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51.
Uzunova Y Lukanov L Filipov I Vladimirov S 《Journal of biochemical and biophysical methods》2008,70(6):883-888
HPLC method was developed for determination of bisphenol A diglycidyl methacrylate (bis-GMA), bisphenol A diglycidyl acrylate (bis-GA), bisphenol A dimethacrylate (bis-DMA), glycidylmethacrylate (GMA) and triethylenglycol dimethacrylate (TEGDMA). Separation was carried out on a reversed phase Omnisphere 5 C18 column with a gradient mobile phase of CH3CN/H2O. UV detection was set at 205 nm and 275 nm parallel. The limits of quantification were found. The method has been applied for quantification of unreacted monomers trapped in polymer network of fillings. 相似文献
52.
Stojan J 《Journal of enzyme inhibition》2001,16(2):89-94
An analysis of sigmoid-shaped progress curves in the reaction between Electric Eel acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7, AChE) and its substrate acetylthiocholine in low concentrations at pH 7 is presented. In order to be able to explain an initial apparent inhibition of the enzyme-substrate reaction, the rate of detection reaction had to be taken into account. The theoretical curves obtained by the fitting of differential equations for the reaction mechanism to the data of six progress curves simultaneously, exactly reproduce the course of the experimental curves. The measurements performed with various concentrations of detection reagent confirm the proposed cause of sigmoidity. 相似文献
53.
Interaction of Drosophila acetylcholinesterases with D-tubocurarine: an explanation of the activation by an inhibitor 总被引:1,自引:0,他引:1
Homotropic cooperativity in Drosophila melanogaster acetylcholinesterase seems to be a consequence of an initial substrate binding to a high-affinity peripheral substrate binding site situated around the negative charge of D413 (G335, Torpedo numbering). An appropriate mutation which turns the peripheral binding site to a low-affinity spot abolishes apparent activation but improves the overall enzyme effectiveness. This contradiction can be explained as less effective inhibition due to a shorter occupation of such a peripheral site. A similar effect can be achieved by an appropriate peripheral inhibitor such as TC, which can in special cases, when less effective heterotropic inhibition prevails over homotropic, acts as an activator. At the highest substrate concentrations, however, these enzymes are always inhibited, although steric components may influence the strength of inhibition like in the F368G mutant (F290, Torpedo numbering). Cooperative effects thus may include a steric component, but covering of the entrance must affect influx and efflux to different extents. 相似文献
54.
17beta-Hydroxysteroid dehydrogenase (17beta-HSD) activity has been described in all filamentous fungi tested, but until now only one 17beta-HSD from Cochliobolus lunatus (17beta-HSDcl) was sequenced. We examined the evolutionary relationship among 17beta-HSDcl, fungal reductases, versicolorin reductase (Ver1), trihydroxynaphthalene reductase (THNR), and other homologous proteins. In the phylogenetic tree 17beta-HSDcl formed a separate branch with Ver1, while THNRs reside in another branch, indicating that 17beta-HSDcl could have similar function as Ver1. The structural relationship was investigated by comparing a model structure of 17beta-HSDcl to several known crystal structures of the short chain dehydrogenase/reductase (SDR) family. A similarity was observed to structures of bacterial 7alpha-HSD and plant tropinone reductase (TR). Additionally, substrate specificity revealed that among the substrates tested the 17beta-HSDcl preferentially catalyzed reductions of steroid substrates with a 3-keto group, Delta(4) or 5alpha, such as: 4-estrene-3,17-dione and 5alpha-androstane-3,17-dione. 相似文献
55.
Ota Fekonja Majda Zorec-Karlovšek Manale El Kharbili Didier Fournier Jure Stojan 《Journal of enzyme inhibition and medicinal chemistry》2013,28(4):407-415
The cholinesterases have been investigated in terms of the effects of methanol and ethanol on substrate and carbamate turnover, and on their phosphorylation. It was found: 1) that at low substrate concentrations the two alcohols inhibit all three tested cholinesterases and that the optimum activities are shifted towards higher substrate concentrations, but with a weak effect on horse butyrylcholinesterase; 2) that methanol slows down carbamoylation by eserine and does not influence decarbamoylation of vertebrate and insect acetylcholinesterase and 3) that ethanol decreases the rate of phosphorylation of vertebrate acetylcholinesterase by DFP. Our results are in line with the so-called ‘approach-and-exit’ hypothesis. By hindering the approach of substrate and the exit of products, methanol and ethanol decrease cholinesterase activity at low substrate concentrations and allow for the substrate inhibition only at higher substrate concentrations. Both effects appears to be a consequence of the lower ability of substrate to substitute alcohol rather than water. It also seems that during substrate turnover in the presence of alcohol the transacetylation is negligible. 相似文献
57.
