in cellular indoleamine 2,3-dioxygenase (IDO) mRNA, a > 90% depletion of tryptophan, and a corresponding > 30-fold increase in the tryptophan metabolite kynurenine in the culture media. Reducing the concentration of tryptophan from 25 microM to 0 markedly diminished the ability of fibroblasts to increase collagenase and stromelysin mRNA and collagenase production in response to IL-1 beta. Addition of exogenous tryptophan (25-50 micrograms/ml) to cultures that had been tryptophan depleted by pretreatment with IFN-gamma for 48 h restored the fibroblast response to IL-1 beta or PMA, but had no effect on IFN-gamma-induced HLA-DR alpha chain mRNA expression. These results indicate that inhibition of collagenase and stromelysin gene expression by IFN-gamma in fibroblasts is associated with activation of IDO and enhanced cellular tryptophan metabolism. Tryptophan degradation and ensuing tryptophan depletion may account, at least in part, for the inhibitory effect of IFN-gamma on metalloproteinase production in dermal fibroblasts.
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PMID:Inhibition of collagenase and stromelysin gene expression by interferon-gamma in human dermal fibroblasts is mediated in part via induction of tryptophan degradation. 761 20

The truncated forms of tissue inhibitor of metalloproteinase-1 and -2 (delta TIMP-1 and -2), comprising the N-terminal active domain, are ideal molecules for structural analysis by intrinsic fluorescence as each contains a single conserved tryptophan residue. In this paper we describe studies on their conformational stability, unfolding/refolding kinetics and the environment of the unique tryptophan as judged by its fluorescence properties in the native state and exposure to an external quencher, acrylamide. Two forms of delta TIMP-2 were studied: delta TIMP-2 T21 derived from the full-length cDNA clone isolated from a mixed-tumour library, and delta TIMP-2 A21 containing the highly conserved V18IRAK22 sequence. In all three delta TIMP proteins the tryptophan environments in the native state appeared to be similar, but substantial differences were seen in their conformational stabilities and refolding kinetics. delta TIMP-1 was approximately twice as stable as delta TIMP-2 T21 and 1.4-fold more stable than delta TIMP-2 A21. This stability difference between delta TIMP-1 and delta TIMP-2 was shown to be independent of N-linked glycosylation. delta TIMP-1 and delta TIMP-2 A21 both showed simple two-state refolding kinetics, whereas delta TIMP-2 T21 refolding was more complex and biphasic in character. These differences between delta TIMP-2 T21 and A21 suggest that residue 21 is a structurally important site in the TIMP protein. All three truncated molecules can be considered as stable independent folding domains ideally suited for further structural analysis.
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PMID:Folding and stability of the active N-terminal domain of tissue inhibitor of metalloproteinases-1 and -2. 780 30

A galactose-binding protein of M(r) = 30,000 previously described in baby hamster kidney cells (Foddy, L., Stamatoglou, S. C., and Hughes, R. C. (1990) J. Cell Sci. 97, 139-148) has been analyzed by the cloning and sequencing of cDNA clones encoding the complete sequence and an amino-terminal fragment. The intact lectin CBP30 contains 245 amino acid residues, including the initiating methionine residue, and is closely homologous to mammalian S-type lectins of similar size characterized in human, rat, and mouse species. The carboxyl-terminal domain contains the carbohydrate binding activity and the amino-terminal domain, which is extremely sensitive to bacterial collagenase, contains a repetitive sequence rich in glycine, tyrosine, and proline. There are 8 repeats in hamster CBP30, as in the human homologue, compared with about 10 in rat and mouse and > 10 in dog homologues. This repeat sequence is also sensitive to the tissue metalloproteinases, gelatinase B and matrilysin, but, unlike the bacterial collagenase, the mammalian enzymes also cause extensive degradation of the carbohydrate binding carboxyl domain. Physical measurements using CD and tryptophan fluorescence spectroscopy indicate that the two domains of CBP30 are structurally, as well as functionally, distinct and independent. Cross-linking studies indicate that the amino-terminal lectin fragment can efficiently self-assemble into oligomeric species, and less efficient but significant aggregation of the intact lectin is also shown. Domain-specific antibodies to hamster CBP30 have been prepared and used to show that only the full-length, undegraded form of CBP30 is present in whole cell lysates.
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PMID:Structure of baby hamster kidney carbohydrate-binding protein CBP30, an S-type animal lectin. 802 86

