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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The trypsin-resistant core protein of the lac repressor was utilized in protecting operator DNA from two types of enzymatic digestion. Core repressor protects and enhances operator DNA digestion by DNase I in the same fashion as intact repressor, though to a lesser degree on the lower strand. DNase I patterns found for the ternary complexes (protein-sugar-operator) were consistent with the expected affinity alterations of the protein species in response to binding these ligands. The 3' boundaries obtained by exonuclease III digestion for the intact repressor-operator complex varied slightly from those reported by Shalloway et al. (1980). Asymmetric binding to operator by the core repressor fragment was suggested by differences in the 3' boundary for the core compared to intact repressor on the promoter-distal side of the complex. A composite picture of repressor structure and function emerges from the protection studies reported here and in the accompanying paper. In light of these and other results, models for repressor binding are examined.
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PMID:Enzymatic digestion of operator DNA in the presence of the lac repressor tryptic core. 639 63

An isoactin analysis was performed on L-[35S]cysteine labeled BC3H1 cells to determine if these smooth muscle-like cells synthesize vascular smooth muscle actin. Three different NH2-terminal peptides were identified on thin layer electrophoretograms of DNase I-purified and trypsin-digested BC3H1 cell actin. Results obtained from secondary digestion with thermolysin or Staphylococcus aureus V8 protease showed that the most acidic NH2-terminal peptide was derived from vascular smooth muscle alpha-isoactin. Treatment of cell monolayers with serum-free medium caused a 3-fold increase in the level of alpha-isoactin expression and a concomitant decrease in the level of non-muscle beta- and gamma-isoactin. Cell-cell contact was required for induction of alpha-isoactin, and the effects of serum depletion on isoactin expression and cell growth were reversible. The intensity of about 11 out of 500 polypeptide spots on two-dimensional gels of BC3H1 cell polypeptides also was influenced by the culture conditions. The finding that smooth muscle isoactin expression was coupled to cell growth conditions indicate the potential usefulness of BC3H1 cells in studies of isoactin expression and utilization during vascular smooth muscle development.
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PMID:Induction of vascular smooth muscle alpha-isoactin expression in BC3H1 cells. 669 10

In HeLa nuclei, 1 microM Ca2+ stimulates 3-fold the phosphorylation of histone H3. Prior treatment of cells with Na butyrate increases the degree of H3 phosphorylation and reveals a correlation between the extents of H3 acetylation and phosphorylation. Acetylation of H3 increases its accessibility to the calcium-dependent kinase. Phosphorylation of H3 occurs at a serine residue located in the trypsin-sensitive region of the protein. Brief digestion of nuclei with DNase I preferentially releases the phosphorylated form of H3 from chromatin.
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PMID:Acetylation and calcium-dependent phosphorylation of histone H3 in nuclei from butyrate-treated HeLa cells. 682

The binding of 3-methylcholanthrene (3-MC), a potent inducer of aryl hydrocarbon hydroxylase activity, to cytoplasmic proteins of a cloned rat hepatocyte culture, RL-PR-C, was studied by sucrose gradient centrifugation. Time course and dose-binding experiments performed on late-passage aryl hydrocarbon hydroxylase-inducible cultures indicate the presence of a saturable pool of high-affinity (average Kd, 3.6 nm) binding sites in the cytosol of these cells. The number of binding sites varied from 20,000 to 80,000 per late-passage hepatocyte with a total capacity of approximately 2.2 pmol of 3-MC bound per mg of cytosolic protein. The complex sedimented at 4.0 +/- 0.2S regardless of the ionic strength of the homogenization buffer or gradient solutions. It was sensitive to denaturation by sodium dodecyl sulfate and trypsin but not by DNase I, RNase A, or the nonionic detergent Nonidet P-40. The binding of 3-MC to the protein was inhibited by 1,2-benzanthracene, benzo(a)pyrene, 5,6-benzoflavone, and 7,8-benzoflavone but not by a series of steroids, aflatoxin B1, phenobarbital, or Aroclor 1254. Elevating the temperature of cultures cells to 37 degrees after the standard ligand-binding incubation at 4 degrees resulted in a rapid decrease in cytoplasmic saturable binding and a concomitant increase in nuclear- and chromatin-associated ligand. A portion of this nuclear-associated ligand was extractable with 400 mM KCl. Adsorption of the [3H]-3-MC binding complex by nuclei in vitro suggested that the 4S binding protein facilitated the entry of 3-MC into the nucleus. The presence of the 4S binding species correlated with the level of inducibility of aryl hydrocarbon hydroxylase throughout its development in RL-PR-C and therefore may be involved in the process of induction of this enzyme.
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PMID:Correlation of induction of aryl hydrocarbon hydroxylase in cultured rat hepatocytes with saturable high-affinity binding of 3-methylcholanthrene to a 4S cytoplasmic protein. 721 46

