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)

A technique is described to detect the activity of protease inhibitors present in sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAG) containing a copolymerized enzyme substrate. The method involved (1) incorporation of substrate (gelatin or casein) into the SDS-PAG at the time of casting; (2) electrophoresis of the protease inhibitors in the presence of SDS; (3) removal of SDS by washing the gel in 2.5% (w/v) Triton X-100; (4) incubation of the gels in a solution containing the proteolytic enzyme at 37 degrees C for 16 h; and (5) staining undigested substrate with amido black. Standard inhibitors such as bovine pancreatic trypsin inhibitor (BPTI), soybean trypsin inhibitor (SBTI), alpha 1-antitrypsin inhibitor, and a protease inhibitor derived from human articular cartilage have been examined by this method and displayed sharp inhibition bands when the gels were treated with bovine trypsin, chymotrypsin, or other enzymes. The technique cannot be used for precise quantification of protease inhibitors. However, there is a relationship between the concentration of inhibitor used and the intensity of staining. By this means, it was possible to estimate the smallest amount of inhibitor that could be detected (against a particular enzyme) under a given set of conditions. Inhibition was detected when 10 ng of SBTI or 20 ng of BPTI were applied to the gels; human alpha 1-protease inhibitor could be detected at a level of 2-3 micrograms. The technique was used to investigate the effectiveness of the human cartilage inhibitor against a variety of proteolytic enzymes, including thermolysin, Pronase, neutral protease, elastase, protease VII, pepsin, bacterial collagenase, protease IV, and papain.
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PMID:Detection of protease inhibitors using substrate-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 635 99

The extracellular matrix of cultured chicken embryo fibroblasts undergoes a number of modifications during the early stages of oncogenic transformation. One alteration is increased production of a small protein (Mr approximately 21,000) which is transiently deposited in the matrix by transforming cells infected with LA24, a temperature-sensitive mutant of Rous sarcoma virus (RSV) (Blenis, J., and Hawkes, S.P. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 770-774). This protein is a major component of substratum-associated material (material which remains attached to culture dishes after removal of cells with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid). Its synthesis is stimulated by transformation of cells with NY68, another ts mutant of RSV, and also by treatment of normal, uninfected cells with the tumor promoter, phorbol myristate acetate. Accessibility of the 21-kDa protein to lactoperoxidase-catalyzed iodination indicates an exposed location within the matrix. The protein binds strongly to the culture dish and/or other matrix components. This interaction can be disrupted by sodium dodecyl sulfate but not by several nonionic detergents, unless beta-mercaptoethanol or KCl (0.5 M) are also present. High concentrations of urea or guanidine hydrochloride also remove the protein from the matrix. The 21-kDa protein is resistant to trypsin, collagenase, and the hydrolytic enzymes associated with cells transformed by the wild-type Prague A RSV but not to Pronase or chymotrypsin. A 21-kDa protein with properties similar to those described above is also detected in the medium and binds to the matrix, suggesting that a potential route of deposition of the 21-kDa protein in the matrix may be via shedding and subsequent interaction with other matrix components.
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PMID:Characterization of a transformation-sensitive protein in the extracellular matrix of chicken embryo fibroblasts. 643 99