The synthesis of a series of N-phosphonalkyl dipeptides 6 is described. Syntheses were devised that allowed the preparation of single diastereoisomers and the assignment of stereochemistry. The compounds were evaluated in vitro for their ability to inhibit the degradation of radiolabeled collagen by purified human lung fibroblast collagenase. Several of the compounds were potent collagenase inhibitors and were at least 10-fold more potent than their corresponding N-carboxyalkyl analogues. Activity was lost when the phosphonic acid group P(O)(OH)2 was replaced by the phosphinic acid groups P(O)(H)(OH) and P(O)(Me)(OH). At the P1 position, (R)- or (S)-alkyl groups, especially ethyl and methyl (e.g., 12a,b, 52a,b, and 53a,b), or an (R)-phenethyl moiety (55a) conferred high potency (IC50 values in the range 0.23-0.47 microM). (S)-Stereochemistry was preferred for the P1' isobutyl side chain. Structure-activity relationships were also investigated at the P2' site, and interestingly, compounds with basic side chains, such as the guanidine 57a, were equipotent with more lipophilic compounds, such as 52a. As with other series of collagenase inhibitors, potency was enhanced by introducing bicyclic aromatic P2' substituents. The most potent phosphonic acid of the series was the bicyclic aromatic P2' tryptophan analogue 59a (IC50 0.05 microM).
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PMID:Synthesis of novel N-phosphonoalkyl dipeptide inhibitors of human collagenase. 828 90

Recombinant interstitial collagenase (rMMP-1) forms insoluble inclusion bodies when over-expressed in Escherichia coli. We surveyed conditions for renaturation of purified rMMP-1 in 6 M guandine hydrochloride (GdnHCl) and found that optimal folding occurred when the denatured protein was diluted at 4 degrees C in approximately 2 M guanidine HCl, 20% glycerol, 2.5 mM reduced and oxidized glutathione, and 5 mM CaCl2, followed by buffer exchange to remove denaturant and thiols. The circular dichroism spectrum and catalytic constants of the refolded enzyme were similar to those of native MMP-1. The propeptide, which comprises approximately 20% of the mass of proMMP-1, was not required for folding to a functional enzyme. Size exclusion chromatography and spectroscopic measurements at intermediate [GdnHCl] revealed two intermediate folding states. The first, observed at 1 M GdnHCl, had a slightly larger Stokes' radius than the folded protein. CD and fluorescence analysis showed that it contained ordered tryptophan residues with a higher quantum yield than the fully folded state. The second intermediate, which appeared between 2 and 4 M GdnHCl, exhibited properties consistent with the molten globule, including secondary structure, lack of ordered tryptophan, exposed hydrophobic binding sites, and a Stokes' radius between that of the folded and unfolded states.
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PMID:Characterization of folded, intermediate, and unfolded states of recombinant human interstitial collagenase. 862 83

