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)

Binding of a denaturated polypeptide chain derived from chick skin collagen, the alpha 1(I) chain, by isolated membranes of human platelets has been demonstrated. The process is reversible, and time- and protein concentration-dependent. The binding is specific, with an association constant of 1.88 X 10(-6) M. Prior treatment of the isolated membranes with trypsin, chymotrypsin, and pronase, resulted in significant inhibition of the 14C-labeled alpha 1 chain binding, but neuraminidase or collagenase treatment had no effect. Dissociation of the bound radioactivity and subsequent chromatographic analyses on carboxymethylcellulose and agarose A-1.5m revealed that the alpha 1 chain was unaltered. Scatchard plot analysis suggested that there are approximately 20,000 binding sites per platelet. The binding of the alpha 1 chain was inhibited by a glycopeptide derived from alpha 1, alpha 1-CB5 and by purified glucosylgalactosyl hydroxylysine, but was not affected by other cyanogen bromide peptides of alpha 1, namely alpha 1-CB3, -CB4, -CB7, and -CB8. Kinetic studies demonstrated that inhibition by the hydroxylysine glycoside is competitive. Dose-response curves of platelet aggregation induced by alpha 1 and the binding of alpha 1 by platelet membranes correlate closely. These results indicate that there are specific binding sites for collagen alpha 1 chain on platelet membranes, and that the carbohydrate moiety of the alpha 1 chain plays a role in the binding. The findings also support the hypothesis that the chick skin alpha 1 chain mediates platelet aggregation and the release reaction by acting on platelet membranes.
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PMID:Binding of chick skin collagen alpha 1 chain by isolated membranes from human platelets. 97 74

Isolated tubules prepared by collagenase treatment of rat renal cortex retained their ultrastructural integrity and responded to added lactate and succinate with an increase in gluconeogenesis and respiration. Inhibition of the mitochondrial respiratory chain with rotenone, or energy conservation sites with oligomycin caused a marked reduction in respiration and ATP content thereby completely inhibiting net gluconeogenesis. Dissociation of gluconeogenesis from respiration was accomplished with quinolinic acid and hydrazine, inhibitors of gluconeogenesis. At 5 times 10(-3) M quinolinic acid, gluconeogenesis from succinate was inhibited approximately 50% and from lactate nearly 100%. This concentration of quinolinic acid did not affect oxygen uptake or the ATP content of tubules in the presence or absence of substrate. Hydrazine at 10(-3) M resulted in approximately 75% inhibition of glucose formation from succinate and complete inhibition from lactate without interfering with respiration or ATP content. The increased mitochondrial energy generation, as manifested by accelerated respiration was independent of gluconeogenesis. The unchanging cell ATP concentration with a higher respiratory rate upon addition of exogenous substrate bespeaks increased ATP turnover. ATP utilization for the substrate-induced enhancement of gluconeogenesis could not account for the increment in ATP hydrolysis.
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PMID:Relationship of energy production to gluconeogenesis in renal cortical tubules. 117 38

In the present study, increasing amounts of the anti-estrogen 1-(p-2-diethylaminoethoxyphenyl)-1-phenyl-2-p-methoxyphenoletha nol (MER-25) were administered to pregnant baboons (Papio anubis) to block the action of endogenous estrogen and to determine effect on placental low-density lipoprotein (LDL) uptake. Pregnant baboons were untreated (n = 8) or received MER-25 orally at a dosage of 25 (n = 10), 50 (n = 8), or 75 (n = 4) mg/kg BW daily on Days 140-170 of gestation (term = 184 days). Placentas were removed on Day 170 of gestation and villous tissue was dispersed with 0.1% collagenase. Placental cells (10(6] were incubated in Medium 199 for 12 h at 37 degrees C with increasing amounts of 125I-LDL, with or without a 100-fold excess of unlabeled baboon LDL. Mean (+/- SEM) placental uptake (ng/micrograms cell protein) of 125I-LDL was 55% (6.4 +/- 1.0), 75% (3.6 +/- 0.7), and 81% (2.7 +/- 0.2) lower (p less than 0.001) in baboons that received MER-25 in doses of 25, 50, and 75 mg/kg BW, respectively, than in untreated baboons (14.2 +/- 1.3 ng/micrograms cell protein). Maximal effect occurred with 50 mg MER-25, because LDL uptake was not further decreased with greater levels of MER-25. Dissociation constants for placental LDL uptake, as determined by Scatchard analysis, were unaltered by anti-estrogen treatment. The amount of 125I-LDL degradation by placental cells of untreated and MER-25-treated baboons was proportional to LDL uptake.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of placental low-density lipoprotein uptake in baboons by estrogen: dose-dependent effects of the anti-estrogen ethamoxytriphetol (MER-25). 187 35

