Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The photosensitive inactivation of trypsin and chymotrypsin by 4-fluoro-3-nitrophenyl azide (FNPA) is described. A dark inhibition was observed at elevated probe concentrations, and was reversible. The enzymes were stable to photolysis in the absence of probe. Photolytic inactivation of trypsin and chymotrypsin with FNPA was found to be irreversible, and occurs in minutes at concentrations of FNPA where dark inhibition is negligible. The photoprobe was equally effective at pH 3 or pH 8. Nonspecific inactivation appears to be low, as evidenced by the stability of glucose oxidase and peroxidase to photolysis with FNPA.
 ...
PMID:Inactivation of trypsin and chymotrypsin with a photosensitive probe. 0 98

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.
 ...
PMID:[Synthesis and properties of carrier-fixed enzymes. VIII. Kinetic studies of the binding velocity of enzymes to macroporous carriers]. 61 87

A device, the enzyme thermistor, is described which is capable of measuring changes in heat due to enzymic reactions. The sensor, a thermistor, is in direct contact with the site of reaction through its placement in a microcolumn filled with an immobilised enzyme preparation. The substrate solution flows past the thermistor tip, and as much as approx. one half of the total heat evolved can be registered as temperature change, deltat. Glass-bound glucose oxidase (EC 1.1.3.4), penicillinase (EC 3.5.2.6), trypsin (EC 3.4.21.4) and urease (EC 3.5.1.5) were used for the determination of glucose, penicillin G, benzoyl-L-arginine ethyl ester and urea respectively. Linear relationships between the deltat recorded and the concentration of substrate were obtained in all cases.
 ...
PMID:Determination of heat changes in the proximity of immobilised enzymes with an enzyme thermistor and its use for the assay of metabolites. 117 49

A striking similarity exists between the pathogenetic properties of group A streptococci and those of activated mammalian professional phagocytes (neutrophils, macrophages). Both types of cells are endowed by the ability to adhere to target cells; to elaborate oxidants, hydrolases, and membrane-active agents (hemolysins, phospholipases); and to freely invade tissues and destroy cells. From the evolutionary point of view, streptococci might justifiably be considered the forefathers of "modern" leukocytes. Our earlier findings that synergy between a streptococcal hemolysin (streptolysin S, SLS) and a streptococcal thiol-dependent proteinase and between cytotoxic antibodies+complement and streptokinase-activated plasmin readily killed tumor cells, led us to hypothesize that by analogy to the pathogenetic mechanisms of streptococci, the mechanisms of tissue destruction initiated by activated leukocytes in inflammatory sites, as well as in tissues undergoing episodes of ischemia and reperfusion, might also be the result of the synergistic effects among leukocyte-derived oxidants, phospholipases, proteinases, cytokines, and cationic proteins. The current report extends our previous synergy studies with endothelial cells to two additional cell types--monkey kidney epithelial cells and rat beating heart cells. Monolayers of 51Cr-labeled cells that had been treated by combinations of sublytic amounts of hydrogen peroxide (generated either by glucose oxidase, xanthine-xanthine oxidase, or by paraquat) and with sublytic amounts of a variety of membrane-active agents (streptolysin S, phospholipases A2 and C, lysophosphatides, histone, chlorhexidine) were killed in a synergistic manner (double synergy). Crystalline trypsin markedly enhanced cell killing by combinations of oxidant and the membrane-active agents (triple synergy). Injury to the cells was characterized by the appearance of large membrane blebs that detached from the cells and floated freely in the media, looking like lipid droplets. Cytotoxicity induced by the various combinations of agonists was depressed, to a large extent, by scavengers of hydrogen peroxide (catalase, dimethyl thiourea, and by Mn2+) but not by SOD or by deferoxamine. When cationic agents were employed together with hydrogen peroxide, polyanions (heparin, polyanethole sulfonate) were also found to inhibit cell killing. It is proposed that in order to effectively combat the deleterious toxic effects of leukocyte-derived agonists on cells and tissues, antagonistic "cocktails" comprised of cationized catalase, cationized SOD, dimethylthiourea, Mn(2+)+glycine, proteinase inhibitors, putative inhibitors of phospholipases, and polyanions might be concocted. The current literature on synergistic phenomena pertaining to mechanisms of cell and tissue injury in inflammation is selectively reviewed.
 ...
PMID:Synergism among oxidants, proteinases, phospholipases, microbial hemolysins, cationic proteins, and cytokines. 142 26

