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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Soybean lipoxygenase 1 was studied using limited proteolysis and active-site labeling to begin the structural characterization of the enzyme in solution. The serine proteases trypsin and chymotrypsin cleaved the large monomeric protein (95 kDa) into two large polypeptides, a C-terminal fragment of about 30 kDa and an N-terminal fragment of about 60 kDa. Under conditions that led to complete cleavage of the protein as judged by SDS-polyacrylamide gel electrophoresis, the catalytic activity of the protein was either reduced slightly (chymotrypsin) or enhanced (trypsin). The characteristics of the cleaved enzymes were the same as for native lipoxygenase 1 in all aspects examined: insensitivity to cyanide, fluoride, and EDTA, regiochemical and stereochemical consequences of catalysis, and EPR spectroscopy upon oxidation by product. The two fragments apparently were tightly associated as they could not be resolved under conditions which preserved the catalytic activity. Both native and protease-cleaved lipoxygenase 1 formed covalent adducts when treated with either 14C-phenylhydrazine or 4-nitrophenylhydrazine. The label was found only in the 60-kDa fragment and following complete trypsin digestion was associated with a peptide beginning after Lys-482 in the primary sequence. Therefore labeling occurred in the vicinity of the conserved histidine cluster which has been postulated as the iron-binding site. From these observations it appears that lipoxygenase 1 exists as a pair of tightly associated domains with the iron-binding site located in the larger of the two.
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PMID:Limited proteolysis and active-site labeling studies of soybean lipoxygenase. 132 20

1. Methacholine relaxed phenylephrine-contracted aorta of the rat with the endothelium intact. This effect was inhibited by haemoglobin, methylene blue, gossypol, phenidone and L-NG-nitroarginine methyl ester (L-NAME). Rat aorta denuded of endothelium failed to relax in response to methacholine, histamine and the peptidoleukotrienes C4, D4 and E4. 2. Methacholine and histamine but not leukotrienes C4, D4 and E4 relaxed phenylephrine-contracted rat aorta without endothelium when surrounded by rabbit epithelium-intact bronchus. The muscarinic antagonist atropine antagonized the methacholine-induced relaxation. 3. Removal of the epithelium either mechanically or chemically, abolished methacholine-induced relaxation of rat aorta in the co-axial bioassay. These data indicate that the epithelium is responsible for the observed relaxant effect to methacholine and histamine. 4. The cyclo-oxygenase inhibitor, indomethacin, the phospholipase A2 inhibitor, mepacrine and the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), failed to inhibit methacholine-induced relaxation of rat aorta in the co-axial bioassay. This indicates that the epithelium-derived inhibitory factor (EpDIF) is not a product of the cyclo-oxygenase or lipoxygenase pathway or a product derived from activation of phospholipase A2. 5. Haemoglobin, methylene blue, phenidone, gossypol and L-NAME failed to inhibit the relaxation of rat aorta in the co-axial bioassay. These results demonstrate that EpDIF detected in the co-axial bioassay is not endothelium-derived relaxing factor (EDRF) or nitric oxide. Similarly, catalase was without effect. 6. EpDIF is unlikely to be a peptide since papain and alpha-chymotrypsin failed to alter the methacholine-induced relaxation of rat aorta in the co-axial bioassay. Furthermore, thiorphan, captopril and aprotinin were also without effect, suggesting that EpDIF is not a substrate for airway peptidases. 7. The results presented in this paper demonstrate the release of a vasoactive epithelium-derived inhibitory factor (EpDIF) from rabbit intrapulmonary bronchi by use of a co-axial bioassay preparation.
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PMID:The release of a non-prostanoid inhibitory factor from rabbit bronchus detected by co-axial bioassay. 185 18

The immobilization of horseradish peroxidase (HRP) onto dissolved agarose by a photochemically initiated graft copolymerization reaction, carried out at room temperature, was studied. Enzyme immobilization parameters such as the catalyst (FeCl3) and the enzyme concentration were considered. Using hexhydro-1,3,5-triacryloyl-s-triazine (HTsT) as vinyl monomer, the agarose/HTsT ratio was the main reaction parameter controlling the copolymer characteristics. By increasing the polymer content of the sample better stability properties were obtained. For the samples with agarose/HTsT ratios of 20/40 and 40/20 (S 20-40, S 40-20) the residual activities after 240 min at 60 degrees C were respectively 47 and 18%. The residual activity in continuous working was 33% for S 40-20 (after 20 h) and 64% for S 20-40 (after 70 h). For both the synthesized copolymers no limitation to substrate diffusion was found but the flexibility of immobilized enzyme decreased with the increase of polymer content as indicated by the Km values that were 0.90 X 10(-4) mol/liter for the sample S 40-20, and 1.50 X 10(-4) mol/liter for the sample S 20-40. Other enzymes (glucose oxidase, alpha-chymotrypsin, and lipoxidase), besides HRP, were immobilized with good yields, showing the wide applicability of the proposed methodology for the preparation of a solid biocatalyst which can be conveniently stored in water suspension or as lyophilized material.
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PMID:Structure and properties of enzyme graft copolymers: effects of using dissolved agarose on horseradish peroxidase immobilization. 311 94

