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)

The low natural abundance of many proteins is a major factor in preventing their development as therapeutic or diagnostic tools. To circumvent this barrier, we have used synthetic oligonucleotide technology to construct a gene based on the sequence of a cDNA for human interleukin 6 (IL-6). The synthetic gene encodes a cysteine-free, bioengineered rIL-6 protein, which is expressed at high concentrations in Escherichia coli as a tripartite fusion protein. Cleavage of the fusion protein with collagenase (EC 3.4.24.8) releases a 23-kDa rIL-6 protein that can be easily purified to homogeneity. This rIL-6 protein displays a range of biological activities similar to those of natural human IL-6, as demonstrated by its ability to (a) protect cells from viral infection and (b) stimulate the synthesis of fibrinogen in rat FAZA cells.
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PMID:Development of a vector system for the expression of bioengineered proteins. 219 36

Cysteine proteinases, particularly cathepsins B and L, have been strongly implicated in fostering metastasis in mice. In this work four different inhibitors of cysteine proteinases have been shown to inhibit the invasion of the human amnion by murine melanoma and mammary carcinoma cells in vitro. Two of the inhibitors are synthetic peptides [ZPhePheCHN2 (benzyloxycarbonyl-L-phenylalanyl-L-phenylalanyldiazomethane) and ZPheAlaCH2F [3-(N-benzyloxycarbonylphenylalanylamido)-DL-1-fluoro-2-butanone]] and two are thiol protease inhibitors (TPIn, TPId) isolated from the skeletal muscle of the hind limbs of normal and dystrophic mice, respectively. The inhibitors (ZPhePheCHN2, TPId), with apparent selectivity for cathepsin L, blocked invasion as effectively as inhibitors (ZPheAlaCH2F, TPIn) effective on both cathepsins. The data reveal that in these cell lines the cysteine proteinases contribute significantly to the invasive capacity of the cells, but to a lesser extent than do the metalloproteinases. We suggest that the cysteine proteinases facilitate the action of metalloproteinases (collagenase, gelatinase, and stromelysin), possibly by activating them, by inactivating the tissue inhibitor of metalloproteinases, and/or by making basement membrane matrix more accessible.
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PMID:Suppression by cathepsin L inhibitors of the invasion of amnion membranes by murine cancer cells. 273 Nov 77

A cDNA library constructed from chick aorta poly(A+) RNA in the expression vector pEX1 was screened with rabbit polyclonal antisera. Additional clones were obtained by DNA-DNA hybridization with subclones from the most 5'- and 3'-ends. The overlapping clones span 4.6 kilobases and code for the entire alpha 1 (VI) chain. The nucleotide sequence reveals a 3057-base pair open reading frame that codes for 1019 amino acids. Analysis of the deduced amino acid sequence predicts that alpha 1 (VI) has one collagenous domain (COL) of 336 residues flanked by three repeated domains of about 200 residues each, one at the amino (A'3) and two at the carboxyl ends (A'2 and A'1), respectively, that are similar to the type A repeats of von Willebrand Factor. The COL domain presents two short interruptions near the carboxyl end of the triple helix and three of the six potential N-asparaginyl-linked carbohydrate attachment sites (Asn-Xaa-Ser/Thr). Furthermore, it contains 1 cysteine at position 89 that could participate in the formation of dimers and 3 Arg-Gly-Asp sequences that might be potential sites for cell adhesion. The COL domain shows an extended region, starting from position 40, within the triple helix, made of 14 Gly-Xaa-Yaa triplets that lack proline in the Y position, suggesting that it might be more flexible than the rest of the domain. At the junction of the COL with the N- and C-terminal domains, there are several cysteines that could confer the well known resistance of type VI collagen to pepsin and collagenase digestion under nonreducing conditions. The present sequence data allow a structural model for type VI collagen assembly to be proposed that is consistent with the structure implied from previous electron microscopic observation by Furthmayr et al. (Furthmayr, H., Wiedemann, H., Timpl, R., Odermatt, R., and Engel, J. (1983) Biochem. J. 221, 303-311).
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PMID:Alpha 1 chain of chick type VI collagen. The complete cDNA sequence reveals a hybrid molecule made of one short collagen and three von Willebrand factor type A-like domains. 278 34