Oval cells and biliary epithelial cells were isolated from livers of rats fed a choline-deficient diet containing 0.1% ethionine and from normal rat livers, respectively. Nonparenchymal cell suspensions prepared from these livers by collagenase perfusion followed by digestion of undissociated tissue with 0.1% collagenase, 0.1% Pronase, and 0.004% DNase I were separated into six fractions by centrifugal elutriation. Cells in each fraction were characterized histochemically for gamma-glutamyl transpeptidase, peroxidase, alkaline phosphatase, and glucose-6-phosphatase activities, and for albumin and alpha-fetoprotein by immunocytochemical methods. Cells from Fraction 5 of the elutriation procedure had various features predicted for oval cells and were selected for further studies. The cell yield in this fraction, from each preneoplastic liver, was 5.7 X 10(7) cells, 93 +/- 2% of which were gamma-glutamyl transpeptidase positive, 6 +/- 1% peroxidase positive, 61% albumin positive, and 29% alpha-fetoprotein positive. Cells in this fraction have a median diameter of 13.1 micron and are diploid and cycling. The majority of these cells has morphological features characteristic of biliary epithelial cells, although some cells display features intermediate between duct cells and hepatocytes. Nucleic acid hybridization using specific probes revealed that these cells contain albumin and alpha-fetoprotein messenger RNAs, while hepatocytes from normal and preneoplastic liver contain only albumin messenger RNA. Biliary cells obtained from normal livers do not contain albumin messenger RNA. The large-scale purification and characterization of cell populations from preneoplastic livers is an important step in elucidating the cellular derivation of liver tumors.
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PMID:Isolation of oval cells by centrifugal elutriation and comparison with other cell types purified from normal and preneoplastic livers. 669 43

Activation of bovine plasma prekallikrein was investigated with several proteinases. Highly purified bovine plasma prekallikrein was rapidly activated to kallikrein [EC 3.4.21.8] by bovine activated Hageman factor, trypsin [EC 3.4.21.4] and Pronase P (proteinases from Streptomyces griseus) and more gradually by papain [EC 3.4.22.2] and ficin [EC 3.4.22.3]. Activation of prekallikrein was also observed with bovine plasmin [EC 3.4.21.7], but not with bovine clotting factors Xa (Stuart factor) [EC 3.4.21.6] and IXa (Christmas factor) or thrombin [EC 3.4.21.5]. Urokinase [EC 3.4.99.26], Reptilase, collagenase [EC 3.4.24.3], elastase [EC 3.4.21.11], alpha-chymotrypsin [EC 3.4.21.1], Nagarse [EC 3.4.21.14], and stem bromelain [EC 3.4.22 4] did not convert prekallikrein to kallikrein. Plasma kallikrein activated to Hageman factor released kinin rapidly from bovine high molecular weight (HMW) kininogen. However, from bovine low molecular weight (LMW) kininogen, liberation of kinin was extremely slow. The kallikrein activity was inhibited by soybean trypsin inhibitor (SBTI), Trasylol, diisopropylfluorophosphate (DFP), and N-alpha-tosyl-L-lysine chloromethylketone (TLCK), but not by egg-white trypsin inhibitor (EWTI), lima bean trypsin inhibitor (LBTI), heparin or hexadimethrine bromide (Polybrene). The kallikrein formed an enzyme-inhibitor complex with SBTI and Trasylol, but not with LBTI. Prekallikrein did not react with SBTI. Prekallikrein consists of a single polypeptide chain of molecular weight about 90,000, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Activation of prekallikrein by Hageman factor was found to involve cleavage of the single peptide bond on the disulfide-bridged polypeptide chain, and no change of molecular weight was observed during the activation. The peptide bond cleaved in prekallikrein by the activation was an Arg-X peptide bond on a disulfide-bridged polypeptide chain.
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PMID:Studies on prekallikrein of bovine plasma. II. Activation of prekallikrein with proteinases and properties of kallikrein activated by bovine Hageman factor. 676 24

Cationized ferritin (CF) was injected interstitially to study the distribution of anionic sites on the basement membrane and abluminal aspect of the endothelium in the fenestrated capillaries of the mouse pancreas and intestinal mucosa. Extensive, but uneven removal of the basement membrane was obtained by collagenase perfusion of the vasculature before CF labeling. In the absence of collagenase treatment, CF label was essentially restricted to the lamina rara externa of the basement membrane and occurred in clusters distributed in a relatively ordered planar lattice. After collagenase digestion, labeling of the lamina rara interna and of the abluminal aspect of the endothelium became possible. In the lamina rara interna, the CF label occurred in clusters with a distribution comparable to that found in the lamina rara externa. On the abluminal aspect of the endothelium, the plasmalemma proper was extensively, though variably, labeled. Coated pits were heavily labeled, whereas the membranes and stomatal diaphragms of plasmalemmal vesicles and transendothelial channels remained free of CF decoration. In contradistinction with the heavy labeling of their luminal aspects, the abluminal surface of the fenestral diaphragms were free of any CF decoration. Pronase treatment removed all anionic sites detectable by CF binding. The findings establish the existence of differentiated microdomains on the abluminal aspect of the endothelial plasmalemma and suggest that the capillary wall selects permeant macromolecules according to charge, in addition to size.
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PMID:Preferential distribution of anionic sites on the basement membrane and the abluminal aspect of the endothelium in fenestrated capillaries. 681 7

