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:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a method for monitoring the N-glycosylation of recombinant glycoproteins directly from conditioned medium samples. Proteins in the conditioned medium are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. After staining the membranes with Coomassie blue, the protein(s) of interest is excised. Oligosaccharides are released from the membrane-bound glycoprotein by digesting with peptide N4-(acetyl-beta-glucosaminyl) asparagine amidase and labeled with the fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS). Labeled oligosaccharides are then separated on polyacrylamide gels which allow for the direct comparison of samples. We have shown that recombinant human lysosomal hydrolase alpha-galactosidase A is N-glycosylated with both sialylated and phosphorylated oligosaccharides. ANTS-labeled oligosaccharide bands from alpha-galactosidase A were isolated from polyacrylamide gels. Sialylated and phosphorylated bands were identified by shifts in their electrophoretic mobility after digesting with neuraminidase or alkaline phosphatase to remove sialic acid or phosphate groups, respectively. Using the ANTS-labeled oligosaccharides from alpha-galactosidase A, we have shown that polyacrylamide gels can be used to resolve sialylated and phosphorylated oligosaccharide structures.
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PMID:A method for monitoring the glycosylation of recombinant glycoproteins from conditioned medium, using fluorophore-assisted carbohydrate electrophoresis. 857 98

Mammalian brain as well as mouse neuroblastoma (N18TG2) and rat basophilic leukaemia (RBL) cells were previously shown to contain "anandamide amidohydrolase', a membrane-bound enzyme sensitive to serine and cysteine protease inhibitors and catalyzing the hydrolysis of the endogenous cannabimimetic metabolite, anandamide (arachidonoyl-ethanolamide). With the aim of developing novel inhibitors of this enzyme, we synthesized three arachidonic acid (AA) analogues, i.e. arachidonoyl-diazo-methyl-ketone (ADMK), ara-chidonoyl-chloro-methyl-ketone (ACMK) and O-acetyl-arachidonoyl-hydroxamate (AcAHA), by adding to the fatty acid moiety three functional groups previously used to synthesize irreversible inhibitors of serine and cysteine proteases. The three compounds were purified and characterized by proton nuclear magnetic resonance and electron impact mass spectrometry. Their effect was tested on anandamide amidohydrolase partially purified from N18TG2 and RBL-1 cells and porcine brain. Pre-treatment of the enzyme with each compound produced a significant inhibition, with ADMK being the most potent (IC50 = 3, 2 and 6 microM) and AcAHA the weakest (IC50 = 34, 15 and 25 microM) inhibitors. The inactivated enzyme regained its full activity when chromatographed by anion-exchange chromatography, suggesting that none of the compounds inhibited the amidohydrolase in a covalent manner. Accordingly, Lineweaver-Burk profiles showed competitive inhibition by each compound. Conversely, the irreversible inhibitor of cytosolic phospholipase As, methyl-arachidonoyl-fluoro-phosphonate (MAFP), covalently inhibited the amidohydrolase. MAFP was active at concentrations 10(3) times lower than those reported for phospholipase A2 inhibition, and is the most potent anandamide amidohydrolase inhibitor so far described (IC50 = 1-3 nM). MAFP, ADMK and ACMK, probably by inhibiting anandamide degradation, produced an apparent increase of the in vitro formation of anandamide from its biosynthetic precursor N-arachidonoyl-phosphatidyl-ethanolamine.
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PMID:Novel inhibitors of brain, neuronal, and basophilic anandamide amidohydrolase. 907 Feb 24

The promoter selectivity of RNA polymerase (RNAP) can be altered by the association with alternative sigma subunits. Bacillus subtilis hosts a multitude of sigma factors, several of which coordinate the complex developmental program culminating in endospore formation. Genome sequencing has revealed an unanticipated seven new sigma factors of the highly divergent extracytoplasmic function (ECF) sub-family. Virtually nothing is known regarding either the promoter selectivity or the target genes for these newly identified sigma factors. We have used saturation mutagenesis to define a promoter consensus for recognition by one such ECF sigma factor, sigma X. The resulting consensus sequence was used to identify candidate sigma X target sites. Three newly identified sigma X-dependent promoters precede genes encoding regulatory proteins: an AbrB homolog (Abh), a putative response regulator aspartate phosphatase (RapD), and a regulator of autolysin expression (LytR). sigma X also contributes to the expression of CsbB, a putative membrane-bound glucosyl transferase that is partially controlled by the sigma B stress response sigma factor. Since LytR modulates the expression of the major autolytic amidase and CsbB may function in peptidoglycan synthesis or modification, we suggest that sigma X participates in the regulation of peptidoglycan synthesis and turnover.
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PMID:Identification of target promoters for the Bacillus subtilis sigma X factor using a consensus-directed search. 963 7

