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)

Fluoride-induced pitting and porosity of teeth have long been observed, but little progress has been made in determining their origin. We have observed, in the trypsin-treated surfaces of enamel, pits that disappear on completion of maturation, following the removal of the protein matrix and full mineralization. Since these pits were considered to be similar to those seen in fluorotic teeth, this scanning electron microscope (SEM) study was undertaken to determine the effect of fluoride on these transient developmental pits during enamel matrix maturation. A group of 20 eight-day-old rats was given daily intraperitoneal injections of NaF (20 mg/kg [9 mg F-/kg] body weight) for five days. Twenty control animals received intraperitoneal injections of isotonic saline. Maxillary and mandibular molars were dissected from the 13-day-old animals, washed in HEPES buffered (Ca2+/Mg2+) free basal medium, Eagle's (BME), incubated in 3% trypsin/BME for 5-10 min at room temperature, then indirectly sonicated in BME for 2-4 min. Clean crowns were fixed in 2.5% glutaraldehyde for three hr, dehydrated, critical-point-dried, and sputter-coated for SEM. Pits in the surfaces of developing enamel were observed in all groups. In control teeth, the pitting was restricted to the cervical margin, whereas in teeth from the fluoride-treated animals, pits were observed on some cuspal surfaces in addition to the cervical margin. These results confirmed that pits in trypsin-treated surfaces of developing enamel are a transient developmental event and showed that, in the presence of a high dose of fluoride, the maturation of enamel is modified with retention of the pits.
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PMID:Scanning electron microscopy of trypsin-treated enamel from fluorosed rat molars. 264 Apr 29

Trypsin, chymotrypsin, and papain stimulate basal adenylate cyclase activity in bovine thyroid plasma membranes in a dose-related, albeit biphasic, fashion. Each of the proteases enhanced TSH-stimulated adenylate cyclase activity over basal activity. The proteases also enhanced GTP-, guanosine 5'-(beta, gamma-imidotriphosphate)-, prostaglandin E1-, and cholera toxin-stimulated adenylate cyclase to varying degrees. Fluoride-stimulated activity was enhanced by chymotrypsin and papain, but not by trypsin. When Mn++ was substituted for Mg++ in the adenylate cyclase assay, no stimulation by the proteases were observed. To see if endogenous membrane proteases are required for optimal thyroid adenylate cyclase response to TSH and other stimulators, studies were performed using the protease inhibitors tosylamide 2-phenylethyl-chloromethyl ketone (TPCK) and p-tosyl-L-arginine methyl ester (TAME), inhibitors of chymotrypsin and trypsin, respectively. TPCK (0.15 mM) had no effect on basal adenylate cyclase activity, but did inhibit TSH-, trypsin-, and chymotrypsin-stimulated activities by approximately 90%. Guanosine 5'-(beta, gamma-imido) triphosphate- as well as cholera toxin-stimulated activities were inhibited by approximately 50%, whereas prostaglandin E1- and fluoride-stimulated activities were inhibited by approximately 25%. TAME (6 mM) produced similar results, except that no effect on fluoride activity was seen, while basal activity was inhibited by approximately 20%. Thus, various serine proteases augment both basal and hormone-stimulated adenylate cyclase in bovine thyroid. Since both trypsin- and chymotrypsin-stimulated as well as TSH-induced enzyme activities were inhibited by TPCK and TAME, it would appear that augmentation of thyroid adenylate cyclase activity may, in part, result from stimulation of endogenous proteases.
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PMID:Effects of proteolytic enzymes and protease inhibitors on bovine thyroid adenylate cyclase activity. 633 11