We have distinguished three fractions of acetylcholinesterase (AcChoE; acetylcholine acetylhydrolase, EC 3.1.1.7) from Torpedo marmorata electric organs, according to their solubilization characteristics. The low-salt-aggregating collagen-tailed forms are soluble in high-salt buffers; their hydrodynamic properties ae not modified in the presence of detergents. They constitute the A fraction, which amounts to about a third of the tissue's AcChoE activity. The low-salt-soluble (LSS) and detergent-soluble (DS) fractions are not sensitive to ionic strength and collagenase. In the presence of nonionic detergents or bile salts, both fractions behave as a monodisperse "6.3S" form, the properties of which have been investigated mostly in the case of Triton X-100. Disulfide bond reduction dissociates the detergent form into a smaller "5S" form. These two forms are thought to be, respectively, detergent-associated dimers and monomers. In the absence of detergent, the LSS fraction is polydisperse: it contains a major 8S component, 11S and 14S components, and faster-sedimenting aggregates, which appear to represent dimers, tetramers, and higher polymers. The heterogeneity of the 8S component in gel filtration suggests that it also contains variable noncatalytic elements. Upon removal of the detergent the DS fraction forms ill-defined aggregates. Trypsin induces quaternary rearrangements of part of the 8S component into 11S and 14S components, which are still convertible into the detergent form; therefore trypsin probably digests noncatalytic elements. Pronase and proteinase K, on the other hand, convert the enzyme into a dimeric form, G2, that does not interact with detergents, probably by cleaving a minor fragment of the subunit that is involved in hydrophobic interactions.
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PMID:Collagen-tailed and hydrophobic components of acetylcholinesterase in Torpedo marmorata electric organ. 693 97

Parenchymal and non-parenchymal cells were isolated from the livers of control, starved, Zn2+-injected and Cd2+-injected rats. Parenchymal cells were prepared by differential centrifugation after perfusion of the liver with collagenase. Non-parenchymal cells were separated from parenchymal cells by unit-gravity sedimentation and differential centrifugation. Yields of 2 x 10(8) non-parenchymal cells with greater than 95% viability and less than 0.2% contamination with parenchymal cells were obtained without exposing cells to Pronase. Metallothioneins-I and -II were identified in parenchymal cells and non-parenchymal cells from Zn2+-treated rats. The metallothionein contents of parenchymal cells, non-parenchymal cells and intact liver were quantified by a competitive 203Hg-binding assay. Administration of heavy-metal salts significantly increased the metallothionein content of both cell populations, although the concentration of the protein was approx. 2.5-fold greater in parenchymal cells than in non-parenchymal cells. Overnight starvation increased the metallothionein content of parenchymal cells without altering that of non-parenchymal cells. The potential significance of this differential response by different liver cell types with regard to the influence of Zn2+ on stress-mediated alterations in hepatic metabolism is discussed.
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PMID:Identification of metallothionein in parenchymal and non-parenchymal liver cells of the adult rat. 711 46

