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)

Immature female Sprague-Dawley rats were primed with 20 IU eCG at 28 days of age and treated with 10 IU hCG 48 h later. Ovulation followed at 12-14 h. Ovaries were extracted at various times after hCG by use of Triton X-100 and 10 mM CaCl2 (Triton extract) followed by heating to 60 degrees C for 6 min with 0.1 M CaCl2 in 50 mM Tris/0.15 M NaCl, pH 7.5 (heat extract). These extracts were tested for their ability to inhibit tissue metalloproteinases by use of the small uterine metalloproteinase (UMP) of the rat. The ovary contains three plasma-derived inhibitors (alpha 1-macroglobulin [alpha 1 M], alpha 2-macroglobulin [alpha 2 M], and alpha 1 inhibitor3 [alpha 1I3]) and one tissue-derived inhibitor of the tissue inhibitor of metalloproteinases (TIMP) family. alpha 1 I3 was shown to inhibit UMP and rat collagenase. The concentration of the tissue inhibitor rose 5-fold from 0.6 micrograms UMP blocked per gram wet tissue in ovaries not primed with eCG to 3.2 micrograms UMP blocked at 8 h after hCG. Over this same time interval, the sum of alpha 1M + alpha 2M per gram of ovary rose 7-fold from 3.2 to 22.4 micrograms UMP inhibited and alpha 1I3 rose 2-fold from 4.4 to 10.7 micrograms UMP inhibited. The increases in the tissue inhibitor are interpreted to be due to increased synthesis by the tissue, whereas the changes in alpha-macroglobulins are postulated to be due to increased vascularity and increased permeability of the vessels.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A tissue inhibitor of metalloproteinases and alpha-macroglobulins in the ovulating rat ovary: possible regulators of collagen matrix breakdown. 172 97

Three-dimensional cellular structures formed by MCF-7 human mammary carcinoma cells within collagen gels were isolated with collagenase and cultivated on plastic substratum to examine whether the cytoskeleton specific for cells forming cellular structures (S-type) changes to that specific for cells grown as monolayers (M-type). The cytoskeleton isolated as 0.05% Triton-insoluble fraction from the cellular structures after culture for 1 day on plastic was exclusively S-type. However, both types of cytoskeletons were observed in the cellular structures cultivated for 7 days on plastic as well as in the cells grown as monolayers for 2 days after dissociation of the cellular structures with trypsin. By use of an antibody raised against a 65-kD polypeptide that was specific for the M-type cytoskeleton, the presence of the polypeptide was found to be restricted to the cells grown out as monolayers from the edge of the cellular structures. In the cells grown for 2 days as monolayers, a mixture of cells both having and lacking the polypeptide was observed. After a 7-day culture of the dissociated cells as monolayers on plastic, however, most of the cells had M-type cytoskeletons. The present results show that the apparent change in the cytoskeleton of MCF-7 cells from S-type to M-type does not occur in cells involved in the three-dimensional cellular structures even in the absence of collagen gels, but that it occurs in cells which are grown as monolayers for at least 7 days on plastic substratum.
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PMID:Regulation of cytoskeletal structure in human mammary carcinoma cells MCF-7 by culture substrata. 208 49

We have developed a procedure which allows the isolation of secretion granules from fresh parathyroid glands. Following collagenase digestion of the tissue, the cells were broken with osmotic shock and a crude granule/mitochondrial pellet was obtained by differential centrifugation. Before loading this fraction onto a metrizamide density gradient it was subjected to brief sonication to disrupt the mitochondria. This procedure was necessary in order to achieve separation of the granules from the mitochondria during ultracentrifugation of the gradient. When the fractionated gradient was analysed for PTH by radioimmunoassay, three bands containing parathyroid hormone were found, at densities of 1.0, 1.05 and 1.18. Upon electron microscopic examination of the gradient fractions, granules were found only in those fractions containing hormone. A typical granule appearance was observed for two of the populations, but the third population (density 1.18), consisted of granules without membranes and which appeared less electron dense than those of populations 1 (density of 1.0) and 2 (density of 1.05). Moreover, the lack of a limiting membrane imparted a fuzzy appearance to the population 3 granules. When fresh tissue sections were examined as control samples, granules with and without membranes were also observed. Standard marker enzyme assays further confirmed that populations 2 and 3 were relatively free of other cellular contaminants, but population 1 contained endoplasmic reticulum and lysosomal material. Because the number of granules contained in this population is very small, we have not been successful in achieving further purification of population 1. Based on radioimmunoassay of extracts of each granule population, PTH was concentrated in population 3, while the other two contained lesser amounts. Interestingly, results obtained with a radioimmunoassay for SP-1 revealed a striking difference in the distribution of SP-1 in the three granule populations. This protein, which is also secreted by the parathyroid gland, was concentrated in population 1 and 2. Only very low levels were found in population 3. Thus, the two major secretory products are localized in different granule populations. The isolated granules were stable to pH changes, cycles of freeze/thaw and sonication. The yields of PTH extracted from each of the granule populations by freezing and thawing in buffer or by Triton containing solutions were low. PTH was completely extracted from each population only by using 8 M urea in HCl. Lower concentrations of urea were less effective. These results indicate that the molecular architecture of the granules is highly resistant to disruption.
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PMID:The isolation and partial characterization of bovine parathyroid secretory granules. 233 91