Fluoride activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] is significantly enhanced (2 to 5 times) by a protein factor isolated from rat brain. The fluoride-dependent adenylate cyclase stimulator (FCS) is nondialyzable, trypsin-labile, and stable at 90 degrees C for 10 min. FCS stimulates adenylate cyclase activity only in the presence of NaF (2-25 mM) and this effect is independent of added GTP, 5'-guanylylimidodiphosphate, or calcium. FCS has been purified roughly 3000-fold from a 12,000 X g supernatant fraction of rat brain homogenate. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and sucrose density gradient sedimentation suggest that FCS is a monomer with an apparent Mr of 59,000. Isoelectric focusing indicates FCS has a pI of 8.9. FCS from rat brain stimulates fluoride-activated adenylate cyclase from a variety of cell types, and FCS can also be isolated from rat liver. The effects of FCS are not reversed by washing membranes when the membranes and FCS are preincubated with NaF. The Km of adenylate cyclase for ATP and the fluoride concentration causing half-maximal activation are unchanged by FCS; however, FCS increases the Vmax by 2.5-fold. FCS may act to increase the catalytic efficiency of fluoride-activated complexes of the GTP-binding unit with adenylate cyclase or to enhance the formation of additional active complexes.
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PMID:Partial purification and characterization of a macromolecule which enhances fluoride activation of adenylate cyclase. 693 10

The measurement of Tryptase by the Fluoro-Immuno-Enzymatic (FEIA) method is nowadays possible on the Pharmacia CAP system (automatic UniCAP). This measurement is more comprehensive as it measures the release of serum tryptase from both the tissue mastocytes (MCTC) as well as the mucosal mastocytes (MCM). Technically the measurements are comparable with those made by the method of radio-immunology (RIA), are absolutely reproducible and surprisingly at 100%. It has also been possible to evaluate the two techniques of FEIA and RIA on negative and positive pools. This new FEIA technique for serum tryptase is applicable: to anaphylactic and/or anaphylactoid accidents at the time of induction of anesthesia, in general conditions such as haemorrhagic recto colitis (RCH), Crohn's disease, and mastocytosis. Finally these measurements can be used during nasal and bronchial provocation tests, as the measurements may be made on nasal and bronchial lavage liquids. The sensitivity and the very good reproducibility of this new technique of FEIA for tryptase is of very great interest and avoids use of radio-active isotopes.
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PMID:[When the fluoro-immuno-enzymatic (FEIA) measurements turn out to be more sensitive than radioimmunologic (RIA) measurements. Application to the measurement of serum tryptase]. 945 35

Double rotational-echo double resonance (double REDOR) has been used to investigate the bound conformations of (13)C,(15)N,(19)F-labeled factor Xa inhibitors to bovine trypsin. Carbon-fluorine dipolar couplings were measured by (13)C{(19)F} REDOR with natural-abundance background interferences removed by (13)C{(15)N} REDOR. The conformations of the bound inhibitors were characterized by molecular dynamics (MD) simulations of binding restrained by double REDOR-determined intramolecular C-F distances. A symmetrical bisamidine inhibitor and an asymmetrical monoamidine-monoamine inhibitor of the same general shape had distinctly different conformations in the bound state. According to the MD models, these differences arise from specific interactions of the amidine and amine groups with the active-site residues of trypsin and nearby water molecules.
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PMID:Conformations of trypsin-bound amidine inhibitors of blood coagulant factor Xa by double REDOR NMR and MD simulations. 1050 39

Compound 1 was identified by high throughput screening as a novel, potent, non-amidine factor Xa inhibitor with good selectivity against thrombin and trypsin. A series of modifications of the three aromatic groups of 1 was investigated. Substitution of chlorine or bromine for fluorine on the aniline ring led to the discovery of subnanomolar factor Xa inhibitors. Positions on the anthranilic acid ring that can accommodate further substitution were also identified.
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PMID:Structure-activity relationships of substituted benzothiophene-anthranilamide factor Xa inhibitors. 1256 61

High-throughput screening is usually the method of drug-lead discovery. It is now well accepted that, for a functional assay, quality is more important than quantity. The ligand-based or protein-based NMR screening methodologies for detecting compounds binding to the macromolecular target of interest are now well established. A novel and sensitive NMR method for rapid, efficient, and reliable biochemical screening is presented. The method named 3-FABS (three fluorine atoms for biochemical screening) requires the labeling of the substrate with a CF(3) moiety and utilizes (19)F NMR spectroscopy for the detection of the starting and enzymatically modified substrates. The method allows for high-quality screening of large compound or natural product extract collections and for measuring their IC(50) values. Applications of this technique to the screening of inhibitors of the Ser/Thr kinase AKT1 and the protease trypsin are presented. In addition, an interesting application of 3-FABS to functional genomics is also presented.
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PMID:A general NMR method for rapid, efficient, and reliable biochemical screening. 1462 13

