Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A procedure for extraction and quantification of fibronectin in human aortic tissue is described in this paper. Dried, defatted samples of human aortic tissue were subjected to sequential extraction with (i) 0.89% NaCl, 10 mmol/l Tris/HCl, pH 7.4, (ii) 5 mg/ml heparin, 2 mol/l urea and (iii) collagenase digestion. More than 75% of hexosamine-containing molecules were solubilized by this procedure. Immunoblotting of extracted proteins separated by SDS-PAGE showed that extracted fibronectin had a mobility in the same range as that of plasma fibronectin. Fibronectin ELISA performed on these extracts gave dilution curves parallel to the standard curve, the sensitivity was 2.7 micrograms/l. Recoveries of a fibronectin standard added to the NaCl, heparin/urea and collagenase solutions during extraction were 97%, 90% and 84% respectively. Normal aortic tissue from 31 patients was subjected to the sequential extraction scheme and fibronectin quantification in the various extracts demonstrated that 4.52 +/- 1.79 micrograms was dissolved in the NaCl extracts, 5.41 +/- 2.28 in the heparin/urea extract and 1.08 +/- 0.43 in the collagenase digest, respectively. (Values are expressed as micrograms fibronectin/10 mg dry, defatted tissue (mean +/- SD]. Our results indicate that the ELISA method can be applied for the measurement of fibronectin in extracts of human aortic tissue. This might be useful in the study of diseases where alterations in arterial fibronectin content may be expected.
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PMID:Quantification of fibronectin in extracts of human aortae by an ELISA. 274 Aug 16

To obtain data concerning the pathology of diabetic arteries, aortas from 23 patients with diabetes mellitus [9 with insulin-dependent diabetes mellitus (IDDM) and 14 with non-insulin-dependent diabetes mellitus (NIDDM)] were collected at autopsy together with aortas from sex- and age-matched nondiabetic persons. A histomorphometric study was performed blindly on antifibronectin PAP-stained sections to determine the distribution of fibronectin-containing space in the vessels. In both IDDM and nonIDDM groups a statistically significant increase of approximately 45% was seen in the amount of stainable material in the tunica media. The increase was not influenced by the presence or absence of overlying plaque. No differences were seen between diabetic and nondiabetic vessels in the tunica intima. The content of extractable fibronectin in intima-media preparations was measured. The samples were extracted sequentially with buffered saline, a heparin-urea solution, and finally collagenase digestion. Fibronectin measured in these extracts showed that statistically significantly more of this glycoprotein was found in vessels from diabetic persons compared with nondiabetic persons, when comparing areas of the vessels without macroscopical visible plaque. However, only among IDDM patients increased amounts were apparent in plaque areas. These results indicate that diabetic patients develop structural alterations in the connective tissue of their arteries, consistent with a hypothesis of a diabetic macroangiopathy.
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PMID:Accumulation of fibronectin in aortas from diabetic patients. A quantitative immunohistochemical and biochemical study. 279 91

The contribution of toxic oxygen (O2) metabolites to ischemic renal injury is unclear because they have not been added directly to the kidney and few ways exist to effectively measure and assess the effect of these highly reactive products in biological systems. Our goal was to determine the effect of hydrogen peroxide (H2O2) or H2O2-derived products on renal function and to determine whether H2O2-mediated renal injury was reflected by consumption of dimethylthiourea (DMTU) (an exogenous O2 metabolic scavenger), depletion of renal cortical total glutathione (an endogenous O2 metabolite scavenger), and/or adenosine triphosphate (ATP). We found that addition of glucose oxidase (GO) or H2O2 to isolated perfused rat kidneys caused injury that was manifested by decreases in glomerular filtration rate, perfusion flow rate, and sodium reabsorption and that was prevented by addition of catalase (CAT) (but not inactivated CAT) or large doses of DMTU (15 mM), but not urea (15 mM). To further ascertain if the protective effect of DMTU was due to reacting with a scavenging H2O2, we conducted parallel experiments in which we measured the consumption of smaller doses of DMTU (1 mM) in kidneys perfused with GO or H2O2. We found that addition of increasing concentrations of H2O2 decreased DMTU concentration. Renal cortical total glutathione and ATP levels were also decreased by addition of GO or H2O2. In contrast to perfusion with GO or H2O2, perfusion with elastase or collagenase also caused renal injury and decreases in ATP but did not decrease DMTU concentration or tissue total glutathione. We conclude that H2O2 or H2O2-derived products are acutely toxic to the kidney and that decreases in perfusate DMTU concentration and tissue total glutathione, but not tissue ATP, may be useful for specifically assessing the presence and/or toxicity of H2O2 in renal and other biological systems.
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PMID:O2 metabolite-mediated injury in perfused kidneys is reflected by consumption of DMTU and glutathione. 282 29

