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 kinetics of binding the enzymes glucose oxidase, trypsin, and peroxidase and also bovine gamma-globulin to the macroporous carriers dialdehyde cellulose, diazotized amino polystyrene, acrolein-acrylamide copolymer, and isothiocyanate groups carrying CPG-glass was studied. It was found empirically that the increase in protein content and--if there occurred no inactivation of enzyme during binding reaction--of enzymatic activity of the enzyme-carrier complex with the reaction time can be described by saturation curves (first order hyperbolas), which represent straight lines after suitable transformation. Thereby it is possible to calculate protein content or specific activity of the enzyme-carrier complex for any moment during binding reaction. From intercepts of the obtained straight lines with the ordinate and with the abscissa, respectively, one can determine the maximum binding capacity and the maximum specific activity and from their slope the reaction rate can be obtained. If there result no straight lines for plotting enzymatic activity of the enzyme-carrier complex against binding time after transformation, then this is an indication for partial inactivation of the free or already insolubilized enzyme. From kinetic measurements it is concluded that in all cases studied by use the preceding stage in chemical fixation of the enzyme to carrier is an adsorption equilibrium. Limitation of reaction rate by diffusion as in loading an ion exchanger could not be observed.
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PMID:[Synthesis and properties of carrier-fixed enzymes. VIII. Kinetic studies of the binding velocity of enzymes to macroporous carriers]. 61 87

We have developed assays for thyroid peroxidase in crude thyroid tissue preparations, in which a linear relationship between activity and amount of tissue could be demonstrated. Linear assays were developed based on the following peroxidase catalyzed reactions in the presence of H2O2:(1) oxidation of I- to I(-3), (2) oxidation of guaiacol, and (3) iodination of human goiter thyroglobulin. To attain satisfactory linearity we found it necessary to solubilize the enzyme beforehand. This was accomplished by a brief treatment of the particulate fraction with trypsin and deoxycholate, followed by centrifugation at 40 000 X g and dialysis. Not only did this treatment facilitate the development of linear assays, but it also resulted in a substantial increase in enzyme activity compared with that in the untreated particulate fraction. The use of a Polytron homogenizer for the initial disruption of the tissue also proved helpful in developing these assay procedures. The three different assays were used to measure peroxidase activities in human thyroid adenomas and in normal tissue derived from adenomatous glands. T he adenomas generally displayed a higher level of peroxidase activity than normal tissue. The greatest difference was observed with the iodination assay and the smallest difference with the guaiacol assay.
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PMID:Improved assay procedures for thyroid peroxidase; application to normal and adenomatous human thyroid tissue. 62 Apr 58

The bovine pancreatic inhibitor of trypsin (trasylol, Bayer) (T) and the soy bean trypsin inhibitor (SBI) were coupled with peroxidase (P). With each one of these coupling products (T and P and SBI and P) the cell distribution of proteolytic enzymes (PE) of ascitic cells of L5178Y murine lymphoma, was localized. The reactions were developed by means of Karnowsky's reaction with diaminobenzidine and H2O2. The preparations were observed under light and electronic microscopy. It was found that L5178Y cells contain intracellular PE in granules measuring 0.1 to 0.8 min diameter, and superficial PE that form a continuous layer of 80 to 120 nm over the cell surface. Superficial PE were not identified in 20 per cent of L5178Y cells, while in every case intracellular granules were found. Both the macrophages and the polimorphonuclear cells present in the ascitic fluid contained intracellular PE granules measuring 0.05 to 0.4 micron in diameter, and did not contain superficial PE.
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PMID:Proteolytic enzymes marking of malignant lymphoblasts, study of the L5178Y murine lymphoma. 63 54

Lysosomal fraction was isolated from rat liver by density gradient centrifugation after pervious loading of lysosomes in vivo with Triton WR-1339. Tritosome preparations were incubated at 37 degrees C and pH 5 for 24 hr with purified human ceruloplasmin or haptoglobin. After this period approximately 20% of total alpha amino nitrogen was released from ceruloplasmin and over 40% from haptoglobin. This was accompanied by loss of peroxidase activity of haptoglobin (in complex with haemoglobin), while enzymatic activity of ceruloplasmin remained unaltered. Removal of sialic acid by neuraminidase had no effect on digestion of ceruloplasmin by rat liver tritosomes. Both glycoproteins were resistant to horse leucocyte proteinases and pancreatic eleastase but were easily inactivated by trypsin and chymotrypsin.
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PMID:Degradation and inactivation of ceruloplasmin and haptoglobin by rat liver lysosomes and some other proteinases. 65 34

