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)

Osteoclastic (OC) and osteoblastic (OB) cells were isolated by sequential collagenase digestions of new-born rat calvaria. Prostaglandin E2(PGE2) did not alter total calmodulin levels after a 5 or 60 min incubation. The calmodulin antagonists, trifluoperazine (TFP) at 10-50 microM and W-7 (50 microM) inhibited PGE2-induced increases in calcium uptake by OC cells, but had no effect on control OC or OB calcium levels. W-5 (50 microM), a chlorine-deficient analogue of W-7 with weak anti-calmodulin activity, had no effect. Compound 48/80 (100-500 micrograms/ml), a highly effective calmodulin antagonist in other systems, had no effect on PGE2-induced calcium levels or control calcium uptake. There was inhibition of PGE2-induced increases in cyclic AMP by compound 48/80 (100 micrograms/ml) in both OC and OB cells but no effect on control levels. TFP at 50 microM inhibited both control and PGE2-induced increases in cyclic AMP but at 10 microM it lessened only the hormone-induced effect. W-7 (100 microM) inhibited PGE2-induced increases in OC and OB cyclic AMP but had no effect on control levels; W-5 (50 microM) had no effect on either of these. Dibutyryl cyclic AMP had no effect on control calcium uptake, PGE2-induced increases or W-7 inhibition of the PGE-2 effect on calcium uptake. The calmodulin antagonists, at doses which had affected only PGE2-induced increases in calcium uptake and/or cyclic AMP production, had no effect on leucine uptake by OC or OB cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effects of calmodulin antagonists on prostaglandin E2-induced responses in rat calvarial bone cells. 301 87

The adrenal gland of the human fetus (HFA) is relatively large compared to that of the adult and exhibits an extremely high rate of steroidogenesis both in vivo and in vitro. The fetal zone cells make up 80-85% of the volume of the HFA and are the major site of steroid production during fetal development. We have recently demonstrated that calcium is involved in the regulation of steroidogenesis in fetal zone cells of the HFA. There is considerable evidence that many actions of calcium within cells are mediated by the calcium-binding protein calmodulin. The purpose of the present investigation was to determine if calmodulin also plays a role in HFA steroidogenesis. To investigate this possibility, the fetal zone was dissected from fetal adrenals of first and second trimester human abortuses. After collagenase digestion of the tissue, dispersed fetal zone cells were maintained in a Krebs-Ringers medium at 37 C for a 3-h incubation. Cells were incubated with and without ACTH (10(-8) M) in the presence of the calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ), and calmidazolium (CAL) at concentrations of 5-100 microM. The media were assayed for contents of dehydroepiandrosterone sulfate (DS), cortisol (F), pregnenolone, and cAMP by RIA. The addition of ACTH stimulated F secretion 5- to 10-fold compared to that in control fetal zone cells. DS secretion increased up to 5-fold and pregnenolone about 2-fold in the presence of ACTH compared to values in control cells. ACTH also stimulated cAMP secretion by 10-fold compared to that in control cells. The addition of TFP, CPZ, and CAL significantly inhibited ACTH-stimulated DS, F, and pregnenolone secretion in a dose-related fashion to near-control levels. We observed that TFP, CPZ, and CAL inhibited cAMP accumulation as well as Bu2cAMP-stimulated steroid secretion. The metabolism of 22R-hydroxycholesterol to pregnenolone was inhibited by TFP and CPZ, but not by CAL. These studies suggest that calmodulin plays a role in regulating steroidogenesis in fetal zone cells of the HFA.
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PMID:The role of calmodulin antagonists on steroidogenesis by fetal zone cells of the human fetal adrenal gland. 302 95

A rat brain cDNA clone containing an open reading frame encoding the neuron-specific protein synapsin I has been sequenced. The sequence predicts a protein of 691 amino acids with a mol. wt of 73 kd. This is in excellent agreement with the size of rat brain synapsin Ib measured by SDS--polyacrylamide gel electrophoresis. Inspection of the predicted primary structure has revealed the probable sites for synapsin I phosphorylation by the cAMP-dependent and Ca2+/calmodulin-dependent protein kinases. All of the biochemically observed intermediates of synapsin I digestion by collagenase can be verified by inspection of the sequence, and the collagenase-resistant fragment has been defined as the amino-terminal 439 amino acids of the molecule. Predictions of sequence secondary structure and hydrophobicity suggest that a central domain of approximately 270 amino acids may exist as a folded, globular core. The carboxyl-terminal domain of the protein (the region sensitive to collagenase digestion) contains sites for Ca2+/calmodulin-dependent protein kinase phosphorylation. These sites are flanked by three regions of repeating amino acid sequence that are proposed to be the synaptic vesicle-binding domain of synapsin I. This region also shares homology with the actin-binding proteins profilin and villin. The characteristics of the synapsin I sequence do not support extensive homology with the erythrocyte cytoskeletal protein 4.1.
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PMID:Determination and analysis of the primary structure of the nerve terminal specific phosphoprotein, synapsin I. 302 73