A preparation of the cuticle of Onchocerca volvulus was obtained by extracting worm fragments in an series of buffers with 1.5% Triton-X-100 and 3% Sodium dodecyl sulfate (SDS). Electron micrographs of worm fragments, treated with the detergents or collagenase showed that our methods had been effective in isolating the cuticle from the other organs of the parasite. The cuticular preparation was found to contain 19 different amino acids with glycine (23.4%); proline (11.23%); hydroxyproline (10%); and glutamic acid (9.4%) being the most abundant. Hydroxylysine was present in small amounts (0.04%). Total reducing sugar was determined to be 5.3 mg per gram dry weight of the preparation. The cuticular preparation was solubilized by boiling in 2-mercaptoethanol and shown by SDS-PAGE to contain at least 10 different polypeptides in the Mr range 17,000-163,000. Five of these polypeptides with apparent Mr respectively of 33,000; 67,000; 74,000, 88,500 and 114,000 were isolated by preparative gel electrophoresis and their amino acid compositions shown to be similar to that of invertebrate collagens. We conclude that the cuticle of O. volvulus contains collagen-like proteins held together by disulfide bridges.
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PMID:Isolation and biochemical composition of the cuticle of Onchocerca volvulus. 284 63

Separation of lung alveolar basement membranes from interstitial connective tissue protein has proved difficult, and a pure preparation of alveolar wall basement membranes (AWBM) is not available. We have modified a technique employing the detergent Triton X-100 for isolating AWBM from rat lungs by adding a step utilizing human skin collagenase (HSC), a highly purified enzyme obtained from skin fibroblasts that specifically cleaves non-basement membrane collagens. Triton extraction of both lungs yields 15-20 mg of basement membrane-enriched material referred to as crude fraction (CF). Ultrastructural studies show that CF includes both epithelial and endothelial basement membranes that appear similar to their in vivo counterparts and contain heparan sulfate proteoglycans. Extraction of type IV collagen is documented by the appearance of highly glycosylated hydroxylysine. This CF contains minimal amounts of contaminating elastin but significant amounts of interstitial collagens. CF was further purified for biochemical studies by incubation with HSC. HSC solubilized 20% of CF hydroxyproline resulting in a final fraction highly enriched in AWBM. Lung minces incubated in tritiated lysine produced a CF extract rich in newly formed type IV collagen, showing that lung tissue synthesizes AWBM collagen in vitro.
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PMID:Isolation and characterization of a rat lung fraction enriched in alveolar wall basement membranes. 298 61

To facilitate investigations on very small fat cell (VSFC) populations in adipose tissue, an alternate method of preparing fat tissue samples was explored. The osmium tetroxide-8M urea method, modified by addition of a 95% ethanol step in tissue processing, centrifugation between steps, and final resuspension in 55% glycerol in 0.01% Triton-saline, was compared with the collagenase method for determination of VSFC populations in Fischer 344 epididymal and Sprague-Dawley retroperitoneal adipose depots. For each method and in both depots, the average histogram of 300 adipocyte diameters, measured by microscopy, was bimodal with the nadir between 30 and 40 micron diameter. The average histogram of fat cells less than 35 micron in diameter showed a separate population of VSFC existed in each depot. The modified osmium-urea method gave better results and was easier to perform than the collagenase method. It has confirmed our earlier results and raises anew questions concerning a role for the natural existence of a VSFC population in the adipose depot.
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PMID:Very small fat cell populations determined by a modified osmium tetroxide-urea method. 299 Feb 29

