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)

1. Electrophysiological properties of the growth hormone-releasing hormone (GRH) receptor were studied in Xenopus oocytes with an intact follicle cell layer (i.e. follicular oocytes) by measuring whole-cell current using the two-electrode voltage-clamp method. 2. A slow transient outward current was elicited in oocytes, clamped at -60 mV, by the application of rat GRH but not bovine, porcine, or human GRH. 3. The response to GRH was not suppressed by blockers known to inhibit other endogenous receptors present in follicular Xenopus oocytes; blockers used were timolol (2 microM; beta-adrenergic blocker), theophylline (0.1 mM; purinergic blocker) and atropine (100 nM; muscarinic blocker). 4. The current response evoked by rat GRH occurred in a dose-dependent manner. The concentrations of GRH for threshold and maximum responses were 1 and 100 nM respectively and the estimated EC50 (half-maximal effective concentration) was approximately 7 nM. The amplitude and conductance of the response became larger and the latency, time-to-peak and half-decay time were shortened when the concentration of GRH was increased. 5. The GRH response was reversibly inhibited by a K+ channel blocker, tetraethylammonium+ (TEA+; 20 mM). The reversal potential for the GRH response was around -100 mV and was compatible with the reported value for a K+ current in Xenopus oocytes. Furthermore, a depolarizing shift of 40 mV in the reversal potential was observed when the external K+ concentration was increased from 2 to 10 mM, agreeing with the Nernst equation. In contrast, no significant shift in the reversal potential was observed by changing the external concentration of Na+ or Cl-. 6. The GRH response was not suppressed in oocytes treated with an acetoxy-methyl ester of bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM; 10 microM) which penetrates the cell membrane and chelates internal Ca2+. 7. The GRH response was potentiated by pre-treatment with forskolin (0.4 microM; 5 min), which stimulates adenylate cyclase and increases the internal concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP). 8. The GRH response was not obtainable when follicle cells surrounding oocytes were removed mechanically with forceps or enzymically with collagenase (i.e. denuded oocytes). The response was also suppressed when gap junctions, which electrically couple follicle cells and the oocyte, were blocked by 1-octanol (1 mM). 9. The first amino acid is considered to be important for the binding of peptide ligands to their receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A potassium current evoked by growth hormone-releasing hormone in follicular oocytes of Xenopus laevis. 182 42

Changes in interstitial collagenase activity in the rat uterine cervix during ripening were clarified in a time-dependent manner. Premature delivery was induced by an antiprogesterone agent, RU486, for rats in late pregnancy. The presence of interstitial collagenase in the extract from the rat cervical tissue was demonstrated, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using the natural and unaffected collagen as a substrate. The collagenase activity was determined as the release of digested peptides from the radio-labeled collagen. Our experiments with RU486 were performed in rats on the 18th day of pregnancy. A single administration of RU486 (15 mg/kg) resulted in the premature delivery of all treated rats within 30 h after the injection (average time was 23.9 h). The marked increase in cervical wet weight was observed up to the time to premature delivery along with a significant acceleration from 18 h after the administration of RU486. In this state, the cervical collagenase activity was enhanced, the highest levels being recorded at 21 h after the administration. The interstitial collagenase in the uterine cervix appears to play a significant role in the regulation mechanisms of cervical ripening in late pregnant rats.
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PMID:Effects of RU486 on the interstitial collagenase in the process of cervical ripening in the pregnant rat. 184 68