Porcine thyroid peroxidase (Iodide: hydrogen-peroxide oxidoreductase, EC 1.11.1.8) was solubilized by proteolytic and non-proteolytic procedures. A kinetic and physical study was undertaken to ascertain the catalytic properties of the peroxidase prepared by the two purported solubilization procedures. Where possible, the properties of the two enzyme preparations were compared with the original microsomal preparation. The n-butanol-solubilized thyroid iodide peroxidase is not truly soluble, but exists as a large molecular weight lipoprotein aggregate. The trypsin-solubilized thyroid iodide peroxidase is truly soluble, active, and contains lipids. The microsomes, butanol-pseudosolubilized enzyme, and trypsin-solubilized enzyme have similar kinetic properties such as pH optima, Km for iodide and H2O2, sigmoid character of the saturation curves, substrate inhibition, and inhibition by 3,5-diiodotyrosine. Since the proteolytic solubilization procedure produced a soluble peroxidase with catalytic properties similar to the microsomal preparation, trypsin-solubilized peroxidase can be studied with reasonable assurance that its properties are essentially unaltered and are not artifacts of the solubilization procedure.
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PMID:The characterization of n-butanol-pseudosolubilized and trypsin-solubilized porcine thyroid iodide peroxidase. 71 42

Lymphocytes from guinea pigs having delayed hypersensitivity to horse-radish peroxidase (HRPO) when cultured in vitro with HRPO produce a large m.w. factor (greater than 100,000 daltons) that causes peritoneal macrophages from nonimmune animals to aggregate. The macrophage aggregation factor (MAF) can be separated from macrophage migration inhibitory factor (MIF) by gel filtration of active lymphocyte supernatants on Sephadex G-150. MAF is heat stable (56 degrees C for 30 min) but inactivated by trypsin. These data suggest that aggregation of macrophages in vitro by lymphokine-rich culture supernatants is not due to MIF but is caused by a separate large m.w. factor.
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PMID:Guinea pig lymphocyte-derived macrophage aggregation factor: its separation from macrophage migration inhibitory factor. 79 15

Peritoneal fluid from the abdominal cavities of guinea pigs having delayed hypersensitivity to horseradish peroxidase (HRPO) was obtained by a lavage technique before and after i.p. challenge with antigen. Macrophage migration inhibitory factor (MIF) and macrophage chemotactic factor activities were measured in peritoneal fluids from each animal. Chemotactic activity for macrophages was maximal 24 hr after i.p. challenge and was absent thereafter. MIF activity was maximal in peritoneal fluid 24 to 48 hr after challenge. Macrophages were present in greatest numbers in peritoneal fluid 24 hr after challenge and returned almost to control levels at 48 hr. Macrophages in 48-hr fluid were larger and exhibited more intense cytoplasmic staining for nonspecific esterases when compared to those in 0-hr fluid. The m.w. of MIF obtained from culture supernatants of HRPO-stimulated guinea pig lymphocytes and 48-hr peritoneal fluid were found to be virtually identical, 58,000 and 54,000 daltons, respectively. MIF from these in vitro and in vivo sources were similarly resistant to heating at 56 degrees C for 30 min but were both destroyed by incubation with isoluble trypsin.
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PMID:Characterization of macrophage migration inhibitory factor activity produced in vivo by a cell-mediated immune reaction in the guinea pig. 79 16

