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)

Parietal cells are a major source of gastric mucosal prostaglandins in various species. We examined cholinergic stimulation of prostaglandin E2 (PGE2) release from human parietal cells; using activators of the protein kinase C we attempted to get an indirect insight into cellular mechanisms which control PGE2 release. Gastric mucosal specimens were obtained at surgery and the cells were dispersed by collagenase and pronase E. Parietal cells were enriched to 65-80% by a Percoll gradient, and were incubated for 30 min. PGE2 release into the medium (radioimmunoassay) was 74-126 pg/10(6) cells/30 min under basal conditions and was 2.6-fold increased by carbachol (10(-5) and 10(-4) M). Similarly, PGE2 release was stimulated by phospholipase C (20-200 mU/ml, 364% above basal), 1-oleoyl-2-acetyl-sn-glycerol (10(-9)-10(-5) M, 229%), 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-9)-10(-5) M, 283%) and calcium ionophore A23187 (10(-7)-10(-5) M, 219%). Simultaneous presence of A23187 and TPA synergistically induced stimulation which was slightly higher than the sum of the individual responses. N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide W-7, a putative calmodulin antagonist, inhibited TPA-induced PGE2 release at concentrations regarded specific for blocking calmodulin (IC50 = 1.5 X 0(-6) M). We conclude that in human parietal cells PGE2 is released upon cholinergic stimulation and that phospholipase C and protein kinase C are involved in the control of PGE2 release. We speculate that calmodulin might interact with a protein phosphorylated by protein kinase C to cause PGE2 release.
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PMID:Potential mediation of prostaglandin E2 release from isolated human parietal cells by protein kinase C. 222 20

We studied PGE2-release from isolated human gastric mucosal cells. Mucosa was obtained at surgery and cells were dispersed by collagenase and pronase. Centrifugation with Percoll yielded a fraction of light density cells (70-75% parietal cells; 2-4% mast cells) revealing maximal rates of PGE2-release. A radioimmunoassay was used to measure PGE2-release into the incubation medium. Calcium ionophore A23187 which aids calcium transport across membranes caused a 3.5-fold increase of PGE2-release; this effect was abolished in calcium-free incubation medium. PGE2-release was also stimulated by phospholipase C (100 mU/ml) which is known to induce phosphoinositol breakdown, as well as by 1-oleyl-2-acetyl-sn-glycerol (OAG; 10 microM) and by 12-O-tetradecanoyl-13-acetate (TPA; 10 microM) which cause direct activation of protein kinase C without preceding induction of phosphoinositol breakdown. The response to TPA was potentiated by A23187. The calmodulin antagonist naphthalene sulfonamide W 7 reduced PGE2-release in response to A23187 and TPA (IC50: 1 microM). Our data indicate that PGE2-release of human gastric mucosal cells is stimulated by calcium influx as well as by indirect (phospholipase C) and direct (OAG, TPA) activation of protein kinase C. Stimulation of PGE2-release involves calmodulin-mediated mechanisms.
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PMID:[Calcium, phospholipase C and protein kinase C stimulate prostaglandin secretion of isolated gastric mucosa cells of the human]. 347 5

Exocytosis of the secondary (2 degree) lysosomal granule is an important process in the activation of human neutrophils. Stored enzymes such as collagenase and gelatinase are released, and adhesion molecules from the granule membrane are inserted in the plasma membrane. This exocytosis is independent of azurophil granule release and respiratory burst activation. We investigated, using kinase and phosphatase inhibitors and activators of adenylate cyclase, common intracellular signalling mechanisms involved in exocytosis (vitamin B12 binding protein release) stimulated by different agonists. Exocytosis in response to tumour necrosis factor alpha (TNF alpha), phorbol myristate acetate (PMA) and the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) was inhibited by the calmodulin antagonist N-(6-amino hexyl)-5-chloro-1-naphthalene sulphonamide (W7). Neither staurosporine, H7 nor genistein was inhibitory. In contrast, the same doses of W7 synergistically enhanced the exocytosis stimulated by the tyrosine phosphatase inhibitor sodium orthovanadate, while kinase inhibition by staurosporine or genistein dose-dependently inhibited the vanadate response. Furthermore, adenylate cyclase activation with prostaglandin E2 or dibutyryl cyclic AMP, inhibited exocytosis in response to TNF alpha and FMLP, while having no effect on the release induced by vanadate or PMA. Thus, 2 degree granule exocytosis stimulated by receptor-bound ligands is calmodulin-dependent, and is independent of protein kinase activity. In contrast, exocytosis in response to tyrosine phosphatase inhibition is antagonised by calmodulin, since the response to vanadate was enhanced synergistically by W7. Thus, depending on the initial stimulus, calmodulin may promote or inhibit 2 degree granule exocytosis by human PMN.
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PMID:Human neutrophil secondary granule exocytosis is independent of protein kinase activation and is modified by calmodulin activity. 892 8