We have recently isolated and heterologously expressed BcPeh28A, an endopolygalacturonase from the phytopathogenic Gram-negative bacterium Burkholderia cepacia. Endopolygalacturonases belong to glycoside hydrolase family 28 and are responsible for the hydrolysis of the non-esterified regions of pectins. The mode of action of BcPeh28A on different substrates has been investigated and its enzymatic mechanism elucidated. The hydrolysis of polygalacturonate indicates that BcPeh28A is a non-processive enzyme that releases oligomers with chain lengths ranging from two to eight. By inspection of product progression curves, a kinetic model has been generated and extensively tested. It has been used to derive the kinetic parameters that describe the time course of the formation of six predominant products. Moreover, an investigation of the enzymatic activity on shorter substrates that differ in their overall length and methylation patterns sheds light on the architecture of the BcPeh28A active site. Specifically the tolerance of individual sites towards methylated saccharide units was rationalized on the basis of the hydrolysis of hexagalacturonides with different methylation patterns. 相似文献
58.
Stojan Maleschlijski Adam Autry Llewellyn Jalbert Marram P. Olson Tracy McKnight Tracy Luks Sarah Nelson 《Metabolomics : Official journal of the Metabolomic Society》2017,13(12):149
Introduction
Infiltrating gliomas are primary brain tumors that express significant biological and clinical heterogeneity in adults, which complicates their treatment and prognosis. Characterization of tumor subtypes using spectroscopic analysis may assist in predicting malignant transformation and quantification of response to therapy.Study objective
To implement an automated algorithm for classification of metabolomic profiles for the classification of glioma pathological grades and the prediction of malignant progression using spectra obtained by high-resolution magic angle spinning (HR-MAS) spectroscopy of patient-derived tissue samples.Methods
237 image-guided tissue samples were obtained from 152 patients who underwent surgery for newly diagnosed or recurrent glioma and analyzed via HR-MAS spectroscopy. Orthogonal projection to latent structures discriminant analysis was used as a classifier and the variable-influence-on-projection values were evaluated to identify signature spectral regions.Results
The accuracy of classifiers developed for discriminating glioma subtypes was 68% for newly diagnosed grade II versus III samples; 86 and 92% for new and recurrent grade III versus IV, respectively; 95% for newly diagnosed grade II versus IV; and 88% for recurrent grade II versus IV lesions. Classifiers distinguished between samples from newly diagnosed vs. recurrent lesions with an accuracy of 78% for grade III and 99% for grade IV glioma.Conclusion
Classifying metabolomic profiles for new and recurrent glioma without prior assumptions regarding spectral components identified candidate in vivo biomarkers for use in assessing changes that are likely to impact treatment decisions.59.
Jure Stojan Marko Golicnik Marie-Thérèse Froment Francois Estour Patrick Masson 《European journal of biochemistry》2002,269(4):1154-1161
Tetraalkylammonium (TAA) salts are well known reversible inhibitors of cholinesterases. However, at concentrations around 10 mm, they have been found to activate the hydrolysis of positively charged substrates, catalyzed by wild-type human butyrylcholinesterase (EC 3.1.1.8) [Erdoes, E.G., Foldes, F.F., Zsigmond, E.K., Baart, N. & Zwartz, J.A. (1958) Science 128, 92]. The present study was undertaken to determine whether the peripheral anionic site (PAS) of human BuChE (Y332, D70) and/or the catalytic substrate binding site (CS) (W82, A328) are involved in this phenomenon. For this purpose, the kinetics of butyrylthiocholine (BTC) hydrolysis by wild-type human BuChE, by selected mutants and by horse BuChE was carried out at 25 degreeC and pH 7.0 in the presence of tetraethylammonium (TEA). It appears that human enzymes with more intact structure of the PAS show more prominent activation phenomenon. The following explanation has been put forward: TEA competes with the substrate at the peripheral site thus inhibiting the substrate hydrolysis at the CS. As the inhibition by TEA is less effective than the substrate inhibition itself, it mimics activation. At the concentrations around 40 mm, well within the range of TEA competition at both substrate binding sites, it lowers the activity of all tested enzymes. 相似文献
60.
A detailed study of the velocity of the reaction between Ellman's reagent and thiocholine was undertaken, in order to test the possibilities of this reaction as a detection method for the earlier stages of cholinesterases reactions. Experiments were carried out on a stopped-flow apparatus with a built-in spectrophotometer. The obtained experimental data were analyzed by fitting the data to theoretical kinetic equations derived for the reaction. In this way, a complete kinetic characterization of the reaction was obtained. An important practical result derived from our investigations is the finding that, under most experimental conditions, the Ellman's reactions is more than sufficiently rapid as a detection method. However, in the case of reactions in the time scale of 200 milliseconds or less, this being 5 times the half life of Ellman's reaction at standard conditions, one has to consider the interference of this reaction with the enzyme reaction itself. 相似文献