Release of peroxidase from secretory cells of rat lacrimal gland upon cholinergic stimulation was studied in vitro with single lobules and isolated cells (lacrimocytes). Isolated lobules, kept in Eagle's medium, remain structurally intact and reaction product of peroxidase is confined to cisternae of rough endoplasmic reticulum, elements of the Golgi apparatus, and all secretory granules. Morphologically, exocytosis occurs by membrane fusion and discharge of granule content. The highest rate of peroxidase released from lobules is observed at 10(-4) M carbamylcholine. The specific activity of peroxidase released into the medium is fourfold higher as compared to the lobules. Release of peroxidase is suppressed by atropine when added before or after the addition of carbamylcholine. At 4 degrees C, no peroxidase release occurs upon cholinergic stimulation. The exocytotic release of peroxidase is dependent on energy supply, as indicated by substantial inhibition (at 37 degrees C) under anoxic conditions or in the presence of dinitrophenol, KCN, or carboxyatractyloside. Furthermore, the process is sensitive to colchicine and vinblastine. Isolated lacrimocytes, consiting of 95% secretory acinar cells, are prepared by digestion with collagenase, hyaluronidase, and trypsin. They retain the characteristic polarity of secretory cells in situ, and localization of peroxidase is the same as in lobules. Since isolated lacrimocytes respond to cholinergic stimulation in the same way as lobules, the receptors are not damaged by the isolation procedure and appear to be associated directly with the exocrine cell. Oxygen uptake by isolated lacrimocytes is about 14 nmol O2 X min-1 X 10(-6) cells; it is about doubled by uncoupling with dinitrophenol. Oxygen uptake rises by 20-30% above the resting rate upon cholinergic stimulation. This additional uptake is suppressed by atropine or by added cholinesterase, indicating that continuous receptor occupancy may be required for the energy demand by exocytosis. On the basis of the specific activity of peroxidase in the medium, the energy demand resulting from cholinergic stimulation is estimated to be 0.08 mumol ATP (or energy-rich phosphate bonds) per microgram of protein released from the lacrimocytes.
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PMID:Exocytosis in secretory cells of rat lacrimal gland. Peroxidase release from lobules and isolated cells upon cholinergic stimulation. 95 71

A system that allows repeated sampling of peritoneal fluid at various time intervals has been adapted to study mechanisms of leukocyte accumulation in vivo. Application of this technique in guinea pigs exhibiting delayed hypersensitivity (DH) to horse radish peroxidase (HRPO) has allowed characterization of some events after i.p.challenge with the sensitizing antigen. Within 24 hr of the administration of HRPO i.p. to such animals there is a significant increase in the number of peritoneal macrophages and in the chemotactic activity (CTX) for macrophages in the sampled peritoneal fluid. At 48 and 72 hr the CTX returns to the pre-challenge level and i.p. macrophages appear to be actively phagocytic. Molecular sieve chromatograms of concentrated peritoneal fluid obtained 24 hr after i.p. challenge with HRPO and of supernatants derived from immune spleen cells cultured in the presence of HRPO in the presence of HRPO in vitro revealed that the major portion of CTX for homologous macrophages eluted in the region of the 12,500 dalton protein marker. The partially purified CTX obtained from peritoneal fluid and supernatants of spleen cell cultures was heat stable (56 degrees C for 30 min) and was destroyed by trypsin digestion. These data demonstrate that a chemotactic factor (LDCF) obtained in vitro, is present in vivo at the site of a cell-mediated immune reaction. Moreover, these observations demonstrate the feasibility of studying the kinetics of leukocyte accumulation and the production of mediators of inflammation at the site of well defined immunologic reactions in vivo.
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PMID:Characterization of chemotactic activity produced in vivo by a cell-mediated immune reaction in the guinea pig. 111 29

Four isoperoxidases of turnip root and isoperoxidase C of horseradish root were digested with trypsin, and their peptide maps, prepared by high-voltage paper electrophoresis, were compared. All five tryptic digests were completely soluble at pH 8. The maps were developed with a variety of general and specific reagents: ninhydrin, histidine, tyrosine, tryptophan and arginine reagents. Cystine peptides and cysteic acid derivatives have also been characterized. All detected half-cystine residues seemed engaged in disulfide bridges. For each individual peroxidase the number of specifically staining peptides agreed very well with the amino acid composition. The two most acidic peroxidases of turnip, P1 and P2, only differ significantly in one peptide. The P2 gene is tentatively proposed to have developed from the P1 gene by a single base mutation, changing an asparagine residue to alysine residue. A less acidic turnip peroxidase, P3, is distinct, although related to peroxidases P1 and P2. Horseradish isoperoxidase C also belongs to this group which appears to be closely related in the amino acid sequences around four disulfide bridges. Peroxidase P7 differs from this group, at least around two of its disulfide bridges, and therefore, may differ from the other four in parts of its three dimensional structure. Sequences of particular importance to peroxidase function must be present in all peroxidases. From the peptide mapping studies we only find two highly homologous sequences present in all five examined peroxidases. Both contain histidine. This finding corroborates previous suggestions of two histidine sequences near the peroxidase heme prosthetic group. The rules applied in relating peptides of different proteins are outlined, and the sources of errors in mapping of glycoproteins of high carbohydrate content (about 20%) are discussed in detail.
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PMID:Similarities and differences of five peroxidases from turnip and horseradish. Peptide mapping studies on glycoproteins. 117 50


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