The work presented here describes an effective method for refolding recombinant tissue inhibitor of metalloproteinases-1 (TIMP-1), a 21-kDa protein with six disulphide bonds. A yield of 30 mg TIMP-1/l culture medium was obtained from a high level bacterial expression system, using a slow removal of denaturant in the presence of 0.5 M guanidine and a suitable redox buffer. This protein is identical to the wild-type species when specific activity and secondary structure (by CD) are compared. The fluorescent, hydrophobic compound 8-anilino 1-naphthalene sulphonate (ANS) was used to quantify hydrophobic binding sites on the surface of both wild-type and recombinant TIMP-1. The wild-type protein has 1 binding site with a mean Kd of 1.3 mM and the recombinant protein has 1.5 binding sites with a mean Kd of 0.39 mM. The presence of surface hydrophobic residues is confirmed by selective broadening of ethyl and aromatic signals in the 1H-NMR spectrum on the addition of the paramagnetic probe 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-N-oxy, OH-TEMPO, to wild-type TIMP-1. When wild-type TIMP-1 is incubated with the N-terminal fragment of human fibroblast collagenase prior to the addition of ANS, the number of binding sites in the system decreases to 0.5 with a Kd of 0.15 mM.
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PMID:Preparation of recombinant tissue inhibitor of metalloproteinases-1 (TIMP-1) in high yield and identification of a hydrophobic surface feature. 983 44

Seven highly informative accounts of drug discovery and design were delivered by members of an international panel of speakers. The evolution of a new drug treatment for schizophrenia commenced with the observation that chlorpromazine possessed "neuroleptic" activity and progressed through studies with various dopamine and serotonin antagonists to ultimately lead to the discovery of risperidone. The 20-amino-acid peptide bivalirudin was rationally derived from hirudin. The alkoxypolyaryl derivative LY-303366 emerged from a second-generation chemical program with the aim of improving the solubility and pharmacokinetic profile of cilofungin. Antiviral activity in the low nanomolar range, combined with oral bioavailability and a clean safety profile, resulted in the development of saquinavir mesylate as the first HIV proteinase inhibitor to become available as a marketed drug. The marimastat drug design program was based on knowledge of the collagenase cleavage site, and molecules incorporated features to allow them to coordinate with the active site zinc atom and side chains that allowed interaction with the enzyme subsites. Initial lead compounds in the anastrozole development program were azoles incorporated onto nonsteroidal estrogen-like scaffolds. Chance synthesis of a nitrile intermediate needed for the naphthalene analogues and further elaboration eventually led to this potent and selective aromatase inhibitor. Rosiglitazone emerged from an SAR program on ciglitazone in which the lipophilic cyclohexyl group was replaced by aromatic and polar groups.
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PMID:Case histories in drug discovery and design. 1561 42

A synthetic collagenase substrate containing the internal peptide sequence--Gly-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Pro--has been synthesized, with an N-terminus 4-((4-(dimethylamino)phenyl)azo)-benzoyl (DABCYL) group and C-terminus 5-[2-(acetamido)ethylamino] naphthalene-1-sulfonic acid (AEDANS) moiety resulting in internal quenching of AEDANS fluorescence. Peptide bond hydrolysis results in a large increase in fluorescence at 490 nm upon excitation at 336 nm. The substrate is cleaved exclusively by Clostridium histolyticum collagenase and is completely resistant to attack by proteases like thermolysin, proteinase K, and trypsin. K(m) and V(max) values for substrate hydrolysis by collagenase have been determined, establishing the peptide as one of the best binding substrates for the enzyme. MALDI mass spectrometry using a derivative of the substrate establishes that the sites of cleavage lie within the collagen like domain. The CD spectrum of an analog peptide lacking the donor and acceptor groups reveals spectral features that are reminiscent of weak polyproline structures.
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PMID:An internally quenched fluorescent substrate for collagenase. 1826 Jan 38