A series of potassium organotrifluoroborates were synthesized. Their stability to hydrolysis was determined in D2O, TRIS and phosphate buffer. It was found that in both D2O and TRIS buffers, these compounds are quite stable, whereas in phosphate buffer rapid hydrolysis occurs. Based on these results, a study was undertaken to determine whether potassium organotrifluoroborates can serve as protease inhibitors. It was found that potassium organotrifluoroborates increased inhibition by at least an order of magnitude over the corresponding boronates. Dixon plots showed that these compounds are reversible competitive inhibitors of alpha-chymotrypsin and trypsin. Based on 19F NMR, we speculate that they inactivate the enzymes as a result of the formation of hydrogen-bonds between fluorine atoms of the inhibitors and the serine protease.
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PMID:Noncovalent inhibition of the serine proteases, alpha-chymotrypsin and trypsin by trifluoro(organo)borates. 1573 82

Two novel series of polyfluorinated amino acids (PFAs) were designed and synthesized according to a very short and scalable synthetic sequence. The advantages and limitations of these moieties for screening purposes are presented and discussed. The potential applications of these PFAs were tested with their incorporation into small arginine-containing peptides that represent suitable substrates for the enzyme trypsin. The enzymatic reactions were monitored by 19F NMR spectroscopy, using the 3-FABS (three fluorine atoms for biochemical screening) technique. The high sensitivity achieved with these PFAs permits a reduction in substrate concentration required for 3-FABS. This is relevant in the utilization of 3-FABS in fragment-based screening for identification of small scaffolds that bind weakly to the receptor of interest. The large dispersion of 19F isotropic chemical shifts allows the simultaneous measurement of the efficiency of the different substrates, thus identifying the best substrate for screening purposes. Furthermore, the knowledge of KM and Kcat for the different substrates allows the identification of the structural motifs responsible for the binding affinity to the receptor and those affecting the chemical steps in enzymatic catalysis. This enables the construction of suitable pharmacophores that can be used for designing nonpeptidic inhibitors with high affinity for the enzyme or molecules that mimic the transition state. The novel PFAs can also find useful application in the FAXS (fluorine chemical shift anisotropy and exchange for screening) experiment, a 19F-based competition binding assay for the detection of molecules that inhibit the interaction between two proteins.
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PMID:Polyfluorinated amino acids for sensitive 19F NMR-based screening and kinetic measurements. 1741 47

Cells resist death induced by the complement membrane attack complex (MAC, C5b-9) by removal of the MAC from their surface by an outward and/or inward vesiculation. To gain an insight into the route of MAC removal, human C9 was tagged with Alexa Fluor 488 and traced within live cells. Tagged C9-AF488 was active in lysis of erythrocytes and K562 cells. Upon treatment of K562 cells with antibody and human serum containing C9-AF488, C9-AF488 containing MAC bound to the cells. Within 5-10 min, the cells started shedding C5b-9-loaded vesicles (0.05-1 mum) by outward vesiculation. Concomitantly, C9-AF488 entered the cells and accumulated in a perinuclear, late recycling compartment, co-localized with endocytosed transferrin-Texas Red. Similar results were obtained with fixed cells in which the MAC was labeled with antibodies directed to a C5b-9 neoepitope. Inhibition of protein kinase C reduced endocytosis of C5b-9. Kinetic analysis demonstrated that peripheral, trypsin-sensitive C5b-9 was cleared from cells at a slower rate relative to fully inserted, trypsin-resistant C5b-9. MAC formation is controlled by CD59, a ubiquitously expressed membrane complement regulator. Analysis at a cell population level showed that the amount of C5b-9-AF488 bound to K562 cells after complement activation was highly heterogeneous and inversely correlated with the CD59 level of expression. Efficient C9-AF488 vesiculation was observed in cells expressing low CD59 levels, suggesting that the protective impact of MAC elimination by vesiculation increases as the level of expression of CD59 decreases.
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PMID:Live cell imaging of outward and inward vesiculation induced by the complement c5b-9 complex. 1764 16


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