AJ-FS9 is one of a new series of monoclonal antibodies (mAbs) raised by immunizing mice with isolated human sperm tail fibrous sheath (FS). Using indirect immunofluorescence (IIF) of human spermatozoa dried onto slides, the AJ-FS9 mAb reacted with the principal piece of occasional spermatozoa. Following their enzymatic treatment with trypsin, dispase or collagenase, but not sulphatase, all the spermatozoa were stained at their principal piece. The ultrastructural localization of the antigens to the FS was established by immunogold electron microscopy, which showed the distribution of gold particles on the FS outer surface of spermatozoa sequentially treated with 1% Triton and dispase; spermatozoa demembranated by Triton alone showed no reaction. For biochemical characterization, spermatozoa were lysed with 1% Triton, and the sperm pellet was run through a reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotted and immunostained with AJ-FS9. The results showed the reaction of the antibody with three protein bands with molecular masses ranging between 46 and 56 kDa. IIF screening of human testicular cryostat sections with AJ-FS9 mAb showed its reactivity with occasional sperm tails; but following their dispase treatment, all spermatozoa were stained. The restricted staining of the assembled FS of maturing sperm tails indicated the late appearance of the antigens during spermatogenesis. The antibody did not react with sperm cell precursors or other cell types within/without the seminiferous tubules. Untreated and dispase-treated frozen sections of skin, oesophagus, tongue, liver, kidney, stomach, ileum or their blood vessels showed no reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:AJ-FS9 monoclonal antibody detects masked antigens within the human sperm tail fibrous sheath. 784 12

The aim of the study was to assess the activities of the collagenases type IV (matrix metalloproteinase type 2 [MMP-2] and matrix metalloproteinase type 9 [MMP-9]), also known as gelatinases, and the local activity of interstitial collagenase (matrix metalloproteinase type I[MMP-1]) in tissue extracts from a case of the botryoid sarcoma, a rare and very malignant tumour of the female genital tract. Zymography revealed that botryoid sarcoma does not express the 92-kDa form of type IV collagenase activity in Triton extract and only weak activity in Heat extract when compared to values found in extracts from striated muscle and fibroma uteri. MMP-1 appeared in the latent form only in the Triton extract of botryoid sarcoma and its activity was lower than those found in the control tissues. These results indicate that the very rapid local invasion and systemic metastases associated with botryoid sarcoma do not depend on the activity of tumour-derived gelatinases.
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PMID:Local activity of matrix metalloproteinases in a case of botryoid sarcoma. 884 7

Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of several proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For ACE we have previously identified a metalloprotease (secretase) responsible for this proteolytic cleavage. The effect of a range of structurally related zinc metalloprotease inhibitors on the activity of the secretase has been examined. Batimastat (BB94) was the most potent inhibitor of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 microM, whereas TAPI-2 was slightly less potent (IC50 18 microM). Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2' substituent decreased the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibitory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is realted to, but distinct from, the matrix metalloproteases. The full-length amphipathic form of ACE was labelled selectively with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was able to cleave the hydrophobic anchoring domain from the bulk of the protein, there was no cleavage of full-length ACE by a Triton X-100-solubilized pig kidney secretase preparation when the substrate was in detergent solution. In contrast, the Triton X-100-solubilized secretase preparation released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipathic form of ACE when it was incorporated into artificial lipid vesicles. Thus the secretase has an absolute requirement for its substrate to be inserted in a lipid bilayer, a factor that might have implications for the development of cell-free assays for other membrane protein secretases. ACE secretase could be solubilized from the membrane with Triton-X-100 and CHAPS, but not with n-octyl beta-D-glucopyranoside. Furthermore trypsin could release the secretase from the membrane, implying that like its substrate, ACE, it too is a stalked integral membrane protein.
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PMID:Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer. 935 32

Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin output by cultures of human amnion cells. This is due to an increase in the expression of type-2 prostaglandin H synthase (PGHS-2), the inducible form of the enzyme, in these cultures. Amnion consists of an epithelial layer of cells and a subepithelial mesenchymal layer of cells. The purpose of the present study was to determine the cell-type(s) responsible for the IL-1beta-induced PGHS-2 expression in amnion cultures. Amnion was obtained at term after elective Cesarean section or vaginal delivery. Tissues were dispersed with collagenase, and cells were plated in multichamber culture slides and cultured for 7 days in media supplemented with 10% fetal bovine serum. Cell types were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Cultures contained both cell types, and the proportion of these varied considerably from one culture to another. Cells were treated with various concentrations of IL-1beta for 6 or 24 h and were then fixed in 4% paraformaldehyde. The fixed cells were permeabilized with Triton and examined by immunohistochemistry for PGHS-2 protein using specific antisera, and PGHS-2 mRNA was localized by in situ hybridization using a specific oligonucleotide probe. The cell type(s) expressing PGHS-2 was characterized using double labeling with antisera to keratin (epithelial cell marker) and vimentin (mesenchymal cell marker). IL-1beta was found to increase expression of immunoreactive PGHS-2 and PGHS-2 mRNA. This increased expression was found to occur only in the vimentin-positive cells and not the epithelial cells. These results highlight the potential importance of the subepithelial cells in the mesenchymal layer of amnion in the formation of prostaglandins during pregnancy and possibly in preterm labor with infection.
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PMID:Cellular specificity of interleukin-1beta-stimulated expression of type-2 prostaglandin H synthase in human amnion cell cultures. 978 Mar 20


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