Activated human neutrophils (PMN) degrade rTNF-alpha resulting in a loss of cytotoxic activity against murine L-929 cells (L cells). This inactivation is mediated through proteases released from activated PMN. Exposure of TNF to H2O2, glucose oxidase, xanthine oxidase, or myeloper-oxidase-H2O2-halide did not affect TNF cytotoxicity for L cells. Exposure to trypsin, chymotrypsin, pronase E, or elastase, however, did diminish TNF bioactivity. FMLP-stimulated PMN in the presence, but not in the absence, of cytochalasin B reduced TNF activity, whereas PMA-stimulated PMN did not affect TNF. Stimulation of PMN with opsonized bacteria also induced TNF inactivation as well as the supernatant of FMLP-stimulated cells. Addition of protease inhibitors to the FMLP-stimulated cytochalasin B-treated PMN abrogated the inactivation of TNF cytotoxicity for L cells, whereas scavengers were not protective. In addition, PMN from a chronic granulomatous disease patient also decreased TNF bioactivity. Inactivation of TNF by activated PMN correlated with granule release and not with superoxide production. Exposure of TNF to proteases and FMLP-activated PMN also resulted in a loss of reactivity with anti-TNF antibodies, as measured by ELISA, and in the formation of an approximately 10-kDa split product from the 17-kDa rTNF molecule. Partial degradation of TNF by proteases released from activated PMN may result in a diminished TNF bioactivity and thereby contribute to the regulation of local inflammatory reactions.
 ...
PMID:Inactivation of recombinant human tumor necrosis factor-alpha by proteolytic enzymes released from stimulated human neutrophils. 194 Mar 72

A new photometric microassay for immune complex (IC) binding to macrophages was developed in a homologous system using glucose oxidase-anti-glucose oxidase complexes (GOAGO) as a model for IC clearance in vitro. Thioglycollate-elicited murine peritoneal cells were incubated with GOAGO solution and then cell-associated glucose oxidase activity was measured after the washing and solubilization of the cell membrane in a microtitre plate. GOAGO binding to macrophages was inhibited in the presence of either IgG or its Fc fragments in a dose-dependent manner, while yeast mannan or IgG Fab fragments had no effect. These results indicated that this binding occurred solely via the Fc receptors on the macrophages. The Fc receptors for GOAGO were eliminated by trypsin digestion of the cells. When the macrophages were cultured with LPS or TPA, GOAGO binding was enhanced compared to that of control, whereas carrageenan treatment suppressed GOAGO binding. The present results suggest that this assay may be of value in the measurement of IC clearance and for studying the expression of Fc receptors on macrophages.
 ...
PMID:A new photometric microassay for the quantitation of macrophage Fc receptor function. In vitro enzyme-containing immune complexes clearance (EIC) assay. 235 41

A 35 kD major surface antigen of Dirofilaria immitis third-stage larvae was characterized biochemically and immunologically. Living larvae were iodinated by using Iodo-gen, iodosulfanilic acid, lactoperoxidase-glucose oxidase, and Bolton-Hunter reagents. Detergent extracts of larvae labeled by the first three methods showed one major 35 kD component and a number of smaller components of about 6 kD, as analyzed by one-dimensional SDS-PAGE. In contrast, extracts from larvae labeled with the Bolton-Hunter reagent showed multiple bands on gels. The 35kD molecule was shown to be exposed on the larval surface, insofar as it was accessible to trypsin-proteolysis on living radiolabeled larvae. Two-dimensional gel electrophoresis resolved the 35 kD band into two components: a major one with a pI of 3.8, and a minor one of pI 7.3. The lower m.w. bands were resolved into about 12 constituents with pI values from 3.5 to 8.0. Of all these surface molecules, the only one that was antigenic was the 35 kD component. It could be immunoprecipitated with sera from dogs carrying an occult experimental D. immitis infection or with sera from dogs immunized with irradiated third-stage larvae of this parasite. Similarly, sera from rabbits immunized repeatedly with normal unirradiated larvae also precipitated the 35 kD antigen. None of these sera, however, contained detectable antibodies to the surface-labeled low m.w. molecules. Sera from rabbits immunized with D. immitis adult worms and microfilariae precipitated the 35 kD antigen, which is therefore not stage specific. In contrast, sera from dogs experimentally infected with Toxocara canis and Ancylostoma caninum or with Uncinaria stenocephala (a canine hookworm) did not contain antibodies to the 35 kD antigen, but did cross-react with many other D. immitis adult and microfilarial antigens. This molecule may therefore be species specific. Evidence for glycosylation of the 35 kD molecule was not found: it did not bind to peanut, wheat germ, lentil, or Ulex europeus lectins, and its electrophoretic mobility was not altered after treatment with endoglycosidase-F or mild alkali solutions.
 ...
PMID:Biochemical and immunologic characterization of a major surface antigen of Dirofilaria immitis infective larvae. 241 45