The purification and cloning of a novel metalloproteinase inhibitor (MI or TIMP-2) related to tissue inhibitor of metalloproteinases (TIMP) has been recently described by our laboratory (DeClerck, Y.A., Yean, T. D., Ratzkin, B.J., Lu, H.S., and Langley, K.E. (1989) J. Biol. Chem. 264, 17445-17453; Boone, T.C., Johnson, M.J., DeClerck, Y.A., and Langley, K.E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2800-2804). We have transfected Chinese hamster ovary cells with a vector containing human MI/TIMP-2 cDNA and purified recombinant-derived MI/TIMP-2 (rMI/rTIMP-2) from the conditioned medium of such cells. We have investigated the inhibitory activity of rMI/rTIMP-2 toward rabbit fibroblast interstitial collagenase. The inhibition of activated collagenase by rMI/rTIMP-2 is stoichiometric and consistent with the formation of a 1:1 molar ratio complex. In addition to blocking the activated enzyme, rMI/rTIMP-2 inhibits the conversion of 52-kDa procollagenase to the 42-kDa active enzyme initiated by organomercurials. When plasmin is used as activator, rMI/rTIMP-2 does not inhibit the plasmin-mediated conversion of the 52-kDa proenzyme to the 46-kDa inactive intermediate but blocks further conversion of the 46-kDa intermediate to the 42-kDa active enzyme. The data indicate that rMI/rTIMP-2 blocks the autoproteolytic activation of procollagenase. Also, rMI/rTIMP-2 forms complexes with the 52-kDa procollagenase, the 46-kDa intermediate, and with the 42-kDa activated enzyme which are stable to sodium dodecyl sulfate (SDS), such that the complexes can be visualized by SDS-polyacrylamide gel electrophoresis. It appears that the formation of a SDS-stable complex with procollagenase requires an initial conformational change of the procollagenase brought about by organomercurials or by plasmin cleavage. The data suggest that MI/TIMP-2 may be able to control the extracellular action of certain metalloproteinases not only at the level of the activated enzyme but also at the level of proenzyme activation.
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PMID:Inhibition of autoproteolytic activation of interstitial procollagenase by recombinant metalloproteinase inhibitor MI/TIMP-2. 184 92

Mouse colon 26 tumor cells were shown to produce collagenase inhibitor in culture. The inhibitor was purified more than 2,000-fold from the culture medium by passage through DE-52 cellulose, CM-52 cellulose, Ultrogel AcA 54, Con A-Sepharose, and Sephadex G-50 Superfine columns. The inhibitor did not bind to Con A-Sepharose as do most other collagenase inhibitors. The inhibitor showed a single band (Mr = 20.5 k) on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and inhibitory activity against interstitial collagenases and gelatinases, except for bacterial collagenase. Double-immunodiffusion analysis using monospecific anti-serum against tissue inhibitor of metalloproteinases (TIMP) from bovine dental pulp showed that colon 26 inhibitor did not cross-react immunologically with the pulp inhibitor. NH2-Terminal protein sequence data were obtained for the first 36 residues of the colon 26 inhibitor, and the first 20 of them exhibited a sequence almost identical with that of a new TIMP recently designated as TIMP-2.
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PMID:Purification and characterization of a new tissue inhibitor of metalloproteinases (TIMP-2) from mouse colon 26 tumor cells. 166 27

Sodium hyaluronate reduces adhesions after tendon repair in rodents and dogs, and has been used in limited clinical trials in people. To evaluate its effect on tendon healing and adhesion formation in horses and to compare these effects with those of a compound of similar visco-elastic properties, a study was performed in horses, using a model of collagenase injection in the flexor tendons within the digital sheath. Eight clinically normal horses were randomly allotted to 2 groups. Adhesion formation between the deep digital flexor tendon and the tendon sheath at the pastern region was induced in the forelimbs of all horses. Using tenoscopic control, a 20-gauge needle was inserted into the deep digital flexor tendon of horses under general anesthesia and 0.2 ml of collagenase (2.5 mg/ml) was injected. The procedure was repeated proximally at 2 other sites, spaced 1.5 cm apart. A biopsy forceps was introduced, and a 5-mm tendon defect was created at each injection site. Group-A horses had 120 mg of sodium hyaluronate (NaHA) gel injected into the tendon sheath of one limb. Group-B horses had methylcellulose gel injected at the same sites. The contralateral limbs of horses in both groups served as surgical, but noninjected, controls. Horses were euthanatized after 8 weeks of stall rest. Ultrasonographic evaluation revealed improved tendon healing after NaHa injection, but no difference in peritendinous adhesion formation. Tendon sheath fluid volume and hyaluronic acid (HA) content were greater in NaHA-treated limbs. Gross pathologic examination revealed considerably fewer and smaller adhesions when limbs were treated with NaHA. However, significant difference in pull-out strengths was not evident between NaHA-treated and control limbs. Histologically, the deep digital flexor tendon from the NaHA-treated limbs had reduced inflammatory cell infiltration, improved tendon structure, and less intratendinous hemorrhage. Treatment with methylcullulose had no significant effect on tendon healing, adhesion size, quantity, or strength or on the volume and composition of the tendon sheath fluid. Sodium hyaluronate, administered intrathecally, appears to have a pharmaceutically beneficial action in this collagenase-induced tendinitis and adhesion model in horses.
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PMID:Effects of sodium hyaluronate on tendon healing and adhesion formation in horses. 185 4

