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)

Ribosomal proteins S1 when associated with the 30-S subunit does not interact with 16-S RNA but its binding is determined mostly by protein-protein interactions. These conclusions are based on the following data. 1. Ultraviolet irradiation (lambda = 254 nm) of the 30-S subunit does not result in the covalent cross-linking of S1 with 16-S RNA at irradiation doses up to 150 quanta/nucleotide, whereas the irradiation under the same conditions of S1 . polynucleotide complexes [S1 . poly(U), S1 . poly(A) and S1 . Q beta phage RNA] induces effective formation of polynucleotide-protein cross-links. 2. Mild treatment of 30-S subunits lacking S-1 with RNase A or with cobra venom endonuclease results in removal of 10--20% of the total nucleotide material but does not affect their sedimentation characteristics of their S1 binding capacity. 3. The association of S1 with S1-depleted 30-S subunits is insensitive to aurintricarboxylic acid, which is known as a strong inhibitor of complex formation between S1 and polynucleotides. 4. Mild trypsin treatment of S1-depleted 30-S subunits greatly reduces their S1 binding capacity.
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PMID:Ribosomal protein S1 associates with Escherichia coli ribosomal 30-S subunit by means of protein-protein interactions. 703 93

The nucleus--centrosome complex from Physarum polycephalum myxamoebae has been purified. The complex contained the centriole pair and pericentriolar material in association with the nucleus. Apart from some unusually stable microtubules, which appeared to be involved in maintaining the nucleus-centrosome association, endogenous microtubule arrays had been stripped from the complex during isolation. When the nucleus--centrosome complex was incubated with purified brain or myxamoebal tubulins the growth of 45-70 microtubules was initiated onto the pericentriolar material. The number and length of the nucleated microtubules was proportional to the tubulin concentration. Pretreatment of the nucleus--centrosome complex with DNase 1, RNase A, antitubulin antibody and anticentriolar antibody did not affect pericentriolar nucleation capacity, although pretreatment with DNase 1 did expose perinuclear nucleation sites that had a much lower minimal tubulin concentration for assembly than the pericentriolar site. After pretreatment with trypsin pericentriolar material and nucleation were destroyed, and microtubule elongation occurred directly onto the centriole microtubules.
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PMID:Microtubule nucleation by the isolated microtubule-organizing centre of Physarum polycephalum myxamoebae. 710 28

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

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

The one-disulfide intermediates formed during the oxidative refolding of ribonuclease A (RNase A) have been characterized. This information is important for understanding the folding pathways of RNase A. The one-disulfide intermediates were blocked with 2-aminoethyl methanethiosulfonate, fractionated using ion-exchange chromatography, and digested with trypsin and chymotrypsin. The resulting peptide fragments were fractionated using reversed phase high-performance liquid chromatography, and identified using mass spectrometry. The relative population of each one-disulfide intermediate was determined from its disulfide bond concentration using a postcolumn disulfide detection system. A total of 24 out of 28 possible one-disulfide intermediates were found to be populated (greater than 0.3%) in the one-disulfide mixture. The population of one-disulfide intermediates displays a nonrandom distribution. All four native disulfide pairings have populations greater than those predicted by loop entropy calculations, suggesting the presence of enthalpic contributions stabilizing these species. The one-disulfide intermediate [65, 72], containing the disulfide bond between cysteines 65 and 72, comprises 40% of the entire one-disulfide population. The interactions that stabilize this intermediate may play an important role in the regeneration pathways of RNase A.
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PMID:Nonrandom distribution of the one-disulfide intermediates in the regeneration of ribonuclease A. 863 87

RNase S is a complex consisting of two proteolytic fragments of RNase A: the S peptide (residues 1-20) and S protein (residues 21-124). RNase S and RNase A have very similar X-ray structures and enzymatic activities. Previous experiments have shown increased rates of hydrogen exchange and greater sensitivity to tryptic cleavage for RNase S relative to RNase A. It has therefore been asserted that the RNase S complex is considerably more dynamically flexible than RNase A. In the present study we examine the differences in the dynamics of RNase S and RNase A computationally, by MD simulations, and experimentally, using trypsin cleavage as a probe of dynamics. The fluctuations around the average solution structure during the simulation were analyzed by measuring the RMS deviation in coordinates. No significant differences between RNase S and RNase A dynamics were observed in the simulations. We were able to account for the apparent discrepancy between simulation and experiment by a simple model. According to this model, the experimentally observed differences in dynamics can be quantitatively explained by the small amounts of free S peptide and S protein that are present in equilibrium with the RNase S complex. Thus, folded RNase A and the RNase S complex have identical dynamic behavior, despite the presence of a break in polypeptide chain between residues 20 and 21 in the latter molecule. This is in contrast to what has been widely believed for over 30 years about this important fragment complementation system.
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PMID:Dynamics of ribonuclease A and ribonuclease S: computational and experimental studies. 889 11

