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)

The binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis. In 5mM-Tris/HCl buffer, pH 7.5, protein C bound 190 nmol of Ca2+/mg of protein. The apparent dissociation constant, K, was determined to be 1.9 x 10(-4)M and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ to protein C apparently depends on groups which ionize above pH 5.0. Ca2+ binding decreased with increased concentration of the dialysis buffer and on addition of SrCL2, MgCl2 and MnCl2 to the dialysis buffer. Digestion of protein C with trypsin or collagenase or heating of the protein to 60 degrees or 100 degrees C had little or no effect on the Ca2+ binding. Digestion of protein C with alkaline phosphatase caused a decrease in the amount of protein-bound Ca2+. This was also found for another salivary phosphoprotein, protein A. In the absence of Ca2+ the S020,w for protein C was 1.29 S and in the presence of Ca2+ it was 1.46S. Ca2+ may cause a conformational change in the protein or an aggregation of the protein molecules. No conformational changes of protein C in the presence of Ca2+ could be detected by circular dichroism or nuclear magnetic resonance.
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PMID:The binding of calcium to a salivary phosphoprotein, protein C, and comparison with calcium binding to protein A, a related salivary phosphoprotein. 1 96

A collagen complex from bovine nasal cartilage was prepared by extraction of the tissue with 3M-MgCl2 solutions, by using two different procedures. When it was compared with calf skin acid-soluble tropocollagen by polyacrylamide-gel electrophoresis, the 3M-MgCl2-soluble cartilage collagen in the complex appeared to be predominantly type I in nature, consisting of both alpha1 and alpha2 chains. The soluble cartilage collagens were digested with purified bacterial collagenase, and the soluble digests were fractionated on Sepharose 4B. Hydroxyproline-free proteoglycan was isolated in the excluded volume of the column eluate, and this was found to be an aggregate which could be dissociated to link proteins and proteoglycan subunit by equilibrium-density-gradient centrifugation in a CsCl-4M-guanidinium chloride gradient. Interaction with calf skin-soluble tropocollagen was studied by CM-cellulose chromatography. The link-protein system did not interact, but proteoglycan from the bottom of the gradient did interact. In addition, when proteoglycan subunit was allowed to interact with collagen, there was a preferential binding to the alpha2 and beta12 components, and this effect was also observed with the proteoglycan material obtained from the collagenase digests of 3M-MgCl2-soluble cartilage collagen complexes. However, specificity for alpha2 and beta12 chains was not exhibited by chondroitin sulphate glycosaminoglycan, and it is therefore concluded that preference for alpha2 and beta12 chains is a function of the intact proteoglycan structure.
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PMID:The isolation of collagen-associated proteoglycan from bovine nasal cartilage and its preferential interaction with alpha2 chains of type I collagen. 17 71

The binding of Ca2+ to a previously described phosphoprotein from human parotid saliva, protein A [Bennick (1975) Biochem J. 145, 557-567] was studied by means of equilibrium dialysis. In 5 mM-Tris/HC1 buffer, pH7.5, protein A bound 664nmol of Ca/mg of protein. Km was determined to be 181 muM and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ apparently occurs to side-chain carboxyl groups in the protein, but protein phosphate is of minor if any importance in calcium binding. Hydrolysis of protein A by trypsin and collagenase or heating of the protein at 60 degrees or 100 degrees C did not affect Ca2+ binding. The Ca2+ binding decreases with increased concentration of the dialysis buffer and on the addition of SrCl2, or MgCl2 or MnCl2 to the dialysis buffer. Protein A does not aggregate in the presence of Ca2+, since the s20,w was identical when determined in the presence (1.30S) and absence (1.35S) of CaCl2. By use of a specific antiserum to protein A it was found that protein C [Bennick & Connell (1971) Biochem. J. 123, 455-464] and perhaps minor related components cross-reacted with protein A. No other salivary proteins showed immunological similarity. Proteins A and C were also present in submandibular saliva. The possible functions of protein A are discussed.
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PMID:The binding of calcium to a salivary phosphoprotein, protein A, common to human parotid and submandibular secretions. 18 Sep 80