The gene for a novel endotype membrane-bound lytic transglycosylase, emtA, was mapped at 26.7 min of the E. coli chromosome. EmtA is a lipoprotein with an apparent molecular mass of 22kDa. Overexpression of the emtA gene did not result in bacteriolysis in vivo, but the enzyme was shown to hydrolyze glycan strands isolated from murein by amidase treatment. The formation of tetra- and hexasaccharides, but no disaccharides, reflects the endospecificity of the enzyme. The products are characterized by the presence of 1,6-anhydromuramic acid, indicating a lytic transglycosylase reaction mechanism. EmtA may function as a formatting enzyme that trims the nascent murein strands produced by the murein synthesis machinery into proper sizes, or it may be involved in the formation of tightly controlled minor holes in the murein sacculus to facilitate the export of bulky compounds across the murein barrier.
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PMID:Membrane-bound lytic endotransglycosylase in Escherichia coli. 964 99

The endogenous cannabinoid anandamide (N-arachidonoylethanolamide) has been shown to possess higher affinity for the cannabinoid CB1 receptor than for the CB2 receptor. Carrier-mediated transport has been proposed to be essential for the termination of the biological effects of anandamide, while hydrolysis of anandamide is performed by a membrane-bound amidohydrolase, fatty acid amidohydrolase (FAAH). As interaction of anandamide with each of these targets occurs in different environments, the conformations of anandamide for interaction with each target may differ. To ascertain what conformations of anandamide, a highly flexible molecule, are favored in polar and nonpolar environments, the new method of Conformational Memories (CM) was used. CM has been shown to achieve complete conformational sampling of highly flexible ligands, to converge in a very practical number of steps, and to be capable of overcoming energy barriers very efficiently (Guarnieri et al. J. Am. Chem. Soc. 1996, 118, 5580). The generalized Born/surface area (GB/SA) continuum solvation models for chloroform and for water were used in the CM calculations. As a means of validation, CM was first applied to arachidonic acid because both experimental and theoretical conformational studies of arachidonic acid have been reported. CM was also applied to sn-2-arachidonylglycerol (2-AG), another endogenous CB ligand; to a 1,1-dimethylheptyl derivative of anandamide, an analogue with higher CB1 affinity than anandamide; and to N-(2-hydroxyethyl)prostaglandin-B2-ethanolamide (PGB2-EA), a prostanoid ligand which does not bind to CB1. Consistent with the literature, arachidonic acid was found to exist in an extended, angle-iron shape and in back-folded conformations which were U, J, or helical in shape. The angle-iron and U-shapes were both highly populated conformations with the angle-iron preferred in CHCl3 and the U-shape preferred in H2O. Results for anandamide and 2-AG paralleled those for arachidonic acid with the exception that anandamide in water does not adopt a pure extended conformation but, rather, favors a hybrid-extended/U-shape. For the dimethyl-heptyl derivative of anandamide, the U-shape was found to be predominant in both environments (42% in CHCl3, 38% in H2O), but the population of the angle-iron shape was still significant (25% in CHCl3, 29% in H2O). For all of these ligands, J-shaped conformers constituted from 7% to 17% of the conformer population, while the helical shape was less than 5%. In both CHCl3 and H2O, the presence of the five-membered ring attenuates the ability of PGB2-EA to adopt an extended conformation. PGB2-EA was found instead to exist predominantly in an L-shape (i.e., distorted U-shape). The low probability of PGB2-EA adopting an extended conformation may be why PGB2-EA shows such low affinity for the CB1 receptor. The conformational information obtained here for anandamide and 2-AG may be useful in the design of rigid analogues which mimic the preferred molecular conformations (shapes) of these ligands. Such rigid analogues may be useful in deducing the bioactive conformation of these endogenous cannabinoids, not only at the CB receptors but also at the FAAH enzyme active site and possibly at the binding site(s) on the newly proposed anandamide transporter.
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PMID:Exploration of biologically relevant conformations of anandamide, 2-arachidonylglycerol, and their analogues using conformational memories. 982 55

