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)

Thrombin, the central regulatory enzyme in coagulation, when incubated in nanomolar concentrations with murine neuroblastoma cells produced a rapid and marked increase in tritiated guanosine 3',5'-monophosphate (cyclic GMP) formation that was blocked by hirudin and competitively antagonized by dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. Diisopropylphosphofluoridate-inactivated thrombin as well as the serine protease trypsin were markedly less potent and less effective than alpha-thrombin in producing this effect. Thrombin-stimulated cyclic GMP formation was inhibited by mepacrine and nordihydroguaiaretic acid but unaffected by indomethacin, suggesting that lipoxygenase metabolites of arachidonic acid are involved in the response. These results suggest that a thrombin-like protease in the brain may be involved with the function of neurons or that thrombin interactions with nerve cells, such as those following cerebral hemorrhage or other trauma of the central nervous system, may be important in the subsequent neuropathology.
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PMID:Thrombin stimulation of guanosine 3',5'-monophosphate formation in murine neuroblastoma cells (clone N1E-115). 630 70

Poly(L-lysine) hydrobromide stimulates arachidonic acid release with concomitant synthesis and release of prostaglandins and lipoxygenase-mediated metabolites (hydroxyeicosatetraenoic acids) in cultures of 3T3 Swiss mouse fibroblasts biosynthetically labeled with [3H]arachidonic acid. The response is rapid, reversible with trypsin and persists for at least 50 min. An evaluation of the calcium dependence of the hydrolytic process was consistent with the rate-limiting step involving a cell-surface, calcium-dependent enzyme. The response involves stimulated hydrolysis of arachidonic acid-containing phospholipids, implying the activation of a phospholipase. Arachidonic acid release is stimulated only by poly(L-lysine) hydrobromide preparations with a molecular weight greater than 30 000, which corresponds to a polypeptide chain of more than 140 lysine hydrobromide residues. A variety of other polycations (Mr greater than 30 000), but not polyanions or neutral polymers, stimulated arachidonic acid release and prostaglandin synthesis. The results are consistent with an activation mechanism involving cross-linking of anionic sites on the cell surface. Poly(L-lysine) hydrobromide is also cytotoxic, but the cytotoxic response occurs at 10-fold higher concentrations than arachidonic acid release.
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PMID:Polycations as prostaglandin synthesis inducers. Stimulation of arachidonic acid release and prostaglandin synthesis in cultured fibroblasts by poly(L-lysine) and other synthetic polycations. 642 14

Dielectric heating at frequencies of 42 and 2450 MHz was applied to whole soybeans of natural moisture content for varies exposure times. The minimum energy absorbed (MEA) was calculated from moisture-loss and temperature-elevation data. Biochemical analyses were performed to determine protein dispersibility index (PDI), nitrogen solubility index (NSI), and trypsin-inhibitor, urease, lipoxygenase, and peroxidase activities. Because the heating rates were different at the two frequencies for the power levels used, plots of the biochemical properties against temperature of exposure time showed an apparent frequency dependence. This dependence on frequency disappeared, however, when MEA was substituted as the independent variable. Chemical analyses revealed that dielectric heating of soybeans at natural moisture levels should be as effective as conventional steam toasting in reducing trypsin-inhibitor activity. PDI and NSI, but not urease, were suitable indicators of trypsin-inhibitor inactivation by dielectric heating. Lipoxygenase was completely inactivated by the dielectric-heating treatments that gave suitable trypsin-inhibitor inactivation, but peroxidase activity remained relatively high, offering possible advantages for bleaching and improved soy product color.
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PMID:Effects of 42- and 2450-MHz dielectric heating on nutrition-related properties of soybeans. 692 Apr 15

We studied vascular sodium pump activity and its regulation by vasoactive agents and endothelium in cultured aortic vascular smooth muscle cells from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Baseline sodium pump activity (ouabain-inhibitable 86Rb+ uptake) was similar in cells from both rat strains. Angiotensin II and endothelin-1 increased ouabain-inhibitable 86Rb+ uptake more in SHR than WKY cells, whereas no effects were obtained with sodium nitroprusside, 8-bromo-cGMP, or iloprost. We examined the influence of endothelium on vascular sodium pump activity either by coculturing smooth muscle and endothelial cells or by using conditioned medium. Both coculture for 24 hours with endothelial cells and treatment with conditioned medium increased smooth muscle cell sodium pump activity, this effect being higher in SHR cells. These results suggest that the endothelium may modulate sodium pump activity in the underlying smooth muscle by releasing a diffusible compound, which is more active on SHR smooth muscle. The conditioned medium obtained in the presence of inhibitors of angiotensin-converting enzyme, endothelin-1-converting enzyme, cyclooxygenase, lipoxygenase, and nitric oxide synthase had no effect on the ability of conditioned medium to increase sodium pump activity, suggesting that angiotensin II, endothelin-1, eicosanoids, and nitric oxide are not involved in this stimulatory effect. The nature of the possible endothelial factor involved is still unknown, but it possesses a molecular weight between 25 and 50 kD, is heat stable, and is sensitive to trypsin treatment. We propose it could be a growth factor.
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PMID:Endothelial stimulation of sodium pump in cultured vascular smooth muscle. 760 21