The polyphenolic compound tannic acid and the cationic stains ruthenium red, Alcian blue and lanthanum chloride have been used to reinvestigate the ultrastructural organization of the tectorial membrane matrix. Tannic acid treatment reveals that the matrix both in between and within the Type A protofibril bundle system has a high degree of structural organization. The basic unit of this matrix is best described as a 'striated sheet'. These striated sheets are formed by alternating 'dark' and 'light' fibrils which run parallel to one another and lie within the plane of each sheet. In sodium based buffers both light and dark fibrils have diameters of approximately 7 nm and the distance between each dark fibril in a sheet varies from 30 to 46 nm. Dark and light fibrils are coupled by periodic, staggered cross-bridges which occur at approximately 12 nm intervals along the fibrils. Fibril diameters in tectorial membranes prepared and fixed in potassium based buffers are from 10-20% greater than they are in tectorial membranes prepared and fixed in sodium based buffers. Fine fibrils can also be resolved in the matrix with the cationic stains lanthanum chloride and ruthenium red, but the organization of these fibrils into a regular matrix structure is most clearly resolved with tannic acid treatment. The striated sheets are largely destroyed by treating the tectorial membranes with neutral trypsin and are insensitive to treatment with bacterial collagenase. In contrast, the Type A protofibril system is trypsin resistant and collagenase sensitive. Treatment of tectorial membranes with salt solutions containing either 5 nM EDTA or 5 mM EGTA and 2 mM MgCl2 results in a complete loss of organized striated sheets and the appearance of randomly dispersed fibrillar material and small particles. Re-addition of Ca2+ ions causes the striated sheets to reform, indicating that the structure can undergo at least one cycle of depolymerization and polymerization in vitro. Reduction of disulphide bonds with beta-mercaptoethanol causes a loss of structural organization similar to that observed after EDTA or EGTA treatment. The results demonstrate that the non-collagenous components of the tectorial form a matrix with a degree of organization that has been previously unrecognised.
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PMID:The ultrastructural organization and properties of the mouse tectorial membrane matrix. 246 Apr 26

We examined the direct effect of magnesium ion on aldosterone production by adrenal cells using collagenase-dispersed zona-glomerulosa cells in rats. The effects of magnesium on aldosterone production stimulated by angiotensin II or ACTH were also investigated. Both magnesium sulphate (MgSO4) and magnesium chloride (MgCl2) (0 to 2 mM) decreased aldosterone production in a dose-dependent manner. In comparison with magnesium-free medium, 2 mM MgSO4 inhibited aldosterone production by 73% and MgCl2 by 65%. In addition, MgSO4 showed an inhibitory effect on aldosterone production stimulated by angiotensin II (10pM to 10nM), whereas it had no significant effect on aldosterone production due to ACTH stimulation (10pM to 10nM). These data suggest that magnesium has an inhibitory action on aldosterone production in vitro and may be a physiological regulator of aldosterone production.
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PMID:Magnesium ion: a possible physiological regulator of aldosterone production. 254 10