Unstimulated (basal) as well as luteinizing hormone (LH)-promoted progesterone production in collagenase-dispersed hen granulosa cells was inhibited in a dose-related manner by two phenothiazines, trifluoperazine (TFP) and chlorpromazine (CP), both of which are known calmodulin antagonists. Using TFP, the more potent antagonist of the two, it was found that LH-stimulated cyclic AMP production was also suppressed. Moreover, TFP attenuated the steroidogenic effects of both 8-bromo-cyclic AMP and isobutylmethylxanthine but had no effect on the conversion of pregnenolone to progesterone. The inhibitory effects of TFP on steroidogenesis were reversible. It is concluded that phenothiazines inhibit steroidogenesis in ovarian granulosa cells by acting at multiple sites both proximal and distal to cyclic AMP generation without influencing the enzyme complex responsible for the conversion of pregnenolone to progesterone. The results are discussed in relation to calmodulin- and non-calmodulin-mediated actions of phenothiazines.
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PMID:Trifluoperazine inhibits progesterone and cyclic AMP production in granulosa cells of the hen (Gallus domesticus). 303 Aug 74

Calmodulin-dependent protein kinase II (CaM kinase II) is associated with microtubule preparations and phosphorylates several endogenous proteins including microtubule-associated protein 2, tubulin, and an 80,000-dalton protein doublet (pp80). We now report that pp80 is identical to synapsin I by all criteria studied including molecular weight, isoelectric point, phosphopeptide mapping of cAMP- and calmodulin-dependent phosphorylated protein, comigration with authentic synapsin I, and sensitivity to digestion with collagenase. Synapsin I and CaM kinase II were found in association with both microtubule preparations and preparations enriched in neurofilaments. Antibodies to synapsin I specifically labeled neurofilaments prepared in vitro. Immunocytochemical studies on rat brain tissue demonstrated synapsin I immunoreactivity specifically associated with the neuronal cytoskeleton as well as synaptic vesicles. The observed synapsin I staining on cytoskeletal elements was considerably diminished or abolished by the inclusion of Triton X-100 in the staining solutions. These results indicate that synapsin I is associated with the cytoskeleton and may be an important link between cytoskeletal elements as well as between the cytoskeleton and membrane.
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PMID:Association of synapsin I with neuronal cytoskeleton. Identification in cytoskeletal preparations in vitro and immunocytochemical localization in brain of synapsin I. 308 74

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

In the present study, human islets were isolated by collagenase digestion from the pancreases of three kidney donors. Maintainance of the islets in tissue culture enabled insulin release, glucose oxidation and Ca2+ -calmodulin-dependent protein phosphorylation to be determined using the same islets. Increasing glucose over a range 0-20 mmol/l resulted in a sigmoidal stimulation of insulin release (28.8 +/- 5.2 to 118.4 +/- 25.8 microU . islet-1 . h-1, n = 10; threshold less than 4 mmol/l). There was a marked correlation between the insulin secretory response of the islets to glucose and their rate of glucose oxidation (5.9 +/- 0.3 at glucose 2 mmol/l up to 25.8 +/- 1.8 pmol . islet-1 . h-1 at 20 mmol/l, r = 0.98). N-acetylglucosamine (20 mmol/l) failed to elicit a secretory response from the islets. Stimulation of insulin secretion by glucose was dependent upon the presence of extracellular Ca2+. Extracts of the islets contained a Ca2+ -calmodulin-dependent protein kinase which phosphorylated a 48-kdalton endogenous polypeptide. Myosin light-chain kinase activity was demonstrated in the presence of exogenous myosin light chains. This report demonstrates for the first time the sigmoidal nature of glucose-stimulated insulin release from isolated human islets, and its correlation with enhanced glucose oxidation. Furthermore, this is the first report of the presence of Ca2+ -dependent protein kinases in human islets.
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PMID:Properties of isolated human islets of Langerhans: insulin secretion, glucose oxidation and protein phosphorylation. 388 20