A subpopulation of cells was derived from the Hs431 connective tissue sarcoma cell line which possessed high affinity (estimated Kd = 0.38-0.55 nM) binding sites for human recombinant [125I]-IL-1 alpha. Binding at 4 degrees C was slow approaching equilibrium by 4 hrs. Dissociation of [125I]-IL-1 alpha was also slow and unaffected by high concentrations of cold ligand. The binding site also underwent ligand-induced internalization at 37 degrees C. An Mr = 83,000 protein was identified in affinity crosslinking studies. Despite these similarities to previously reported IL-1 receptors, Hs431 cells did not exhibit biological responses to IL-1 which have been observed in other cell lines. IL-1 did not induce PGE2 or collagenase synthesis. IL-1 also failed to induce ornithine decarboxylase activity (ODC) or stimulate [3H]-thymidine incorporation. In contrast, the Hs431 cells did contain a functional epidermal growth factor (EGF) receptor as determined from binding studies, protein kinase activity, induction of ODC, and stimulation of [3H]-thymidine incorporation. Thus, the refractoriness of Hs431 cells to IL-1 was fairly specific and did not result from a generalized defect associated with cell transformation.
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PMID:Characterization of a high affinity interleukin-1 (IL-1) specific binding site in a human synovial sarcoma (Hs431) cell line. 216 29

Dissociation of adult cardiac myocytes by collagenase perfusion techniques requires separation of the junctional contacts that link the cells physically, electrically and metabolically in the intact heart. Gap junctions, one of three types of intercellular junction present at the cardiac intercalated disc, are not split into their component membranes when myocytes are dissociated; they are ripped from the plasma membrane of one cell, to be retained by its neighbour. Partitioning of junctions in this way might be expected to constitute a serious threat to the ionic integrity of dissociated myocytes, but in practice, high yields of functionally intact cells, suitable for experimental studies, are routinely obtained. To explain this apparent paradox, repair mechanisms, operating to seal the membrane lesions caused by gap junction tearing, have been hypothesized, but evidence for their existence has previously been lacking. Using freeze-fracture electron microscopy, the present study identifies repair sites as smooth membrane domains that are continuous with the neighbouring plasma membrane, thus forming intact seals. That these structures are not chemically-induced artefacts is demonstrated by their presence in myocytes that were frozen directly from the living state. Subsarcolemmal vesicle clusters, detected in thin sections and freeze-fracture replicas, are associated with the smooth sealing domains. These structures may represent either rounded-up fragments of mechanically disrupted membrane or structures concerned with the synthesis of new lipid. From their freeze-fracture morphology, the sealing domains appear to be lipid-rich and protein-poor. Cytochemical studies using Ruthenium Red, cationized ferritin and lectins show in addition that they have a lower content of negatively-charged membrane components than the neighbouring plasma membrane, and that the carbohydrate residues normally associated with plasma membrane glycolipids and glycoproteins are absent.
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PMID:Integrity of the dissociated adult cardiac myocyte: gap junction tearing and the mechanism of plasma membrane resealing. 235 53

A procedure was developed for purifying the globular domain NC1 of basement membrane collagen from collagenase digests of a variety of tissues. The globule (Mr = 170,000) is a hexameric structure originating from two collagen IV molecules that are cross-linked at their COOH-terminal ends. Dissociation into subunits derived from alpha 1(IV) and alpha 2(IV) chains occurs at a pH below 4 and after denaturation (8 M urea). The subunits obtained include monomers (Mr = 28,000) and two different dimers (Da,Db) which are connected by disulfide bonds (Db) and/or nonreducible bonds (Da). Almost perfect reconstitution to hexamers is obtained in neutral buffer with mixtures of the subunits or purified dimers but not with purified monomers. Stabilization by dimer formation and other physical data suggest conformationally distinct segments within the subunits, which is also supported by a repeating subdomain structure deduced from cDNA sequences. Monocline crystals of NC1 give a sufficiently detailed X-ray diffraction pattern that should permit elucidation of the three-dimensional structure of the hexamer. Antibodies raised against the globular domain react with all subunits and mainly recognize epitopes stabilized by internal disulfide bridges and/or the hexameric assembly. Immunoprecipitation tests with these antibodies demonstrated a slightly larger subunit size of NC1 in PYS-2 cell culture and the rapid release of precursor-specific segments prior to secretion from the cells. Autoantibodies against mouse tumor NC1 were produced in mice and were detected both in the blood and as tissue-bound forms (kidney, lung). The autoantibody response is accompanied by certain pathological alterations mimicking Goodpasture's syndrome. The possible relationship between the two diseases is substantiated by reaction of Goodpasture antisera with the globular domain obtained from various tissue sources.
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PMID:Structure and biology of the globular domain of basement membrane type IV collagen. 242 28

The application of flow cytometry (FCM) to solid human tumors has been hindered by the difficulty in producing high yield, viable, unaltered single cell suspensions. Carcinomas containing a high desmosomal content, such as well-differentiated squamous cell (SCC) cancers of the head and neck (H&N) region, are particularly difficult to prepare. The desire to employ FCM to study cellular DNA parameters of these tumors led to the use of a 3-methylcholanthrene induced murine SCC for the comparative testing of preparative techniques. Dissociation techniques, including mechanical, enucleation, chemical, single and combination enzymes methods, were comparatively tested. Of these, the combination enzyme treatment employing trypsin and collagenase produced the highest cell yields in the shortest time with the highest dye exclusion viability and the least expense. Several fixation systems including glutaraldehyde, paraformaldehyde, acetic acid, and ethanol were comparatively tested using percent of cell loss and quality of the DNA histograms produced as end points. Ethanol-water systems with added fetal calf serum provided minimal cell loss and high quality histograms which were stable for extended periods of time. A murine tumor, closely mimicking the histology of the human tumor of interest, may be used as a model for the determination of optimum techniques of solid tumor preparation for flow cytometric analysis.
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PMID:Solid tumor preparation for flow cytometry using a standard murine model. 244 98