The asymmetric forms of acetylcholinesterase were purified from the electric organs of the electric rays Narke japonica and Torpedo californica, and their properties were compared. Asymmetric acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. The MgCl2 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted by lowering the pH of the eluent to 2.8. The purified asymmetric acetylcholinesterases gave peaks of 17 S (A12) and 13 S (A8) on sucrose density gradients. The enzyme from N. japonica contained more A8 than A12, while that of T. californica contained more A12. After treatment with collagenase, the enzymes gave three peaks on sedimentation; 20 S, 16 S and 11 S for N. japonica, and 19 S, 15 S and 11 S for T. californica, indicating the presence of collagen-like tails. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the asymmetric acetylcholinesterase from N. japonica gave bands of Mr 140 000, 100 000, 70 000 and 60 000, while that from T. californica gave bands of Mr 140 000, 100 000, 70 000 and 55 000. The bands of Mr 70 000 and 140 000 were monomers and non-reducible dimers, respectively, of the catalytic subunits. The bands of Mr 60 000 and 55 000 were the tail subunits, since collagenase treatment of the purified enzymes markedly decreased the amounts of these components. The Mr 100 000 subunit constituted less than 3% of the total asymmetric acetylcholinesterase from N. japonica but 18% of that from T. californica. The tail subunits constituted 6-8% of the two preparations. The catalytic subunits and the Mr 100 000 subunits bound concanavalin A, indicating that they are glycoproteins. The amino acid compositions of the enzymes from N. japonica and T. californica were very similar. Both contained hydroxyproline and hydroxylysine, characteristic of the collagen-like tails. The enzyme required divalent metal ions for activity, but only Mn2+, Mg2+ and Ca2+ were effective. Mn2+ was effective at the lowest concentrations, while Mg2+ gave the highest activity.
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PMID:Comparison of asymmetric forms of acetylcholinesterase from the electric organ of Narke japonica and Torpedo californica. 300 Jul 81

Vasoactive intestinal peptide (VIP)-responsive adenylate cyclase and VIP binding sites were investigated in membranes prepared from ciliary processes dissected from albino rabbit eyes. High-affinity binding sites for VIP (Kd, 0.95 nM; 607 fmol/mg of protein), in addition to beta adrenergic sites labeled by dihydroalprenolol (Kd, 0.48 nM; 123 fmol/mg of protein), were present. Activation of adenylate cyclase by VIP had a Ka of 65 nM, and the maximal response was 3.3-fold greater than that for I-isoproterenol (Ka, 102 nM). A peptide fragment of VIP (sequence 10-28) was inactive in all assays and did not inhibit VIP-stimulated adenylate cyclase at 10 microM. Responses to VIP and isoproterenol in combination were additive at lower doses but less than additive at maximal doses. Responses to VIP in combination with a low dose of forskolin (0.1 microM) were potentiated at all dose levels, whether assays were done in presence of MgCl2 or MnCl2. VIP- and forskolin-activated adenylate cyclase was associated with the nonpigmented epithelial cell fraction and not with pigmented epithelial cells separated on Percoll density gradients after dissociation of cells from processes by collagenase digestion. Intravitreous injection of 10 nmol of VIP into the rabbit eye caused a maximal reduction in intraocular pressure at 40 to 50 hr lasting beyond 72 hr. VIP-responsive and beta adrenergic-responsive adenylate cyclase are present on the same cell type (nonpigmented epithelial cells) and appear to share components of the adenylate cyclase system in the same membrane. VIP may participate in the physiologic regulation of aqueous humor secretion at the level of the epithelial cell membrane.
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PMID:Vasoactive intestinal peptide and intraocular pressure: adenylate cyclase activation and binding sites for vasoactive intestinal peptide in membranes of ocular ciliary processes. 303 1

Procedures for isolation of single smooth muscle cells from taenia coli of guinea pigs have been developed. The preparation was performed with a combination of highly purified collagenase prepared by Amano Pharmaceutical Co. (Japan) and papain obtained from Sigma Chemical Co. (Type III). This combination resulted in very high yield of the single cells (39.2 +/- 4.5 X 10(3) cells/mg tissue wet wt) and less cell debris. In the ordinary procedure, commercially available collagenase preparations contaminated with various peptidases have been used. With these enzyme preparations, however, the yield of single cells was dependent on the batch of the preparations, and a large amount of cell debris was contaminated. Combination of the highly purified collagenase and papain resulted in higher yields constantly. Cells, isolated with these enzymes in a medium consisting of 140 mM KCl, 1.0 mM MgCl2, 4.2 mM Hepes, and 5.6 mM glucose (pH 7.4), were spindle shaped. The length of the cells was 185.9 +/- 5.2 micron (n = 90) and the diameter was approximately 12.6 micron. The diameter was not dependent on the cell length. More than 80% of the single cells were viable when examined by trypan blue exclusion technique. Under the depolarized condition, cells remained viable longer because of lower energy consumption, and these cells were contracted by Ca dose dependently. The dose-response relationship was similar to that obtained with intact tissue. Because the cells are constantly available with higher yield, the preparation might be applicable for biochemical research such as ion flux. Details of cell properties under the physiological conditions are under investigation.
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PMID:Preparation of single smooth muscle cells from guinea pig taenia coli by combinations of purified collagenase and papain. 304 Nov 21