A rapid inexpensive method is presented for detecting peripheral blood lymphocyte chromatin activation by the neutral red "topo-optical" reaction, which causes strong and easily measurable birefringence in the lymphocyte nuclei. This reaction can be enhanced by fixing the cells with 150 mM/l NaCl in 70% ethanol and/or by treating the unfixed cellular suspensions with 0.2 M/l HCl to remove histones. In histone-removed preparations, 30 min DNase I treatment almost completely abolished the birefringent reaction, whereas RNase treatment resulted in only 18% loss. Chromatin activation induced by enzyme inhibition increased chromatin birefringence significantly. The same phenomenon could be induced in sensitive subjects' lymphocytes by specific antigens or haptens much more rapidly. The monocytes were not activated to a significant extent. In non-sensitive subjects different kinetics of antigen or hapten-dependent activation and no cytotoxic effects have been observed. Depletion of T-lymphocytes in vivo in SLE patients or by in vitro treatment with 0.5 mM/l KCN as well as with 0.02% trypsin has caused a significant drop in the mean chromatin birefringence. The effect of trypsin was reversible.
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PMID:Measurement of lymphocyte activation by a chromatin topo-optical reaction. Mechanism and specificity of the test. 723 31

Histone-depleted nuclei were prepared from isolate HeLa nuclei by extracting the histones and other proteins with polyanions (dextran sulphate and heparin) or with high salt concentrations as used previously. The particles were characterized by sucrose density gradient sedimentation, thin sectioning and electron microscopy, and by polyacrylamide gel electrophoresis. The general result of the experiments is that the DNA in the histone-depleted nuclei is highly organized, and that this residual, higher-order structure is maintained by a reproducible subset of nuclear proteins, and perhaps by RNA. Furthermore, the residual proteins remain associated, in some conditions, as rapidly sedimenting structures even when the DNA is digested with nucleases. These nuclear scaffolds can resemble extracted nuclei. Histone-depleted HeLa nuclei sediment in sucrose density gradients as well defined peaks with sedimentation coefficients of around 12 000 S, when 2M NaCl is used to extract the histones, or 6 000 S, when dextran sulphate is used. The rate of sedimentation is drastically decreased by treating the particles with trypsin, and reduced to a lesser extent with RNase A. Thin sectioning and electron microscopy show that histone-depleted nuclei possess the nuclear periphery and that internal material is also present. These general features are also seen in thin sections of nuclear scaffolds, which are prepared by treating the nuclei with micrococcal nuclease of DNase I in addition to extracting the histones. Two groups of major proteins are associated with histone-depleted HeLa nuclei and the nuclear scaffolds: One group has molecular weights of 50 000-55 000 Daltons. The major species of this latter group of proteins have mobilities that are similar to the proteins of the metaphase chromosomal scaffold.
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PMID:Organization of chromosomes in HeLa cells: isolation of histone-depleted nuclei and nuclear scaffolds. 740 Feb 38

Human pancreatic Deoxyribonuclease I (DNase I), inhibitor was partially purified from duodenal juice of healthy subjects collected in the Pancreozymin-Secretin test, by a procedure which included ammonium sulfate fractionation, DEAE cellulose fractionation, Sepharose 4B affinity chromatography, and gel filtration. The final preparation inhibited DNase I only, and had no inhibitory activity on pancreatic RNase, and trypsin. The inhibitor had a molecular weight of approximately 40,000, as determined by gel filtration, and showed the same mobility as skeletal muscle actin on SDS gel electrophoresis. Then clinical studies were made on the DNase I inhibitor in duodenal juice obtained after administration of Pancreozymin and Secretin to patients with various pancreatic diseases. In patients with suspected chronic pancreatitis with whom the ordinary test, containing the assay of the total volume, amylase output and maximum bicarbonate concentration of duodenal juice had produced normal results, the DNase I inhibitor output was observed to be higher than that in control subjects. While it was lower in patients with confirmed chronic pancreatitis than in control subjects. There results imply that DNase I inhibitor output may be an indicator of the pancreatic inflammation state and be useful for the early detection of pancreatic diseases.
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PMID:Biochemical and clinical studies on human pancreatic deoxyribonuclease I inhibitor. 745 Mar 90