We show that a simple cell-free translation system from Escherichia coli, programmed with phage MS2 RNA, is able to infect F+ E. coli cells. The plaques appearing on the E. coli host strain are morphologically indistinguishable from those derived from normal phage MS2 infection. This effect is strictly translation-dependent, since an incomplete translation system or the system inhibited by antibiotics leads to no infection. The cell-free based infection is maximal under conditions favouring the highest synthesis of maturation protein (one of the four phage-encoded proteins). The infection is abolished when RNase A or trypsin treatment is included before addition of cells. Similarly, due to RNA and maturation protein degradation, the continued incubation of the translation mixture under protein synthesis conditions significantly decreases infectivity. These findings suggest the formation of 'minimal infectious units', simple complexes of MS2 RNA and maturation protein. Here we describe the first example of bacteriophage infectious unit formation directly performed in a cell-free translation system. A possible application of this phenomenon might be the construction of newly designed RNA vector delivery systems and, moreover, could be an approach for molecular evolution studies.
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PMID:Formation of bacteriophage MS2 infectious units in a cell-free translation system. 895 35

The thermal stabilities of ribonuclease A (RNase A) and ribonuclease B (RNase B), which possess identical protein structures but differ by the presence of a carbohydrate chain attached to Asn34 in RNase B, were studied by proteolysis and UV spectroscopy at pH 8.0. Proteolysis was quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. Increasing protease concentrations led to a hyperbolic increase of the rate constants of proteolysis. With thermolysin, which attacks the unfolded molecules only, the thermal unfolding constants were determined by extrapolating the rate constants of proteolysis to infinite concentration of protease. With trypsin, the unfolding constants of RNase A could be confirmed. Subtilisin attacked even the native RNases, where RNase B was more stable toward proteolytic degradation. Kinetic stabilities (deltaG++) calculated from the unfolding constants for temperatures between 52.5 and 65 degrees C revealed a higher kinetic stability of RNase B, which results from enthalpic effects only, whereas entropic effects counteract stabilization. delta deltaG++ at the transition temperature of RNase A (60.4 degrees C) was 2.2 +/- 0.3 kJ mol(-1). Thermodynamic stabilities (deltaG) were estimated from the thermal transition curves at 287 nm for the temperature range from 55 to 70 degrees C. For 17.5-25 degrees C, deltaG values were determined from transition curves of unfolding induced by guanidine hydrochloride and extrapolation of the free energy values to those in the absence of denaturant. At all temperatures, RNase B proved to be more stable than RNase A with essentially the same enthalpy and entropy of unfolding. delta deltaG was 2.5 +/- 0.2 kJ mol(-1) at 60.4 degrees C and 2.3 kJ mol(-1) at 25 degrees C.
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PMID:Kinetic and thermodynamic thermal stabilities of ribonuclease A and ribonuclease B. 904 16

Previous studies have implicated the heat shock cognate (hsc) protein of 73 kD (hsc73) in stimulating a lysosomal pathway of proteolysis that is selective for particular cytosolic proteins. This pathway is activated by serum deprivation in confluent cultured human fibroblasts. We now show, using indirect immunofluorescence and laser scanning confocal microscopy, that a heat shock protein (hsp) of the 70-kD family (hsp70) is associated with lysosomes (ly-hsc73). An mAb designated 13D3 specifically recognizes hsc73, and this antibody colocalizes with an antibody to lgp120, a lysosomal marker protein. Most, but not all, lysosomes contain ly-hsc73, and the morphological appearance of these organelles dramatically changes in response to serum withdrawal; the punctate lysosomes fuse to form tubules. Based on susceptibility to digestion by trypsin and by immunoblot analysis after two-dimensional electrophoresis of isolated lysosomes and isolated lysosomal membranes, most ly-hsc73 is within the lysosomal lumen. We determined the functional importance of the ly-hsc73 by radiolabeling cellular proteins with [3H]leucine and then allowing cells to endocytose excess mAb 13D3 before measuring protein degradation in the presence and absence of serum. The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected. The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3. These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.
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PMID:An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation. 915 85

Syncephapepsin is a fungal aspartic proteinase from Syncephalastrum racemosum. By using the property of syncephapepsin after having increased activity at higher temperature, two rapid purification protocols were developed. In the former case, a crude extract was initially diluted fivefold with an activity assay buffer and heated at 50 degrees C overnight. In this situation, syncephapepsin would digest most of the proteins that the crude extract contained. Subsequently, syncephapepsin of the crude extract was precipitated from 50 to 70% of ammonium sulfate and the preparation was then directly applied to the Superdex 200 HR FPLC column. In this manner, syncephapepsin was rapidly purified to apparent homogeneity within 24 h. In this report, an alternative method of purification is also provided. Compared with the procedure mentioned above, the heating step was proceeded after FPLC chromatography through which the same result was obtained. Using cytochrome c and RNase A as substrates, the cleavage sites of both substrates were identified by HPLC peptide mapping. The results showed that syncephapepsin had a broad specificity. Residues recognized to be cleaved were primarily those of trypsin and chymotrypsin and Lys was the most susceptible.
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PMID:Single-column purification of syncephapepsin--an aspartic proteinase from Syncephalastrum racemosum. 953 8


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