In the present study we investigated the effect of nordihydroguaiaretic acid (NDGA), indomethacin, and cortisone on trypsin-induced acute inflammation in the hamster cheek pouch. Permeability changes, evaluated by fluorescence microscopy after injection of FITC-dextran (MW 150,000), induced by trypsin (2.5 microM) and trypsinated serum (2.5 microM) were significantly suppressed by pretreatment with NDGA (20 mg/kg) and indomethacin (20 mg/kg). Pretreatment with cortisone (40 mg/kg) reduced the permeability changes induced by trypsinated serum but had no significant effect on trypsin-induced leakages. Accumulation of polymorphonuclear leukocytes, as calculated by a whole tissue histological technique, induced by trypsin or trypsinated serum, was significantly reduced by pretreatment with cortisone, NDGA, or indomethacin. These results indicate a role of both cyclooxygenase and lipoxygenase products in trypsin-induced acute inflammation in the hamster cheek pouch.
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PMID:Anti-inflammatory agents inhibit leukocyte accumulation and vascular leakage induced by trypsin and trypsin-digested serum in hamster cheek pouch. 768 36

Mammalian 15-lipoxygenases undergo a characteristic self-inactivation. The oxygenation of a single methionine to methionine sulfoxide, by 13(S)-hydroperoxyoctadecadienoic acid (13-HPODE), was previously suggested as the cause of the inactivation of rabbit reticulocyte lipoxygenase. The site of oxygenation is potentially near the enzyme's active site; however, the specific location of the modified amino acid residue has not been identified. To determine which of the methionine residues is oxygenated, we inactivated both human and rabbit 15-lipoxygenases with 13-HPODE and sequentially denatured, reduced, carboxymethylated, and digested the enzymes with trypsin. The digested mixtures were analyzed by reverse-phase HPLC chromatography. Mass spectrometric analysis of each of the methionine-containing fractions enabled us to locate the peptide segments containing the oxidized methionine in both enzymes separately. Tandem electrospray mass spectrometry identified the oxidized methionine residues to be amino acid 590 in the human enzyme and 591 in the rabbit enzyme. To investigate the significance of this oxygenation, Met590 in human 15-lipoxygenase was substituted with leucine by site-directed mutagenesis. The mutant protein was inactivated by 13-HPODE, yet no oxygenated peptide or other modified peptide could be identified by HPLC-MS analysis. We also found that human 15-lipoxygenase was inactivated during arachidonate oxidation and by the reaction product 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), and no modified peptide was detected. Thus, methionine oxygenation is not essential for the inactivation of human 15-lipoxygenase. We suggest, however, that Met590 is an amino acid in the substrate binding pocket of human 15-lipoxygenase and interacts with the enzyme product 13-HPODE.
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PMID:Identification of a specific methionine in mammalian 15-lipoxygenase which is oxygenated by the enzyme product 13-HPODE: dissociation of sulfoxide formation from self-inactivation. 776 17

Various reports have described that amino acid substitutions can alter substrate, positional, inhibitory, and target gene specificities of proteins. By using the method of Chou and Fasman, the present work predicts that critical amino acids for converting these substrate specificities of trypsin, L-lactate dehydrogenase, aspartate aminotransferase, beta-lactamase, and cytochrome P-450 are found to exist within regions predicted as beta-turns. The ratios of hydroxylation and oxygenation positions of substrates by cytochrome P-450 and lipoxygenase, respectively, are varied by changes of the protein structures, probably around turn conformations. Inhibitory specificities of bovine pancreatic trypsin inhibitor and alpha 1-antitrypsin and target gene specificity of glucocorticoid receptor are converted by changing turn structures. Occurrence of beta-turn probabilities can be predicted around the amino acid alteration positions of an evolutionally antecedent protein of a nylon degradation enzyme. These findings will have relevance to work on protein engineering and enzyme evolution.
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PMID:Critical amino acids responsible for converting specificities of proteins and for enhancing enzyme evolution are located around beta-turn potentials: data-based prediction. 813 29