To clarify the relationship of the 290 and 145 kDa chains of the epidermolysis bullosa acquisita (EBA) antigen, we subjected urea extracts of skin basement membrane zone (BMZ) proteins and isolated 290 and 145 kDa chains of the EBA antigen cut out of sodium dodecyl sulfate polyacrylamide gels to treatment with clostridial collagenase. When the reaction products were electrophoresed, transblotted, and reacted with EBA patient sera or two monoclonal antibodies to the EBA antigen, the 290 kDa chain was degraded into the 145 kDa band that was resistant to cleavage with collagenase. The 145 kDa domain, isolated after collagenase treatment of the whole BMZ extract, was resistant to degradation by hyaluronidase, chondroitinase ABC, heparinase, and heparitinase but was readily degraded by V-8 protease. These data suggest that the EBA antigen consists of collagen and noncollagen domains of identical size (Mr 145,000), and that the 145 kDa noncollagen domain is generated via degradation of the native 290 kDa species by collagenase.
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PMID:Epidermolysis bullosa acquisita antigen: relationship between the collagenase-sensitive and -insensitive domains. 282 79

A method is described for the cleavage of collagenous molecules by bacterial collagenase in the presence of sodium dodecyl sulphate (SDS) and urea. Comparison of three commercially available preparations of bacterial collagenase showed that the most efficient cleavage under these conditions was by the enzyme isolated from Achromobacter iophagus (E.C. 3.4.24.8). No non-specific proteinase activity was apparent in conditions where all collagen types showed some susceptibility to attack. This method represents a simple one-stage identification of collagenous molecules in complex mixtures of proteins and where limited amounts of a protein are available.
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PMID:Bacterial collagenase and collagen identification. 283 4

To clarify interactions between carcinoma and mesenchymal cells, we examined the extracellular matrix-substance remaining on culture dishes after confluent growths of gastric carcinoma cells were removed with EDTA. The matrix synthesized by poorly differentiated adenocarcinoma cells (cell lines KATO-III and MKN-45) cultivated in serum-free medium has a fibroblast (cell line WI38)-attachment activity, which is not detected in the matrix synthesized by a well differentiated adenocarcinoma (cell line MKN-28). This activity was not observed in KATO-III-matrix extracted with 6 M urea, but could be detected in a 1% SDS extract from the remaining matrix on the culture dishes after 6 M urea extraction. The activity was abolished by treatment with pronase (16 micrograms/ml), trypsin (0.005%) or alkali, but was unaffected by collagenase (80 micrograms/ml, 4 h) or chondroitinase ABC (1 U ml, 1 h). It is conceivable that the fibroblast-attachment activity of the matrix produced by poorly differentiated adenocarcinoma cells is related to the proliferation of interstitial connective tissue in vivo.
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PMID:Extracellular matrix of cultivated, poorly differentiated human gastric adenocarcinoma cells promotes attachment and spreading of mesenchymal cells. 290 Nov 69