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases which are secreted from cells as zymogens and can be activated by treatment with organomercurial reagents or limited proteolysis. The proenzyme forms of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are found in complex with tissue inhibitor of metalloproteinases (designated proMMP-2/ TIMP-2 and proMMP-9/TIMP-1, respectively). The proposed mechanism of activation by mercurial compounds involves the induction of a conformational change in the zymogen which leads to propeptide autoprocessing. To investigate the possibility of conformational differences in MMPs, solute quenching of MMP intrinsic fluorescence was used to probe the relative exposure of tryptophan residues in latent and mercurial-activated MMPs. Our data demonstrate that fluorescence quenching of the proMMP-2/TIMP-2 complex by either acrylamide or iodide is significantly increased following mercurial activation. In contrast, no significant change in tryptophan accessibility accompanies mercurial treatment of either proMMP-2 or TIMP-2 alone, or mercurial-activated MMP-2 mixed with TIMP-2. To determine whether the enhanced fluorescence quenching was unique to the activated proMMP-2/TIMP-2 complex, similar experiments were performed using MMP-1, MMP-3, and MMP-9/TIMP-1 complex. In all cases, both latent and mercurialtreated MMPs exhibited similar fluorescence quenching profiles, suggesting that there are no significant conformational differences between the zymogen and activated forms of MMP-1, -2, -3, or -9/TIMP-1. The enhanced fluorescence quenching observed with mercurial-treated proMMP-2/TIMP-2 is indicative of increased exposure of a previously buried tryptophan residue(s), providing evidence for a structural rearrangement of the activated complex. These data, together with our previous biochemical observation that mercurial treatment of proMMP-2/TIMP-2 exposes the MMP-2 active site without propeptide processing (Y. Itoh et al. (1995) Biochem. J. 308, 645-651), suggest that the activated proMMP-2 in the complex may represent a transitional conformational intermediate in MMP activation.
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PMID:Fluorescence quenching studies of matrix metalloproteinases (MMPs): evidence for structural rearrangement of the proMMP-2/TIMP-2 complex upon mercurial activation. 880 67

Collagenase, a prototypic matrix metalloproteinase, plays a major role in the degradation of the extracellular matrix. The essential amino acid L-tryptophan was recently shown to stimulate the expression of collagenase gene in human dermal fibroblast cultures. In this study, we focused our attention on the mechanisms responsible for activation of collagenase transcription by L-tryptophan. Incubation of fibroblasts with L-tryptophan resulted in a dose- and time-dependent elevation of collagenase and tissue inhibitor of metalloproteinase mRNA levels. The maximum enhancement in collagenae mRNA was approximately 50-fold. This effect was not abolished by cycloheximide, suggesting independence from ongoing protein synthesis. Transient cell transfections with a promoter/reporter gene construct containing 3.8 kb of 5' flanking DNA of the human collagenase gene linked to the chloramphenicol acetyl transferase (CAT) gene or a construct containing three phorbol ester-responsive AP-1 binding sequences (12-O-tetradecanoyl-phorbol-13-acetate-responsive element) in front of the thymidine kinase promoter linked to the CAT gene indicated enhancement of promoter activity by L-tryptophan. Furthermore, electrophoretic DNA mobility shift assays demonstrated enhanced DNA-protein complex formation specific for an AP-1 binding site probe with nuclear extracts prepared from cells incubated with L-tryptophan. These results collectively suggest that activation of collagenase gene expression in dermal fibroblasts by L-tryptophan is mediated through AP-1 binding elements in the collagenase gene promoter that are sufficient for gene response.
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PMID:L-tryptophan induces expression of collagenase gene in human fibroblasts: demonstration of enhanced AP-1 binding and AP-1 binding site-driven promoter activity. 886 82

Interleukin-1 beta (IL-1 beta) is a potent signal for the induction of the matrix-degrading enzymes collagenase and stromelysin. These metalloproteinases (MMP) play a critical role in physiologic and pathologic connective tissue remodeling, and are potential targets for therapeutic manipulation. Treatment of human dermal fibroblasts with interferon-gamma inhibited. Type I collagen gene expression, and abrogated the effect of IL-1 beta on MMP expression. Interferon-gamma also caused a dramatic dose-dependent increase in indoleamine 2,3-dioxygenase mRNA, with consequent depletion of tryptophan and accumulation of kynurenine in the culture media. To examine the role of tryptophan metabolism in the effects of interferon-gamma on matrix-degrading enzymes, exogenous tryptophan was added to tryptophan-depleted media, followed by stimulation of the cultures with IL-1 beta. Supplementation with tryptophan completely overcame the inhibitory effects of interferon-gamma on MMP mRNA expression and metalloproteinase secretion into the media. In contrast mRNA levels for Type I collagen remained profoundly depressed in interferon-gamma-treated cultures in spite of addition of exogenous tryptophan. These results indicate that oxidative tryptophan metabolism mediates the effects of interferon-gamma on MMP gene expression in human fibroblasts.
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PMID:Control of extracellular matrix degradation by interferon-gamma. The tryptophan connection. 890 57


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