Intrathoracic injection of endotoxin lipopolysaccharide, LPS into rats induced a dose-dependent increase in the number of eosinophils recovered from the pleural cavity. The pleural eosinophil accumulation peaked within 24-48 h, and returned to basal levels within 120 h. This phenomenon was accompanied by mononuclear cell infiltration, and preceded by massive neutrophil accumulation. Pretreatment with indomethacin, BW 755C (a dual cyclo/lipoxygenase inhibitor), BW A4C (a specific lipoxygenase inhibitor) or the platelet activating factor (PAF) antagonists WEB 2086 and PCA 4248 failed to inhibit the endotoxin-induced pleural eosinophilia, whilst dexamethasone (5-10 micrograms/cavity) or cycloheximide (14-28 micrograms/cavity) abolished this phenomenon. Transfer of the cell-free pleural washing from LPS-treated donor rats to normal recipient rats led to a two-fold increase in the eosinophil counts. Treatment of donors, but not recipients, with cycloheximide or dexamethasone inhibited the eosinophil accumulation induced by the pleural washings, indicating that the generation of the eosinophilotactic activity, but not its effects, depends on protein synthesis. This eosinophilotactic activity was maintained after lyophilization and heating (100 degrees C for 30 min), but was destroyed by trypsin. This substance has a molecular weight ranging between 10 and 50 kDa. The available data suggest that the late eosinophil accumulation induced by LPS is independent of arachidonic acid metabolites and PAF, and probably depends on a newly generated heat-stable soluble protein.
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PMID:Pharmacological modulation of lipopolysaccharide-induced pleural eosinophilia in the rat; a role for a newly generated protein. 833 53

The serine proteases thrombin and trypsin are both powerful platelet agonists that act by cleaving the terminal portion of the thrombin receptor and allowing the new C-terminal to auto-stimulate the receptor. Synthetic peptides, termed thrombin receptor-activating peptides (TRAPs), have been shown to mimic many of the effects of thrombin. Here we have compared the effects of inhibitors on platelet aggregation and [14C]-arachidonic acid release in response to thrombin, trypsin and TRAP. Pretreatment of human platelets with BW755C (80 microM), which inhibits both cyclooxygenase and lipoxygenase, blocked trypsin (15-20 nM)- or TRAP (4-6 microM)-induced aggregation, but not thrombin (0.06-0.1 U/ml)-induced aggregation. The protease inhibitor leupeptin (10 micrograms/ml) abolished trypsin-induced aggregation and returned [14C]-arachidonic acid release from [14C]-arachidonic acid-prelabeled platelets to control levels. In contrast, leupeptin did not affect either aggregation or [14C]-arachidonic acid release in platelets stimulated by TRAP. Thrombin-induced aggregation and [14C]-arachidonic acid release were only partially inhibited by leupeptin. These data are consistent with the activation of platelets by both trypsin and TRAP occurring via the proteolytic receptor, whereas thrombin-induced platelet activation appears to occur by a dual mechanism of action. One component of thrombin-induced platelet activation is by a proteolytic action on the moderate-affinity receptor. This effect is sensitive to inhibition by leupeptin and is mimicked by trypsin and TRAP. The other component of thrombin is nonproteolytic and may occur by an action at a high-affinity receptor such as glycoprotein lb.
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PMID:Thrombin receptor-activating peptide releases arachidonic acid from human platelets: a comparison with thrombin and trypsin. 915 95

In the present investigation, we determined whether A549 cells, a type II pneumocyte cell line, might release mediators that are responsible for monocyte chemoattractant activity (MCA) constitutively. To test this hypothesis, A549 cell supernatant fluids were harvested and evaluated for monocyte chemotaxis. A549 cell supernatant fluids showed MCA in a time-dependent manner (P < 0.001). Checkerboard analysis of 24- and 72-h supernatant fluids showed that the activity was chemokinetic rather than chemotactic. Partial characterization of 24- and 72-h supernatant fluids revealed that the mediator was composed of lipid-soluble activity that was blocked by lipoxygenase inhibitors and trypsin-sensitive activity blocked by cycloheximide. Molecular sieve column chromatography identified four molecular weight peaks. Two of four peaks were blocked by anti-monocyte chemoattractant protein-1 (MCP-1) and anti-transforming growth factor-beta (TGF-beta) polyclonal antibodies. MCP-1 and TGF-beta were detected by enzyme-linked immunosorbent assay. Leukotriene B4 (LTB4) receptor antagonist attenuated the lowest-molecular-weight peak chemotactic response, and the concentration of LTB4 was high enough for chemotactic activity. These findings suggest that type II pneumocytes may modulate the recruitment of monocytes into the alveolar space by releasing MCP-1, TGF-beta, and LTB4 constitutively.
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PMID:Type II pneumocytes release chemoattractant activity for monocytes constitutively. 917 45


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