1. Single smooth muscle cells from the fundus region of the guinea-pig stomach, which showed contractile responses to acetylcholine (ACh) at concentrations greater than or equal to 10(-7) mol/l, were obtained by enzymatic digestion using highly purified collagenase and papain. They were studied by recording membrane currents under voltage clamp with the patch pipette technique in the whole-cell configuration at 25-28 degrees C. 2. By applying voltage jumps from negative holding levels (-70 to -60 mV) to more positive levels, we identified two major activating currents: an initial inward Ca2+ current (ICa) was followed, and partly overlapped, by an outward K+ current (IK). 3. Cholinergic effects on membrane currents were investigated in the range of negative membrane potentials by determining current-voltage relations in the absence of ACh and during its continuous presence in the bathing fluid. 4. ACh induced a decrease in the steady-state conductance which was reversibly blocked by atropine. At physiological external K+ concentration [( K+]o = 6 mmol/l), the reversal potential (Erev) of the current suppressed by ACh (3 x 10(-6) mol/l) was about 20 mV more positive than the calculated K+ equilibrium potential (EK). 5. When [K+]o was increased, Erev was shifted positively; but at each [K+]o, Erev was more positive than EK. 6. Like ACh (10(-6) mol/l), tetraethylammonium (TEA, 1 mmol/l) also suppressed a current with a reversal potential that was, at physiological [K+]o, 20 mV more positive than EK. ACh (10(-5) mol/l) applied in the presence of 1 mmol/l TEA suppressed a pure K+ current (Erev = EK), which was also suppressed by 10 mmol/l TEA. 7. When K+ in the pipette and in the bathing solution was completely replaced by Na+, both ACh (10(-5) mol/l) and TEA (1 mmol/l) caused a reduction of the membrane conductance that appeared to be identical. TEA added to the bathing solution in the presence of ACh did not produce a significant additional conductance decrease. These results did not depend on whether Cl- was present as a charge carrier or not. 8. It is concluded that in fundus muscle of the guinea-pig stomach a major mechanism underlying muscarinic activation is a decrease of a K+ conductance. In addition the results indicate a suppression of a small Na+ conductance which is made up by a population of channels that are also blocked by TEA.
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PMID:Suppression of steady membrane currents by acetylcholine in single smooth muscle cells of the guinea-pig gastric fundus. 188 57

The macroscopic and single-channel properties of sodium currents and membrane potential were studied in intact extensor digitorum longus (EDL) muscle fibers from mdx (C57BL/10ScSn-mdx) and normal (C57BL/10SnJ) mice. The voltage dependence of activation and inactivation were determined and the associated gating charges were calculated to determine if the lack of dystrophin associated with the mdx condition has any influence on sodium channels either directly or by effects on the membrane environment of the channel. Sodium currents were recorded from cell-attached patches on EDL muscle fibers isolated by collagenase treatment and manual dissection. Both macroscopic and single-channel currents were studied. We found no apparent difference in the sodium channel properties from the two types of muscle. In addition, microelectrode measurements in both mdx and normal muscle fibers indicated similar resting membrane potentials (Vm around -95 mV), which suggests that the normal behavior of sodium channels in the muscle sarcolemma is unaffected by the X-linked gene defect.
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PMID:Sodium current and membrane potential in EDL muscle fibers from normal and dystrophic (mdx) mice. 192 32