The biochemical properties of G-actin, and the kinetics of polymerization of G-actin into F-actin, are dependent upon whether Mg2+ or Ca2+ is bound at the high-affinity metal-binding site in actin. Three-dimensional reconstructions from electron micrographs show that a bridge of density, that we interpret as arising from a major shift of the C terminus, exists between the two strands of the filament in Ca(2+)-actin that is absent in Mg(2+)-actin. This bridge is also absent in models of F-actin built from an atomic structure of G-Ca(2+)-actin. The cleavage of the DNase I-binding loop in actin between residues 42 and 43, with the non-covalent association of the 42 cleaved residues with the remainder of the actin, induces an even larger bridge of density between the two strands. When the bridge is absent, the two C-terminal residues in F-actin are easily cleaved by trypsin, while these residues become increasingly resistant to tryptic cleavage as the bridge becomes more prominent. Conversely, cleavage of the two C-terminal residues leads to a conformational change in the DNase I-binding loop. Since both the DNase I-binding loop and the metal-binding site are quite distant from the C terminus, large allosteric effects must exist in F-actin. The conformational change in F-actin that results from the creation of this bridge may be induced by myosin binding, since this movement generates changes in actin's diffraction that are very similar to the changes in the muscle X-ray pattern during activation that are associated with the binding of myosin to the thin filament.
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PMID:Structural dynamics of F-actin: I. Changes in the C terminus. 784 28

The ability of myosin subfragment 1 to induce actin polymerization was reinvestigated using the DNase I inhibition assay, by electron microscopy after negative staining, cosedimentation, measurement of viscosity and the fluorescence increase of pyrenyl-labeled actin. Using these techniques we demonstrate that rabbit skeletal muscle myosin subfragment 1 containing either the alkali light chain 1 (S1A1) or the alkali light chain 2 (S1A2) is able to promote actin polymerization even in the absence of divalent cations or salt. In the presence of ATP the rate of induction of actin polymerization by S1A2 is slower than by A1A1. In contrast, in the absence of free ATP, both subfragment 1 variants exhibit equal ability to induce actin polymerization. Evidence is given that the slower rate of induction of actin polymerization by S1A2 in the presence of free ATP is due to a slower rate of ATP-hydrolysis by S1A2 and thus to a slower rate of ATP depletion. We therefore assume that the formation of rigor type complexes involving the subfragment 1 heavy chain is necessary for the induction of actin polymerization. The ability of subfragment 1 to induce actin polymerization is retarded by a synthetic heavy chain mimetic peptide which inhibits its actin binding or after proteolytic cleavage of the subfragment 1 heavy chain by trypsin.
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PMID:Both isoforms of skeletal muscle subfragment 1 (S1A1 and S1A2) can induce actin polymerization with equal speed in the absence of ATP. 792 79

The aim of this work was to re-examine, on a quantitative basis, the relationship between banding pattern after Giemsa staining and the amount (and distribution) of DNA along the length of the chromatid arms. To do this, we investigated by cytofluorometric methods the occurrence of possibly different extraction of chromosome DNA after some alternative G-banding procedure, i.e. treatment of chromosomes with saline solutions, or DNasi I digestion in situ. The G-banding procedure entailing trypsin pretreatment is known to be difficult to standardize; in the present investigation, it was also found that trypsin induced a massive, although quantitatively variable, extraction of DNA from fixed metaphase chromosomes. G-banding-like patterns may be obtained, by treating chromosomes preparations with saline solutions. Both PBS and Tris-HCl treatment for the times considered induced a G-banding-like pattern after Giemsa staining, regardless of the age of chromosome preparations; no banding was observed after staining DNA with PI, nor extraction of DNA was found to occur. DNase I digestion initially induced a G-banding in both human and mouse chromosomes after Giemsa staining, with concomitant extraction of DNA (but without apparent G-banding-like pattern after PI staining); after 30 min digestion, a C-banding-like pattern was observed after both Giemsa and PI staining. Exposure to PBS or Tris-HCl buffer at room temperature may therefore be recommended as a G-banding inducing treatment, since it allows the classification of single chromosomes after Giemsa staining, without determining significant displacement of genomic DNA, which can be submitted to further analysis in situ.
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PMID:Treatments with saline solutions and DNase I have different effects on DNA content and distribution in human and in mouse chromosomes. 883 3


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