The arachidonic acid pathway plays an important role in many inflammatory reactions. Current evidence suggests that platelets can play a central part in host inflammation. Since microfilariae are mobilized into the bloodstream following diethylcarbamazine (DEC) treatment, we have studied the effects of Onchocerca cervicalis cuticle preparations on equine platelet aggregation. The authors have found that O cervicalis cuticular preparations can induce platelet aggregation in vitro. Furthermore, this activity was abrogated by treatment with collagenase and not hyaluronidase, elastase, or alpha-chymotrypsin. When this evidence is viewed collectively with the evidence for in vivo parasite cuticular damage following DEC treatment, it becomes entirely plausible that the cuticular damage may indeed reveal a platelet-reactive surface, thus permitting platelet-parasite binding to occur. This binding would result in platelet aggregation and the generation and release of platelet-derived arachidonate metabolites. These metabolites may play a very critical role in the development of the described pathologic sequelae observed following DEC treatment. Field studies using cyclooxygenase and lipoxygenase inhibitors might therefore be very efficacious in decreasing the frequency of side effects due to DEC or other potentially effective drug regimens.
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PMID:Aggregation of equine platelets by Onchocerca cervicalis collagen. 629 6

We offer a novel graphical method for determining the number of essential sites in enzymes that contain multiple binding sites for a ligand. This method is applicable both to monomeric enzymes containing multiple "unspecific" sites (for protons, metal ions, etc.) and to oligomeric enzymes containing multiple "specific" sites (for substrates and their cognate analogues). The overall procedure is based on some of the intrinsic properties of the general rate equation for enzyme-catalyzed reactions involving multiple binding sites for ligands as elaborated by W. G. Bardsley and R. E. Childs (Biochem. J., 149, 313-328, 1975). The experimental protocol involves measurement of initial rates of enzyme-catalyzed reactions either at varying concentrations of the substrate or at a fixed concentration of the substrate and varying concentrations of effectors (activator or inhibitor). The data are analyzed by plotting 1/v[L]p versus [L] (for inhibition) and [L]p/v versus 1/[L] (for activation) for different (integral) increments of p (such as 0, 1, 2, 3, ... etc.). According to the analytical procedure developed herein, the magnitude of p that yields a horizontal asymptote on these plots serves as the quantitative measure of the number of essential sites in enzyme molecules. By employing this procedure, we have been able to quantitatively ascertain the number of essential sites required for the activation or inhibition of a variety of monomeric and oligomeric enzymes. Among monomeric enzymes we have established that: (i) of the three binding sites for linoleic acid in the lipoxygenase molecule, one site is essential for catalysis, and the other two sites are inhibitory; (ii) of several plausible protonation sites in the alpha-chymotrypsin molecule, only one protonation site is required for the activation of the enzyme; and (iii) there are two inhibitory sites for guanidine-HCl per ribonuclease A molecule; the enzyme is fully inhibited upon binding of guanidine-HCl at any of these two sites. Among oligomeric enzymes, we have discerned that: (i) the individual subunits of LDH and phosphorylase b are catalytically active, and (ii) the catalytic/functional unit of the creatine kinase molecule is likely to be the dimeric subunit. The theoretical details leading to the graphical analysis and its usage in delineating the functional stoichiometry of enzyme-ligand complexes are discussed.
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PMID:A graphical method for determining the number of essential sites in enzymes with multiple binding sites for a ligand. 813 45

Spectroscopic techniques (UV absorbance, circular dichroism, fluorescence emission and anisotropy, and light scattering) were used to investigate enzyme solubilization in Aerosol-OT (AOT) reversed micelles in which a bile salt, sodium taurocholate (NaTC), is used as a novel cosurfactant. NaTC significantly increases the water capacity and size of the reversed micelles through surfactant reorganization. The solubilization of several enzymes, including lysozyme, chymotrypsin, lipase, lipoxidase, carbonic anhydrase, and ribonuclease A, was demonstrated. These enzymes, ranging in mass from 10(4) to 10(5) Da, are incorporated in the micelles in stable, optically transparent solutions. Several other proteins were not successfully solubilized. The presence of NaTC in the reversed micelles significantly altered the conformations of the solubilized enzymes, apparently by promoting unfolding of the enzyme through interactions with the interior micellar interface. Lysozyme and lipase respond to solubilization in the AOT/NaTC micelles by altering their conformations to accommodate the micellar structure. The effect of NaTC is greatest for lysozyme, inducing a higher degree of order and helicity in the enzyme structure. Chymotrypsin, on the other hand, disrupts the micellar structure and reorganizes the surfactants to accommodate its own preferred conformation. Addition of NaTC to the reversed micelles causes a 3-fold increase in the enzymatic activity of solubilized chymotrypsin. Copyright 1997Academic Press
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PMID:Enzyme Solubilization in a Reversed Micellar Microreactor with a Bile Salt Cosurfactant 929 86