The in vitro succinate dehydrogenase inhibition (SDI) test was adapted to be used with microtiter plates and this microtiter SDI (mSDI) test was evaluated for clinical use of chemosensitivity testing, as compared to findings with the SDI test. The optimal conditions of the mSDI test were determined: (1) 2-5 x 10(4) cells/well; (2) enzymatic disaggregation of solid tumors with the use of a mixture of 0.2% pronase, 0.25% collagenase, 0.1% DNase for 20 min at 37 degrees C; (3) addition of 10 mM sodium succinate in the colorimetric reaction; and (4) use of dimethyl sulfoxide (DMSO) as a solvent for extraction of formazan product. Good correlations were observed between the mSDI and the SDI tests when S-180 cells (r = 0.890-0.996) or 16 human fresh tumor cells (r = 0.731-0.999) were exposed to six anti-cancer drugs (carboquone, adriamycin, mitomycin C, aclacinomycin A, cisplatin, 5-fluorouracil). Thus, the mSDI test facilitates testing of a large number of drugs with minimal amounts of specimens, and is expected to replace the SDI test for chemosensitivity testing of clinical tumor cells.
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PMID:The microtiter SDI test is more advantageous than the SDI test for assessing the chemosensitivity of human tumor cells. 195 59

PGE2 production by glomeruli is increased in a variety of glomerular diseases. Potentially, this process may affect mesangial cell protein synthesis and mesangial cell growth. Thus studies have been undertaken, using cultured human mesangial cells, to assess the effects of PGE2 on proline uptake, protein synthesis and cell proliferation. In the presence of 140 mM NaCl, incubation of mesangial cells with 0.01 to 1 microM PGE2 for 72 hours resulted in a marked decrease of 14C proline uptake, but did not modify 14C leucine uptake. Substitution of choline to sodium inhibited 14C proline uptake by 85% which became independent of PGE2, indicating that this PG specifically altered sodium-dependent proline uptake. Inhibition of this component reached 35 to 50% with 1 microM PGE2. The inhibitory effect of PGE2 on sodium-dependent proline uptake required a lag time of 48 hours, and was suppressed by ouabain, an inhibitor of Na+, K+ ATPase activity. PGE2 did not modify the Vmax of the transport system (1.007 vs. 1.023 nmol/mg/min) but increased (P less than 0.01) its Km (1.179 vs. 0.823 mM). 8-bromo-cyclic AMP also inhibited sodium-independent proline uptake, and PGE2 markedly increased cyclic AMP production. Taken together, these results suggested that PGE2 acted via cyclic AMP stimulation. PGE2 under identical conditions (1 microM, 72 hr incubation) produced a decrease in collagen synthesis estimated by the relative rate of collagen production after incubation of mesangial cells with 14C proline (percentage of 14C radioactivity in collagenase-sensitive proteins over total proteins). PGE2 also diminished the intracellular free proline pool. More generally, PGE2 inhibited cell proliferation and cell total proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of prostaglandin E2 on proline uptake and protein synthesis by cultured human mesangial cells. 196 49

Latent collagenase has been isolated in pure form from the rheumatoid synovial fluid. The final preparation, activated by trypsin, yielded a collagenase of specific activity 2,227 units/mg. Electrophoresis in sodium dodecyl sulfate polyacrylamide gels revealed a protein doublet of 54 and 50 kDa. Trypsin or HgCl2 activation resulted in disappearance of the doublet and emergence of a new doublet of 47 and 43 kDa. The latent collagenase could also be activated by leucocyte cathepsin G or plasmin. Neither the latent nor the active collagenase from synovial fluid showed any cross-reactivity with the antibodies against leucocyte collagenase. The trypsin activated collagenase degraded collagen type I, II, III giving typical cleavage products but did not degrade type IV and V collagen.
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PMID:Some properties of latent collagenase from human synovial fluid. 196 84


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