Hepatocytes were isolated by application of the two-step collagenase technique to pieces of human liver. 125I-labelled alpha 2-macroglobulin-trypsin complex bound to hepatocytes at 4 degrees C with a half time of approximately 4.5 h. At near equilibrium half of the receptors were saturated at an alpha 2-macroglobulin-trypsin complex concentration of about 60 pmol 1(-1) and the Scatchard plot was linear. Dissociation of the labelled complex was slow (T1/2 = 24 h) at low receptor occupancies. At high receptor occupancies dissociation was biphasic with a rate constant (K-1) for the initial rapid phase of about 2.4 x 10(-2) min-1. Labelled alpha 2-macroglobulin-trypsin complex bound at 4 degrees C was rapidly internalized at 37 degrees C (T1/2 = 1.9 min), and in 3.5 h approximately 10% of the label was released into the medium in a trichloroacetic acid-soluble form. At 37 degrees C, 125I alpha 2-macroglobulin-trypsin was taken up by hepatocytes and trichloroacetic acid soluble radioactivity appeared in the medium following a sigmoidal curve. Similar results were obtained with 125I-pregnancy zone protein-chymotrypsin complex. At 4 degrees C, hepatocytes bound nearly equal amounts of labelled alpha 2-macroglobulin-trypsin and pregnancy zone protein-chymotrypsin complex, and a large excess (100 nmol 1(-1) of one of the macroglobulins could almost completely abolish binding of trace amounts (5-20 pmol 1(-1] of the other. The present findings strongly suggest that the hepatocyte is of major importance for removal of alpha 2-macroglobulin- and pregnancy zone protein-proteinase complex in humans, in agreement with previous results in rats and mice.
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PMID:Human hepatocytes exhibit receptors for alpha 2-macroglobulin and pregnancy zone protein-proteinase complexes. 245 24

The glomerular basement membrane is a complex extracellular matrix formed of various molecules which build a supramolecular network. The major structural components are collagen IV, laminin, heparan sulfate proteoglycan, and nidogen/entactin. Cross-reacting antibodies against laminin, nidogen, and collagen IV may occur after several infectious diseases. They are however of doubtful pathogenetic significance. The pathogenetic relevant autoantibodies in Goodpasture's syndrome and rapidly progressive glomerulonephritis with linear immunofluorescence pattern are directed against epitopes which are located on the collagenase resistant C-terminal globule NC1 of collagen IV. The human NC1 globule appears as a hexamer which dissociates into monomers and dimers under various experimental conditions. Dissociation is paralleled by a significant increase in available epitopes. Immunisation with the dissociated NC1 globule initiates a pulmo-renal syndrome in rabbits similar to the human Goodpasture's syndrome. In hereditary nephritis one of the alpha-chains which form the triple-helix of collagen IV seems to be altered within the NC1 region. This may possibly explain the typical morphologic findings in this disease as well as the reduced binding of antiglomerular basement membrane antibodies to basement membranes of kidneys in Alport's syndrome.
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PMID:[Structure and antigenicity of the glomerular basement membrane]. 248 35

Because little is known of the phagocytes of the human colon we enumerated these cells in mucosal suspensions and studied their phagocytic activity. Phagocyte rich suspensions were made by EDTA collagenase dissociation followed by elutriation centrifugation. Phagocytosis was evaluated by measuring cellular radioactivity after incubation of phagocytes with 3H-adenine labelled E coli ON2 and checked microscopically. Dissociation of normal mucosa from colorectal neoplasms yielded means of 1.9 X 10(6) eosinophils, 1.4 X 10(6) macrophages and 2 X 10(5) neutrophils per gram of mucosa. Visually normal mucosa of inflammatory states yielded 2.2 X 10(6) eosinophils, 2.3 X 10(6) macrophages and 7 X 10(5) neutrophils per gram of mucosa. Phagocyte rich suspensions of normal mucosa from tumour patients phagocytosed 21.8% of a pool of opsonised tritiated E coli ON2 and by microscopy 100% of mucosal neutrophils ingested bacteria, 83% of eosinophils were phagocytic, and 53% of macrophages contained bacteria. These results suggest that in the human colonic mucosa, the eosinophil is more abundant than the macrophage and the per cent of those cells exhibiting phagocytosis is intermediate between that of the macrophage and the neutrophil. Thus these three types of cells are actively phagocytic and share the potential for a major role in host defence against invasive enteric bacteria.
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PMID:Phagocytes in cell suspensions of human colon mucosa. 366 66


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