The time course of accumulation and the composition of proteinase-inhibiting proteins in diffusates from potato tubers treated with elicitors such as salicylic, jasmonic, and arachidonic acids were studied. The 40-kDa reserve protein patatin and the chymotrypsin inhibitors, among which proteins of 24.6, 22.0, and 16.0 kDa were prevalent, accumulated in diffusates from potato tubers. Jasmonic and arachidonic acids activated the accumulation of the chymotrypsin inhibitors in tubers in response to the injury stress, whereas salicylic acid inhibited this process. The effects of jasmonic and arachidonic acids increased when their concentrations decreased to 10(-6) M. The data suggest an important role of the lipoxygenase metabolism in signal transduction of the anti-injury defense system in the dormant potato tubers.
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PMID:[Effect of elicitors on accumulation of protease inhibitors in injured potato tubers]. 1160 75

Mechanical damage or infection of potatoes with Phytophthora infestans caused an accumulation of only serine protease inhibitors in exudates of potato tubers. Among them, proteins prevailed that are structurally similar to those present in healthy tubers: a 22-kDa trypsin inhibitor, a 21-kDa serine protease inhibitor consisting of two polypeptide chains, and a 8-kDa potato chymotrypsin I inhibitor produced de novo. The accumulated proteins inhibited the growth of hyphae and germination of zoospores of P. infestans. Treatment with elicitors, jasmonic and arachidonic acids, intensified the accumulation of these inhibitors in tubers in response to the wound stress, whereas salicylic acid blocked this process. These results suggest that the lipoxygenase metabolism plays a substantial role in signal transduction of the protective system of resting potato tubers.
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PMID:[Role of proteinase inhibitors in potato protection]. 1460 5

The role of phospholipase C (an enzyme involved in the metabolism of inositol-containing phospholipids), cyclooxygenase and lipoxygenase (the enzymes of arachidonic acid metabolism), and adenylate cyclase and phosphodiesterase (the enzymes of cyclic adenosine 3,5-monophosphate (cAMP) metabolism) in the mechanisms of the aggregation of human platelets induced by the serine protease in low concentrations (thrombin 0.5 mkg per ml, trypsin 1 mkg per ml, and alpha-chymotrypsin 10 mkg per ml) have been investigated with the use of the inhibitor analysis. The effect of neomycin sulfate (phospholipase C inhibitor), indometacin (cyclooxygenase inhibitor), and nordihydrogvayaretic acid (lipoxygenase inhibitor) on protease-induced increase in the content of calcium cations in platelet plasma has been studied. The results of the inhibitor analysis indicated that the enzymes of metabolism of inositol-containing phospholipids, arachidonic acid, and cAMP are involved in the mechanisms of the protease-induced platelet aggregation. The increase in the content of calcium ions, associated with the protease-induced activation of phospholipase C, in cytoplasm may play an important role in the mechanisms of platelet aggregation.
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PMID:[Mechanisms of the protease-induced aggregation of human platelets]. 1790 8

Chalcones and Mannich bases have been reported to present antiinflammatory activities as well as inhibitory activities on several factors implicated in inflammation disorders. A series of chalcones and some related Mannich bases were prepared by Claisen-Schmidt condensation of appropriate acetophenones with appropriate aromatic aldehyde. Mannich bases were derived from chalcones, with formaldehyde and the corresponding amine. The compounds were tested in vitro for their ability to inhibit various enzymes involved in the arachidonic acid cascade, for their antioxidant behaviour and in vivo for anti-inflammatory activity. Some chalcones and Mannich bases present strong anti-inflammatory and antioxidant activities. Almost all the tested compounds present high inhibitory activity on lipid peroxidation. Some compounds showed potent inhibitory effect on superoxide anion formation. Among the tested compounds 5 and 6 showed the highest lipoxygenase (LO) inhibitory activity. All the tested compounds inhibit both the proteolytic and esteratic activities of trypsin and chymotrypsin. The results indicated that the anti-inflammatory effects of the compounds were partially mediated, through their antioxidant activity. Attempts to correlate quantitatively structure with activity revealed that lipophilicity and molar refractivity influence the biological response.
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PMID:Synthesis and anti-inflammatory activity of chalcones and related Mannich bases. 1899 44


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