The coupling of iodotyrosine (coupling reaction) is one of the least studied in the formation of thyroid hormone, particularly in human thyroid diseases. This paper describes a method of measuring iodotyrosine coupling catalyzed by human thyroid peroxidase (TPO) in vitro. There were two important requirements to demonstrate the coupling reaction: 1) thyroglobulin with a low thyroid hormone content, and 2) partially purified TPO. Thyroglobulin with low thyroid hormone content was obtained from Grave's and follicular adenoma tissues after propylthiouracil (PTU) therapy and L-T4 therapy, respectively. TPO was prepared from Graves' thyroid by solubilizing the 100,000 X g pellet of thyroid homogenate with sodium deoxycholate and trypsin, followed by Sephacryl S-300 gel filtration. Before the coupling reaction, thyroglobulin was iodinated with chloramine-T and potassium iodide, followed by dialysis. The coupling reaction was carried out by incubating newly iodinated thyroglobulin with TPO, diiodotyrosine, a coupling stimulator, and a H2O2-generating system (glucose and glucose oxidase) for 20 min at 37 C. After thyroglobulin was digested with Pronase, the thyroid hormone content of the thyroid digest was measured by RIA. Coupling activity was measured by the amount of newly formed T3 (nanograms of T3 per mg thyroglobulin). The time course of coupling reaction showed a progressive increase in coupling activity up to 30 min, and the reaction was temperature and pH dependent, with a pH optimum of 7.0. Coupling activity in the presence of H2O2 and TPO was 43 +/- 5.0 ng T3/mg thyroglobulin (mean +/- SD of triplicate samples), and addition of diiodotyrosine to the H2O2-TPO system caused a nearly 3-fold increase in coupling activity. This method has potential utilization for measurement of peroxidase coupling activity, since there was a linear relationship between the measured coupling activity and the amount of added TPO when the TPO concentration was over 3 micrograms/300 microliter. Methimazole (MMI) and PTU had similar potencies in inhibiting the TPO-catalyzed coupling reaction, whereas MMI was distinctly more potent than PTU as an inhibitor of TPO-mediated iodination in vitro. The different potencies of MMI in the two reactions suggest that different inhibitory mechanisms may be involved in iodination and coupling. The reducing agent, sodium metabisulfite, was also found to be a more potent inhibitor of the TPO-mediated coupling reaction than of the TPO-mediated iodination reaction. The method of iodotyrosine coupling described here may be useful to investigate the coupling step of thyroid hormone formation in human thyroid diseases.
 ...
PMID:Coupling of iodotyrosine catalyzed by human thyroid peroxidase in vitro. 383 97

Medium conditioned by tumor cells (TCM) and certain nonmalignant cells contains a trypsin-sensitive factor that suppresses macrophage oxidative metabolism. Because the killing of intracellular pathogens such as Toxoplasma gondii and Leishmania donovani by macrophages is largely oxygen-dependent, we tested the effect of TCM on the antiprotozoal activity of mouse peritoneal macrophages. After 24 hr of cultivation with TCM, in vivo and in vitro activated macrophages could no longer kill toxoplasmas or inhibit their replication. In vivo administration of TCM resulted in similar impairment. The leishmanicidal activity of resident and activated macrophages, when measured 6 hr after infection, was markedly suppressed by in vitro exposure to TCM. The addition of exogenous H2O2 in the form of glucose-glucose oxidase reconstituted the capacity of TCM-exposed macrophages to kill L. donovani promastigotes as quickly as control cells. Thus, TCM appears to deactivate macrophages by the functional criteria of suppressed antitoxoplasmal and antileishmanial activity, as well as by the biochemical criterion of suppressed oxidative metabolism.
 ...
PMID:Suppression of macrophage antimicrobial activity by a tumor cell product. 640 80

beta-Galactosidase and other enzymes were immobilized on p-amino-carbanilated derivatives of cellulose and methylol cellulose using the diazo method and through glutaraldehyde. The optimum conditions for coupling cellulose tri-(p-amino-carbanilate) (CTAC) to beta-galactosidase were established. The diazo coupling method with CTAC gave greater activity than with glutaraldehyde when coupled to beta-galactosidase (Escherichia coli). The stability of the CTAC-beta--galactosidase system was examined. The disubstituted p-amino-carbanilate derivative (CDAC) gave a lower activity, whereas the methylol analog (MCTAC) gave slightly greater activity. The CTAC was also used to immobilize glucose oxidase, trypsin, pepsin, and papain.
 ...
PMID:Immobilization of beta-galactosidase and other enzymes onto p-amino-carbanilated cellulose derivatives. 676 31


1 2 3 4 Next >>