1. Rates of fluid endocytosis of rat liver, spleen, hepatocytes and sinusoidal liver cells have been determined, by using 125I-labelled poly(vinylpyrrolidone) as marker. Poly(vinylpyrrolidone) was injected intravenously into rats, and plasma clearance and uptake by liver and spleen were estimated. From these data, rates of fluid endocytosis of 1.2 and 1.8 ml of plasma/g of protein per day were calculated for liver and spleen respectively. Essentially the same results were found in nephrectomized rats. 2. Hepatocytes and sinusoidal cells were separately isolated by the collagenase/Pronase method, and sinusoidal cells were further fractionated by centrifugal elutriation. Hepatocytes, sinusoidal cells, Kupffer cells and endothelial cells showed rates of fluid endocytosis of 0.96, 9.0, 19 and 13 ml of plasma/g of cell protein per day respectively. Total-body X-irradiation did not influence uptake of poly(vinylpyrrolidone) by spleen, indicating that spleen lymphocytes are not significantly involved in fluid endocytosis. 3. For liver a rate constant of exocytosis of 5% per day was found, whereas for spleen no significant loss of accumulated label could be demonstrated during a 21-day period. 4. Distribution of label over a great number of organs and tissues was measured 9 days after the injection. Liver, skin, bone and muscle together contained about 70% of the label present in the carcass; only spleen and lymph nodes contained more label per g fresh weight of tissue than liver.
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PMID:Fluid endocytosis by rat liver and spleen. Experiments with 125I-labelled poly(vinylpyrrolidone) in vivo. 723 29

The formation of adducts between methyltetrahydrophthalic anhydride (MTHPA), an important industrial chemical and potent allergen, and collagen from guinea pig lung tissue was investigated. Collagen peptides were obtained from the lung tissue by homogenization, defatting, washing, and digestion with collagenase. In experiments in vitro, lung tissue was exposed to 8.4 mumol (50 microCi) of 14C MTHPA. The amount of adducts was 97 nmol MTHPA/g of wet tissue as determined from the bound radioactivity. In a study in vivo, four guinea pigs were injected intratracheally with 8.4 mumol of 14C MTHPA each. The amount of adducts was 0-1.2 nmol MTHPA/g of wet tissue (determined by bound radioactivity). N epsilon-methyltetrahydrophthaloyl-L-lysine (MTHPL) was synthesized and characterized by NMR, UV, and mass spectrometry (MS). A method to analyze MTHPL, after derivatization with methanol and pentafluorobenzoyl chloride, using gas chromatography-MS was developed. Analysis of Pronase-digested MTHPA-exposed lung tissue showed a concentration of 19 nmol MTHPL/g wet lung in vitro and between 0 and 0.15 nmol MTHPL/g wet lung in vivo. Thus, 20% in vitro and 12-15% in vivo of the bound radioactivity was found as adducts with lysine. These results are a first step toward studies of allergenic epitopes in proteins and methods for biological monitoring of exposure to acid anhydrides.
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PMID:Lysine adducts between methyltetrahydrophthalic anhydride and collagen in guinea pig lung. 748 35

Tritiated N alpha-acetyl-L-lysine chloromethyl ketone (ALCK) was synthesized on a laboratory scale for use as an active-site-directed affinity label in the fluorographic detection of proteases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The synthesis involved acetylation of N epsilon-benzyloxycarbonyl-L-lysine chloromethyl ketone with [3H]acetic anhydride just before the removal of the benzyloxycarbonyl group. By this method, [3H]ALCK with a specific activity of 250 mCi/mmol was obtained as a crystal. Trypsin, thrombin, plasmin, papain, and clostripain were inactivated by ALCK according to first-order kinetics. For fluorographic detection of proteases, enzyme samples were allowed to react with [3H]ALCK and then resolved by SDS-PAGE. Proteases that reacted with [3H]ALCK could be detected with a sensitivity equivalent to or higher than that of Coomassie brilliant blue R-250 staining. A trypsin-like protease in Pronase, clostripain as a contaminant in a commercial preparation of Clostridium histolyticum collagenase, and cysteine proteases in Porphyromonas gingivalis could be detected.
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PMID:Synthesis of N alpha-[3H]acetyl-L-lysine chloromethyl ketone and its use in the fluorographic detection of proteases. 825 Feb 26


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