Search results for "Substrate Specificity"
showing 10 items of 217 documents
2′,5′-oligoadenylate synthetase from a lower invertebrate, the marine sponge Geodia cydonium, does not need dsRNA for its enzymatic activity
2002
AbstractRecently, the presence of 2′,5′-linked oligoadenylates and a high 2′,5′-oligoadenylate synthetase activity were discovered in a lower invertebrate, the marine sponge Geodia cydonium. It has been demonstrated that mammalian 2–5A synthetase isozymes require a dsRNA cofactor for their enzymatic activity. Our results show that, unlike mammalian 2–5A synthetases, the 2–5A synthetase from the sponge acts in a dsRNA-independent manner in vitro. A prolonged incubation of the G. cydonium extract with a high concentration of a micrococcal nuclease had no effect on the activity of the 2–5A synthetase. At the same time, the micrococcal nuclease was effective within 30 min in degrading dsRNA nee…
The diversity of GABAA receptors. Pharmacological and electrophysiological properties of GABAA channel subtypes.
1998
The amino acid gamma-aminobutyric-acid (GABA) prevails in the CNS as an inhibitory neurotransmitter that mediates most of its effects through fast GABA-gated Cl(-)-channels (GABAAR). Molecular biology uncovered the complex subunit architecture of this receptor channel, in which a pentameric assembly derived from five of at least 17 mammalian subunits, grouped in the six classes alpha, beta, gamma, delta, sigma and epsilon, permits a vast number of putative receptor isoforms. The subunit composition of a particular receptor determines the specific effects of allosterical modulators of the GABAARs like benzodiazepines (BZs), barbiturates, steroids, some convulsants, polyvalent cations, and et…
Efficient Expression and Crystallization System of Cancer-Associated Carbonic Anhydrase Isoform IX.
2015
Human carbonic anhydrase IX (CA IX) is overexpressed in a number of solid tumors and is considered to be a marker for cellular hypoxia that it is not produced in most normal tissues. CA IX contributes to the acidification of the extracellular matrix, which, in turn, favors tumor growth and metastasis. Therefore, CA IX is considered to be a promising anti-cancer drug target. However, the ability to specifically target CA IX is challenging due to the fact that the human genome encodes 15 different carbonic anhydrase isoforms that have a high degree of homology. Furthermore, structure-based drug design of CA IX inhibitors so far has been largely unsuccessful due to technical difficulties regar…
Midgut aminopeptidase N isoforms from Ostrinia nubilalis: Activity characterization and differential binding to Cry1Ab and Cry1Fa proteins from Bacil…
2013
Aminopeptidase N (APN) isoforms from Lepidoptera are known for their involvement in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis. These enzymes belong to a protein family with at least eight different members that are expressed simultaneously in the midgut of lepidopteran larvae. Here, we focus on the characterization of the APNs from Ostrinia nubilalis (OnAPNs) to identify potential Cry receptors. We expressed OnAPNs in insect cells using a baculovirus system and analyzed their enzymatic activity by probing substrate specificity and inhibitor susceptibility. The interaction with Cry1Ab and Cry1Fa proteins (both found in transgenic insect-resistant maize) was …
Active Site Mapping of Xylan-Deconstructing Enzymes with Arabinoxylan Oligosaccharides Produced by Automated Glycan Assembly
2017
Xylan-degrading enzymes are crucial for the deconstruction of hemicellulosic biomass, making the hydrolysis products available for various industrial applications such as the production of biofuel. To determine the substrate specificities of these enzymes, we prepared a collection of complex xylan oligosaccharides by automated glycan assembly. Seven differentially protected building blocks provided the basis for the modular assembly of 2-substituted, 3-substituted, and 2-/3-substituted arabino- and glucuronoxylan oligosaccharides. Elongation of the xylan backbone relied on iterative additions of C4-fluorenylmethoxylcarbonyl (Fmoc) protected xylose building blocks to a linker-functionalized …
Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis.
2003
The plant enzyme arbutin synthase isolated from cell suspension cultures of Rauvolfia serpentina and heterologously expressed in Escherichia coli is a member of the NRD1beta family of glycosyltransferases. This enzyme was used to prove, by site-directed mutagenesis, suggested catalytic domains and reaction mechanisms proposed for enzyme-catalyzed glycosylation. Replacement of amino acids far from the NRD domain do not significantly affect arbutin synthase activity. Exchange of amino acids at the NRD site leads to a decrease of enzymatic activity, e.g. substitution of Glu368 by Asp. Glu368, which is a conserved amino acid in glycosyltransferases located at position 2 and is important for enz…
Oxidative DNA base damage induced by singlet oxygen and photosensitization: recognition by repair endonucleases and mutagenicity.
2000
We have analyzed the recognition by various repair endonucleases of DNA base modifications induced by three oxidants, viz. [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), a photochemical source of tert-butoxyl radicals, disodium salt of 1,4-etheno-2,3-benzodioxin-1,4-dipropanoic acid (NDPO(2)), a chemical source of singlet oxygen, and riboflavin, a type-I photosensitizer. The base modifications induced by BCBT, which were previously shown to be mostly 7,8-dihydro-8-oxoguanine (8-oxoGua) residues, were recognized by Fpg and Ogg1 proteins, but not by endonuclease IIII, Ntg1 and Ntg2 proteins. In the case of singlet oxygen induced damage, 8-oxoGua accounted for only 35% of…
Distinct Site Specificity of Two Pea Histone Deacetylase Complexes
2001
We report on the site specificity of two intact pea histone deacetylase complexes. HD1 deacetylates lysines 5 and 16 of H4 in the order K16 > K5, while in the case of H3 the preferred order is K4 >> K18 approximately K9. The specificity of the HD2 complex is markedly different. The preferred residues in H4 are K8 approximately K5 > K16, while in H3 deacetylation, the complex HD2 prefers sites 4 and 18. To obtain these results, we have used a novel procedure based on the SPOT technique, a method to synthesize peptides on membrane supports. Different sets of membranes with sequentially overlapping histone peptides containing acetylated lysines in the sites corresponding to all in vivo acetyla…
Characterization of Acylating and Deacylating Activities of an Extracellular Phospholipase A2 in a Water-Restricted Environment
1994
The behavior of porcine pancreatic phospholipase A2 (ppPLA2) in monophasic low-water media has been explored, for the first time, in a systematic manner. It has been investigated how a number of variables can modulate both acylating and deacylating activities of the enzyme, and several interesting, unexpected results are presented. Among the most relevant, when placing ppPLA2 in the water-restricted environment, are the following: (i) it displays a remarkable alteration of its specificity toward the substrate polar head relative to all-water medium; (ii) it is quite severely inhibited by lysophosphatidylcholine (LPC), which has important implications, particularly concerning its acylation a…
Study and characterization of polyphenol oxidase from eggplant (Solanum melongena L.)
2011
ABSTRACT: In this study the catecholase and cresolase activities of eggplant polyphenol oxidase (PPO) were investigated. Enzyme activity was determined by measuring the increase in absorbance using catechol as substrate and 3-methyl-2- benzothiazolinone hydrazone (MBTH) as coupled reagent. The effects of substrate specificity, heat inactivation, temperature, pH, and inhibitors were investigated to understand the enzymatic alteration of ready-to-eat preparations. Browning of vegetables was determined through a colorimeter. Decrease of lightness (L*) and increase of color difference values (ΔE*) were correlated with tissue browning. Antibrowning agents were tested on PPO under the same condit…