The effects of specific inhibitors of cysteine-proteinases ((Z-Phe-Ala-CHN2: benzyloxycarbonyl-phenyl-alanyl alanyl diazomethane and E-64: trans-epoxy-succinyl-L-leucylamido(4-guanidino)-butane) and collagenase and collagenase ((Cl-1: N-(3-N-benzyloxycarbonyl amino-1-R-carboxypropyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide) have been tested on the osteoclastic resorption of dentine. Chick osteoclasts were cultured in the presence or absence of 12.5 microM Z-Phe-Ala-CHN2, 40 or 60 microM E-64, or 40 or 100 microM Cl-1 for 1 or 2 days. In addition, osteoclasts were cultured on oyster shell calcitostracum with or without 12.5 microM Z-Phe-Ala-CHN2. Specimens were studied by light microscopy to count cells and resorption features and by scanning electron microscopy (SEM) stereophotogrammetry for the measurement of the depths, plan-areas and volumes of resorption pits. The numbers, depths and volumes (but not the plan-areas) of the resorption pits in dentine were significantly reduced by Z-Phe-Ala-CHN2 and E-64. Thus, for a given plan-area, the volumes and the depths of resorption pits were smaller in these experimental groups compared with control dentine specimens. The overall inhibition of resorption was at least 75%. Cl-1 did not have this inhibitory effect on the numbers or sizes of resorption pits in dentine. When the oyster calcitostracum was used as a substrate for the osteoclasts, Z-Phe-Ala-CHN2 did not reduce the numbers or volumes of pits, but increased the plan-areas and prevented the formation of deeper pits.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effects of inhibitors of cysteine-proteinases and collagenase on the resorptive activity of isolated osteoclasts. 282 13

The hexameric complex of globular domains of type IV collagen was isolated after collagenase digestion of human placenta and the different monomers and dimers present were chromatographically separated. The ratio of alpha 1(IV)NC1 to alpha 2(IV)NC1 was 2:1. About 50% of the NC1 domains were connected to dimers. Predominantly alpha 1-alpha 1 dimers were found. Only 12% were alpha 2-alpha 2 dimers and no alpha 1-alpha 2 dimers could be detected. The majority (88%) of the intermolecular bonds was found to be disulfide bridges. The remainder could not be cleaved by reduction. To elucidate the arrangement of the disulfide bonds, the unreduced alpha 1(IV)NC1 monomers were treated with cyanogen bromide, the disulfide-bridged peptides isolated and characterized by Edman degradation. Each of the two homologous subdomains within a monomer is stabilized by an identical set of three disulfide bonds. In subdomain I, cysteines at positions 20 and 53 are connected with the C-terminal cysteine pair 108 and 111. Thus formed, the disulfide knot stabilizes two interconnected loops of 32 and 54 residues, respectively. A smaller loop of five residues occurs due to a disulfide bond between the cysteines 65 and 71. A similar disulfide arrangement is indicated for subdomain II which is separated from subdomain I by a segment of 20 amino acid residues. The same arrangement of disulfide bonds has been strongly suggested for the alpha 2(IV)NC1 monomer by the isolation and characterization of its disulfide-bridged tryptic fragments. Similar investigations on the dimeric alpha 1(IV)NC1 domain established the arrangement of the intermolecular disulfide bonds. They are formed by a complete disulfide exchange between corresponding disulfide knots of two monomeric NC1 domains.
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PMID:The arrangement of intra- and intermolecular disulfide bonds in the carboxyterminal, non-collagenous aggregation and cross-linking domain of basement-membrane type IV collagen. 284 31

Since its discovery, vertebrate collagenase is postulated to be the key enzyme for collagen degradation, but the evidence for its participation in bone resorption was, until recently, very weak. The osteoclast, which is the major bone resorbing cell, does apparently not produce collagenase but lysosomal acid proteinases, including collagenolytic cysteine proteinases, whose involvement in bone resorption is supported by many observations. Recent reports indicating 1) that osteoblasts produce and secrete collagenase in response to bone resorbing hormones and 2) that bone resorption is inhibited by a specific collagenase inhibitor, have reintroduced collagenase as a plausible participant in the mechanisms of bone resorption. Moreover bone resorbing agents do not act directly on osteoclasts, but on osteoblasts which, by an unknown mechanism, induce the osteoclasts to excavate bone matrix. Collagenase could participate in the control of bone resorption by degrading the unmineralized collagen which seems to shield the underlying bone from osteoclastic attack. In addition, collagenase, stored as a latent proenzyme in the mineralized matrix, might cooperate with the lysosomal cysteine proteinases in the subosteoclastic lysis of demineralized collagen.
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PMID:The role of collagenase in bone resorption. An overview. 285 68

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.
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PMID:Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes. 290 50