Most effects of thyroid hormones appear to be mediated by the binding of triiodothyronine (T3) to nuclear triiodothyronine binding sites (NT3BS). Although thyroid hormones influence adipocyte metabolism, NT3BS have not been described in mature adipocytes yet. This report describes T3 nuclear binding in isolated nuclei from rat epididymal fat pad adipocytes. Nuclei were isolated by exposing collagenase-dispersed adipocytes to STM (sucrose, 0.25 mol/L; TRIS, 20 mmol/L; MgCl2, 1.1 mmol/L, pH 7.85) containing 0.5% (vol/vol) Triton X-100. Incubation of nuclei suspended in STM/EDTA (2 mmol/L)/DTT (5 mmol/L) with 125I-T3 and varying concentrations of unlabeled T3 at 37 degrees C for one hour revealed the presence of high-affinity, low-capacity NT3BS. Their MBC was 0.39 +/- 0.04 (SD) ng of T3 per milligram of DNA and their Kd was 1.4 +/- 0.5 (SD) X 10(-10) mol/L T3. Specific binding reached a plateau between 30 minutes and two hours of incubation. The addition of 5 X 10(-7) mol/L T3 to nuclei incubated for one hour with 2 X 10(-11) mol/L T3 completely displaced the specifically bound 125I-T3 within 30 minutes. Thyroxine (T4) and 3, 3', 5'-triiodothyronine (rT3) could displace 125I-T3 from the NT3BS but were less than 10% and 1% as effective, respectively, as T3. Rat epididymal fat pad adipocytes contain NT3BS, the binding characteristics of which are similar to those of rat hepatic NT3BS.
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PMID:Nuclear triiodothyronine binding sites in rat adipocytes. 632 15

We have previously shown that inositol-1,4,5-trisphosphate (IP3) releases Ca2+ from an intracellular calcium store in permeabilized acinar cells of rat pancreas (H. Streb et al., 1983, Nature (London) 306:67-69). This observation suggests that IP3 might provide the missing link between activation of the muscarinic receptor and Ca2+ release from intracellular stores during stimulation. In order to localize the intracellular IP3-sensitive calcium pool, IP3-induced Ca2+ release was measured in isolated subcellular fractions. A total homogenate was prepared from acinar cells which had been isolated by a collagenase digestion method. Endoplasmic reticulum was separated from mitochondria, zymogen granules and nuclei by differential centrifugation. Plasma membranes and endoplasmic reticulum were separated by centrifugation on a sucrose step gradient or by precipitation with high concentrations of MgCl2. IP3-induced Ca2+ release per mg protein in the total homogenate was the same as in leaky cells and was sufficiently stable to make short separation procedures possible. In fractions obtained by either differential centrifugation at 7000 X g, sucrose-density centrifugation, or MgCl2 precipitation there was a close correlation of Ip3-induced Ca2+ release with the endoplasmic reticulum markers ribonucleic acid (r = 0.96, 1.00, 0.91, respectively) and NADPH cytochrome c reductase (r = 0.63, 0.98, 0.90, respectively). In contrast, there was a clear negative correlation with the mitochondrial markers cytochrome c oxidase (r = -0.64) and glutamate dehydrogenase (r = -0.75) and with the plasma membrane markers (Na+ + K+)-ATPase (r = -0.81) and alkaline phosphatase (r = -0.77) in all fractions analyzed. IP3-induced Ca2+ release was distributed independently of zymogen granule or nuclei content of the fractions as assessed by electron microscopy. The data suggest that inositol-1,4,5-trisphosphate releases Ca2+ from endoplasmic reticulum in pancreatic acinar cells.
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PMID:Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas. 633 62


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