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide and oleamide. FAAH's primary structure identifies this enzyme as a member of a diverse group of alkyl amidases, known collectively as the "amidase signature family". At present, this enzyme family's catalytic mechanism remains poorly understood. In this study, we investigated the catalytic features of FAAH through mutagenesis, affinity labeling, and steady-state kinetic methods. In particular, we focused on the respective roles of three serine residues that are conserved in all amidase signature enzymes (S217, S218, and S241 in FAAH). Mutation of each of these serines to alanine resulted in a FAAH enzyme bearing significant catalytic defects, with the S217A and S218A mutants showing 2300- and 95-fold reductions in k(cat), respectively, and the S241A mutant exhibiting no detectable catalytic activity. The double S217A:S218A FAAH mutant displayed a 230 000-fold decrease in k(cat), supporting independent catalytic functions for these serine residues. Affinity labeling of FAAH with a specific nucleophile reactive inhibitor, ethoxy oleoyl fluorophosphonate, identified S241 as the enzyme's catalytic nucleophile. The pH dependence of FAAH's k(cat) and k(cat)/K(m) implicated a base involved in catalysis with a pK(a) of 7.9. Interestingly, mutation of each of FAAH's conserved histidines (H184, H358, and H449) generated active enzymes, indicating that FAAH does not contain a Ser-His-Asp catalytic triad commonly found in other mammalian serine hydrolytic enzymes. The unusual properties of FAAH identified here suggest that this enzyme, and possibly the amidase signature family as a whole, may hydrolyze amides by a novel catalytic mechanism.
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PMID:Chemical and mutagenic investigations of fatty acid amide hydrolase: evidence for a family of serine hydrolases with distinct catalytic properties. 1043 86

The endocannabinoids, a family of endogenous lipids that activate cannabinoid receptors, are released from cells in a stimulus-dependent manner by cleavage of membrane lipid precursors. After release, the endocannabinoids are rapidly deactivated by uptake into cells and enzymatic hydrolysis. Endocannabinoid reuptake occurs via a carrier-mediated mechanism, which has not yet been molecularly characterized. Endocannabinoid reuptake has been demonstrated in discrete brain regions and in various tissues and cells throughout the body. Inhibitors of endocannabinoid reuptake include N-(4-hydroxyphenyl)-arachidonylamide (AM404), which blocks transport with IC50 (concentration necessary to produce half-maximal inhibition) values in the low micromolar range. AM404 does not directly activate cannabinoid receptors or display cannabimimetic activity in vivo. Nevertheless, AM404 increases circulating anandamide levels and inhibits motor activity, an effect that is prevented by the CB1 cannabinoid antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A). AM404 also reduces behavioral responses to dopamine agonists and normalizes motor activity in a rat model of attention deficit hyperactivity disorder. The endocannabinoids are hydrolyzed by an intracellular membrane-bound enzyme, termed anandamide amidohydrolase (AAH), which has been molecularly cloned. Several fatty acid sulfonyl fluorides inhibit AAH activity irreversibly with IC50 values in the low nanomolar range and protect anandamide from deactivation in vivo. alpha-Keto-oxazolopyridines inhibit AAH activity with high potency (IC50 values in the low picomolar range). A more thorough characterization of the roles of endocannabinoids in health and disease will be necessary to define the significance of endocannabinoid inactivation mechanisms as targets for therapeutic drugs.
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PMID:Mechanisms of endocannabinoid inactivation: biochemistry and pharmacology. 1140 19

Endocannabinoids (endogenous ligands of cannabinoid receptors) such as anandamide (N-arachidonoylethanolamine) and 2-arachidonoylglycerol (2-AG) are inactivated upon enzymatic hydrolysis. Recent progress in the enzymological and molecular biological studies on the 'endocannabinoid hydrolases' is reviewed in this article. Anandamide is hydrolyzed to arachidonic acid and ethanolamine by a membrane-bound amidase generally referred to as fatty acid amide hydrolase (FAAH). This enzyme has a broad substrate specificity, hydrolyzing oleamide (an endogenous sleep-inducing factor) and 2-AG as well as anandamide. cDNA cloning revealed that FAAH is composed of 579 amino acids and belongs to the amidase signature family. A serine residue functioning as a catalytic nucleophile and several other catalytically important residues were identified in its primary structure. Furthermore, recent generation and analysis of the FAAH gene-deficient mice demonstrated the central role of this enzyme in the metabolism of anandamide. Alternatively, an amidase, which is distinct from FAAH but also hydrolyzing anandamide and other N-acylethanolamines at acidic pH, was identified in human megakaryoblastic cells and rat organs such as lung and spleen. As for the 2-AG hydrolysis, in addition to the known monoacylglycerol lipase, other esterases and FAAH may be involved.
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PMID:Endocannabinoid hydrolases. 1243 41