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

A novel gelatin-binding 21 kDa protein was identified in the culture medium of fibroblastic and sarcoma cells by affinity chromatography on gelatin-Sepharose. Its affinity for gelatin was lower than that of the other gelatin-binding proteins, fibronectin and the 70 kDa protein, as judged by stepwise elution by urea and arginine. The protein bound also to spermine and to some extent to heparin but not to staphylococcal protein A, bovine serum albumin, concanavalin A or plain Sepharose 4B. In gel filtration chromatography the protein eluted in fractions differing from those of fibronectin and the Mr 70,000 protein and retained its ability to bind to gelatin-Sepharose, indicating that the binding was not mediated by the two other gelatin-binding proteins. It contains intrachain disulfide bridges, as judged by analysis under nonreducing and reducing conditions. The protein is composed of two major subtypes with pI values of 5.85-6.10 and 6.55-6.75. It was sensitive to trypsin but not to collagenase or thrombin. Antiserum was raised in rabbits against the gelatin-binding proteins isolated from serum-free conditioned fibroblast culture medium. The antiserum reacted with fibronectin, the Mr 70,000 protein and the Mr 21,000 protein in immunoprecipitation experiments. Absorption of the antiserum with human plasma fibronectin did not decrease its reactivity with the Mr 70,000 and 21,000 proteins. However, absorption with the Mr 70,000 protein abolished also the reactivity against the Mr 21,000 protein, suggesting immunological cross-reactivity. The protein was synthesized independently from the Mr 70,000 protein, as shown by pulse-chase labeling experiments of cells. The production of the Mr 21,000 protein in cultured cells was enhanced by transforming growth factor-beta.
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PMID:Characterization of a novel gelatin-binding 21 kDa protein secreted by cultured adherent cells. 301 29

The effects of osmolality on prostaglandin E2 (PGE2) biosynthetic capacity and the interaction between endogenous PGE2 synthesis and vasopressin (AVP)-dependent cyclic AMP generation were examined in papillary collecting ducts (PCD) microdissected from collagenase-digested rat kidneys. Increasing medium osmolality with NaCl:urea (1:2 molar ratio) progressively increased PGE2 synthesis in PCD up to 1,500 mOsm. Addition of NaCl:urea or NaCl alone were equally effective in stimulating PGE2 biosynthetic capacity in PCD. In contrast, addition of urea alone had a much smaller stimulatory effect on PGE2 synthesis. Inhibition of endogenous PGE2 synthesis with naproxen (10(-5)M) suppressed AVP-dependent cAMP formation in PCD when incubated in 300 mOsm medium but had no effect when incubated in 1,500 mOsm medium. Addition of 2.5 X 10(-5) M PGE2 also suppressed AVP-dependent cAMP formation in PCD only when incubated in 300 mOsm medium. The present study suggests that the PCD is a site of active PGE2 synthesis that is modulated by osmolality. Our results do not support the concept that endogenous PGE2 antagonizes vasopressin action via inhibition of AVP-dependent cAMP formation.
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PMID:Prostaglandin E2 synthesis in the inner medullary collecting duct of the rat: implications for vasopressin-dependent cyclic AMP formation. 302 64

Interleukin 1 (IL-1) possesses multiple biological activities that may be blocked selectively by different inhibitors. Some known inhibitors block the lymphocyte activating factor (LAF/IL-1) but not the mononuclear cell factor (MCF/IL-1) measured by its capacity to stimulate prostaglandin E2 (PGE2) and collagenase production. The presence of IL-1 in vivo may be difficult to detect due to the presence of inhibitor(s) and the level of the inhibitor(s) may vary depending upon pathological conditions. We have found that urine from three patients with monocytic leukemia (M5) contained high levels of inhibitor(s) of MCF/IL-1, whereas urine of normal subjects did not contain significant amounts. Urine from two patients with other blood neoplasic diseases also contained little inhibitory activity. The MCF/IL-1 inhibitor(s), which also acts on human recombinant IL-1 beta, is approximately 25-35 kD, is not retained on concanavalin A-Sepharose column and can be partially destroyed with urea and boiling. At this stage of the purification the fraction containing the MCF/IL-1 inhibitor(s) also inhibits the LAF/IL-1 assay. However, this inhibitor(s) is probably distinct from other inhibitors already described.
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PMID:Prostaglandin E2 and collagenase production by fibroblasts and synovial cells is regulated by urine-derived human interleukin 1 and inhibitor(s). 302 90


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