To characterize the role of Ca2+ in cholinergic stimulation of lacrimal gland protein secretion, the effects of inhibitors of cellular Ca2+ handling on protein secretion were investigated. Protein secretion was measured from rat exorbital glands using either pieces of gland in perifusion or acini isolated by collagenase digestion. Peroxidase was used as a measure of protein secretion. An inhibitor of Ca2+ influx via voltage sensitive Ca2+ channels (verapamil) at 10(-5) and 5 X 10(-5) M did not alter protein secretion stimulated by the cholinergic agonist carbachol at 10(-5) M. Inhibition of Ca2+ efflux via Na+/Ca2+ exchange by removal of extracellular Na+ or by inhibition of Na+-K+-ATPase activity using ouabain (10(-3) M) or extracellular K+ removal did not stimulate protein secretion. In contrast, inhibition of Ca2+ release from intracellular stores with TMB-8 at 100 micron completely blocked protein secretion stimulated by carbachol at 10(-5) M. Similarly, the Ca2+/calmodulin (CaM) antagonists W-13 and W-12 decreased carbachol-induced protein secretion with potencies similar to those which inhibit Ca2+/CaM dependent processes. We conclude that cholinergic agonists stimulate lacrimal gland protein secretion primarily by mobilizing Ca2+ from intracellular stores and that one mechanism by which this Ca2+ could activate secretion is in conjunction with calmodulin.
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PMID:Role of calcium in cholinergic stimulation of lacrimal gland protein secretion. 401 36

Cultured adherent rheumatoid synovial cells with fibroblast properties release large amounts of collagenase and prostaglandin E2 (PGE2) into the medium. With age in culture and passage of the cells, the levels of collagenase and PGE2 decrease, but can be increased by a factor (MCF; mononuclear cell factor) released by cultured human blood monocyte-macrophages. The magnitude of the stimulation varies with different synovial cell strains. To determine some of the mechanisms which regulate the collagenase response, synovial cells were exposed to a cyclooxygenase inhibitor (indomethacin) and substances which alter the cytoskeleton (cytochalasin B or colchicine) or interact with Ca2+ X calmodulin (trifluoperazine). The collagenase response was retained even when PGE2 synthesis was totally blocked with indomethacin. The collagenase response, however, was blunted at high indomethacin concentrations (greater than 10 microM) and paradoxically augmented at lower indomethacin concentrations (0.001 microM). In some synovial cell strains, the blunting effect of 10 microM indomethacin was reversed by the addition of low concentrations of exogenous PGE2 (10 ng/ml). Preincubation of synovial cells for 1 or 24 hr with colchicine or cytochalasin B (1-10 microM) resulted in an augmented collagenase and PGE2 response to MCF. Cells preincubated or incubated with 1-50 microM trifluoperazine, a phenothiazine, also augmented collagenase stimulation by MCF, but, in contrast to colchicine or cytochalasin B, trifluoperazine suppressed the PGE2 response. Thus, although PGE2 and collagenase production by synovial cells may be dissociated, altering ambient PGE2 levels affected basal collagenase production and modulated the collagenase response to MCF.
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PMID:Effects of prostaglandin E2, indomethacin, trifluoperazine and drugs affecting the cytoskeleton on collagenase production by cultured adherent rheumatoid synovial cells. 608 39

Protein I, a specific neuronal phosphoprotein, has previously been shown, using rat brain synaptosome preparations, to contain multiple sites of phosphorylation which were differentially regulated by cAMP and calcium. In the present study, Protein I was purified to homogeneity from rat brain and its phosphorylation was investigated using homogeneous cAMP-dependent protein kinase and a partially purified calcium-calmodulin-dependent protein kinase from rat brain. Employing various peptide mapping techniques, a minimum of three phosphorylation sites could be distinguished in Protein I; the phosphorylated amino acid of each site was serine. One phosphorylation site was located in the collagenase-resistant portion of Protein I and was the principal target for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. This site was also phosphorylated by calcium-calmodulin-dependent protein kinase. The other two phosphorylation sites were located in the collagenase-sensitive portion of Protein I. These latter sites were markedly phosphorylated by calcium-calmodulin-dependent protein kinase, but not by cAMP-dependent protein kinase in concentrations sufficient to phosphorylate maximally the site in the collagenase-resistant portion. Thus, the phosphorylation of purified Protein I by purified cAMP-dependent and calcium-calmodulin-dependent protein kinases provides an enzymological explanation for the regulation of phosphorylation of endogenous Protein I in synaptosome preparations by cAMP and by calcium observed previously. The studies suggest that certain of the synaptic actions of two distinct second messengers, cAMP and calcium, are expressed through the distinct specificities of cAMP- and calcium-dependent protein kinases for the multiple phosphorylation sites in one neuron-specific protein, Protein I.
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PMID:Differential phosphorylation of multiple sites in purified protein I by cyclic AMP-dependent and calcium-dependent protein kinases. 625 98


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