We have determined the nucleotide sequence of a cDNA clone encoding the amino-terminal portion of human alpha 2(V) procollagen and found that the structure of the 186-residue amino-terminal propeptide closely resembles those of the fibril-forming procollagens. Juxtaposed to a 26-residue leader peptide, pro-alpha 2(V) exhibits a characteristic cysteine-rich globular region followed by 24 Gly-X-Y repeats which are interrupted by two short non-collagenous sequences. Upon closer examination, each of these two sequences was noted to display structural motifs characteristic of either pro-alpha 1(I) and pro-alpha 1(III) collagens or pro-alpha 1(II) collagen, respectively. Finally, within the amino-terminal telopeptide, a putative amino-terminal proteinase cleavage site, Ala-Gln, was identified. This latter finding strongly suggests that the alpha 2(V) amino-terminal propeptide can be potentially processed and thus leaves unresolved the issue pertaining to the nature of the collagenase-resistant sequence that is retained by mature type V collagen molecules.
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PMID:Amino-terminal propeptide of human pro-alpha 2(V) collagen conforms to the structural criteria of a fibrillar procollagen molecule. 291 27

Collagenolytic activity in ovarian follicles was previously demonstrated by using synthetic peptides and reconstituted collagen fibers. However, attempts to demonstrate degradation of ovarian collagen and to correlate collagenase activity with ovulation were not successful. By administration of L-(5-3H) proline, we have labeled ovarian and follicular collagen and followed collagenolytic activity by separation of 3H-hydroxyproline (3H-Hyp) from acid hydrolyzates of ovarian tissue by HPLC. The level of ovarian and follicular 3H-Hyp decreased by about 40% on the afternoon of proestrus or after exogenous stimulation of ovulation by human CG (hCG), and this decrease was abolished by blocking the surge of gonadotropins with Nembutal. To verify that the observed reduction in 3H-Hyp was due to the action of a typical collagenase, the collagenous fraction was prepared from ovarian tissue and from preovulatory follicles before and after the ovulatory stimulus. The extracts were treated with trypsin (25 min, 25 C, 0.01 mg/ml) plasmin and p-amino-phenyl-mercuric acetate to fully activate the collagenase extracted along with collagen. Both, enzymatic and chemical activation of collagenase in vitro resulted in degradation of collagen. This degradation could be inhibited by cysteine and EDTA; both are classic inhibitors of mammalian collagenases. The activity of ovarian collagenase increased within 3 h after hCG-stimulation, peaked at 5-fold 6 h after hCG, and declined afterwards. Administration of cysteine (0.001-0.01 mmol) into the bursal cavity of proestrous rats blocked ovulation and breakdown of ovarian collagen in a dose-dependent manner. Cysteine effectively inhibited ovulation even when injected 7 h after the hCG stimulus. Inhibitors of arachidonic acid metabolism prevent ovulation. Indomethacin (inhibitor of cyclooxygenase) and nordihydroguaiaretic acid (inhibitor of lipoxygenase) blocked ovulation and inhibited hCG-induced ovarian collagenolysis. Collectively, these results corroborate the essential role of collagenolysis in follicular rupture in mammals.
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PMID:The involvement of collagenolysis in ovulation in the rat. 298 65

An assay for the degradation of type IV (basement membrane) collagen was developed as a biochemical marker for neoplastic cells from chemically transformed human skin fibroblasts. Type IV collagen was isolated from basement membrane of Syrian hamster lung and type I collagen was isolated from rat tails; the collagens were radioactively labelled by reductive alkylation. Both collagens were in the native form as shown by their low sensitivity to degradation by trypsin. The abilities of normal (KD) and chemically transformed (Hut-11A) human skin fibroblasts to degrade the collagens were studied. The normal cells released a collagenase into the medium which after activation by trypsin or oxidized glutathione degraded type I collagen. Hut-11A cells also produced a collagenolytic activity that degraded type I collagen; however, no activation of the medium was required for this activity. Type IV collagen was not degraded by medium conditioned with the normal (KD) cells with or without activation. In contrast, Hut-11A cells secreted an active collagenase into the medium that degraded type IV collagen extensively. Treatment of the medium from Hut-11A cells with trypsin resulted in only a loss of activity while treatment with oxidized glutathione was without effect. The degradation of type IV collagen by Hut-11A conditioned medium was linear for up to 1 h and the extent of degradation increased linearly with increasing amounts of conditioned medium. A cell-associated assay was performed by growing either normal or transformed cells in the presence of radioactively labelled type IV collagen and measuring the released soluble peptides in the medium. This assay also demonstrated that KD cells failed to synthesize an activity capable of degrading type IV collagen whereas Hut-11A cells degraded type IV collagen in a linear manner for up to 4 h. Human serum at very low concentrations, EDTA and L-cysteine inhibited the enzyme activity, whereas protease inhibitors like phenylmethyl sulfonyl fluoride, N-ethyl maleimide or soybean trypsin inhibitor did not inhibit the enzyme from Hut-11A cells. These results suggest that the ability to degrade specifically type IV collagen may be an important marker for neoplastic human fibroblasts and supports a role for this collagenase in tumor cell invasion.
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PMID:Degradation of type IV collagen by neoplastic human skin fibroblasts. 298 12


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