N-Acylethanolamines (NAEs) are endogenous constituents of plant and animal tissues, and in vertebrates their hydrolysis terminates their participation as lipid mediators in the endocannabinoid signaling system. The membrane-bound enzyme responsible for NAE hydrolysis in mammals has been identified at the molecular level (designated fatty acid amide hydrolase, FAAH), and although an analogous enzyme activity was identified in microsomes of cotton seedlings, no molecular information is available for this enzyme in plants. Here we report the identification, the heterologous expression (in Escherichia coli), and the biochemical characterization of an Arabidopsis thaliana FAAH homologue. Candidate Arabidopsis DNA sequences containing a characteristic amidase signature sequence (PS00571) were identified in plant genome data bases, and a cDNA was isolated by reverse transcriptase-PCR using Arabidopsis genome sequences to develop appropriate oligonucleotide primers. The cDNA was sequenced and predicted to encode a protein of 607 amino acids with 37% identity to rat FAAH within the amidase signature domain (18% over the entire length). Residues determined to be important for FAAH catalysis were conserved between the Arabidopsis and rat protein sequences. In addition, a single transmembrane domain near the N terminus was predicted in the Arabidopsis protein sequence, similar to that of the rat FAAH protein. The putative plant FAAH cDNA was expressed as an epitope/His-tagged fusion protein in E. coli and solubilized from cell lysates in the nonionic detergent, dodecyl maltoside. Affinity-purified recombinant protein was indeed active in hydrolyzing a variety of naturally occurring N-acylethanolamine types. Kinetic parameters and inhibition data for the recombinant Arabidopsis protein were consistent with these properties of the enzyme activity characterized previously in plant and animal systems. Collectively these data now provide support at the molecular level for a conserved mechanism between plants and animals for the metabolism of NAEs.
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PMID:Molecular identification of a functional homologue of the mammalian fatty acid amide hydrolase in Arabidopsis thaliana. 1282 67

Fatty acid amidohydrolase, a membrane-bound enzyme found in a variety of mammalian cells, is responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide. In an earlier study we reported that Tetrahymena pyriformis was able to secrete a FAAH-like activity in starvation medium (Karava V., Fasia L., Siafaka-Kapadai A., FEBS Lett. 508 (2001) 327-331). In this study the endocannabinoid anandamide, was found to be metabolized by T. pyriformis homogenate by the action of a FAAH-like enzyme, in a time- and concentration-dependent manner. The main metabolic products of [3H]anandamide hydrolysis were [3H]arachidonic acid and ethanolamine. Amidohydrolase activity was maximal at pH 9-10, it was inhibited by phenylmethylsulfonyl fluoride and arachidonyltrifluoromethyl ketone and was Ca2+ and Mg(2+)-independent. Kinetic experiments demonstrated that the enzyme had an apparent K(m) of 2.5 microM and V(max) of 20.6 nmol/min mg. Subcellular fractionation of T. pyriformis homogenate showed that the activity was present in every subcellular fraction with highest specific activity in the microsomal as well as in non-microsomal membrane fraction. Immunoblot analysis of selected subcellular fractions, using an anti-FAAH polyclonal antibody, revealed the presence of an immunoreactive protein with a molecular mass approximately 66 kDa similar to the molecular mass of the mammalian enzyme. In conclusion, this study demonstrates that a FAAH similar to the mammalian enzyme is present in a unicellular eukaryote, indicating the importance of FAAH activity throughout evolution. It also supports the notion that Tetrahymena species may be a suitable model for metabolic studies on endocannabinoids, as well as for the study of drugs targeted towards FAAH.
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PMID:Anandamide metabolism by Tetrahymena pyriformis in vitro. Characterization and identification of a 66 kDa fatty acid amidohydrolase. 1595 Oct 97


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